What is pharmaceutical organic chemistry pdf?

Pharmaceutical organic chemistry is the main branch of organic chemistry deals with the study of preparation, structure and reactions of organic compounds.

What is pharmaceutical chemistry laboratory?

This laboratory is meant for synthesis, purification, physicochemical studies, identification, and qualitative tests of organic, inorganic, and medicinal compounds .

What is the difference between pharmaceutical chemistry and medicinal chemistry?

The primary distinction between medicinal chemistry and pharmaceutical chemistry is that the former focuses on the research, development, and design of novel chemical compounds with the intention of employing them in the pharmaceutical industry, whereas the latter concentrates on the analysis of existing medications ...

What is the difference between pharmaceutical chemistry and analytical chemistry?

Analytical chemistry deals with the great variety of methods used to identify and quantify the chemical components of materials, while pharmaceutical chemistry focuses on aspects of drug design, synthesis, and manufacture.

Is pharmaceutical chemistry hard?

Studying medicinal or pharmaceutical chemistry can be difficult and you may need help with it. Fortunately, there are several ways to make it easier or more manageable.

What's the difference between pharmacy and pharmaceutical chemistry?

In the pharmaceutical industry, chemists develop combinations of compounds that can have medicinal value (in contrast, pharmacists mainly focus with safe and correct dosages of the compound ). And that's just one industry where you'll find chemists.

What is the difference between a pharmacist and a pharmacologist?

A pharmacist dispenses prescription medications and advises patients on their use. Pharmacology, a biomedical science, focuses on chemical drugs and how they affect the people and organisms that consume them. Pharmacologists are responsible for developing the drugs that pharmacists safely dispense to patients.

What is the role of pharmaceutical organic chemistry?

Organic chemistry is so vital in pharmacy that scholars argue it's the backbone of pharmacy. It forms the basis for understanding drug structure and function, crucial in developing and designing new drugs . Developing medications to treat various illnesses and diseases would be impossible without organic chemistry.

What is the difference between organic chemistry and pharmaceutical chemistry?

Medicinal chemistry aims to identify and develop new drugs that can be used to treat a variety of diseases and medical conditions, while organic chemistry is concerned with the synthesis and study of organic molecules, including natural products and synthetic compounds [7].

Is pharmaceutical organic chemistry hard?

Organic chemistry is a difficult subject ... and a challenging course . It covers a lot of demanding material. It uses its own language, and employs many very precise concepts, yet without referring to mathematical models. You can, however, become quite good at it in a semester or two.

What is the best definition of pharmaceutical chemistry?

Pharmaceutical chemistry is the study of drugs, and it involves drug development . This includes drug discovery, delivery, absorption, metabolism, and more. There are elements of biomedical analysis, pharmacology, pharmaco*kinetics, and pharmacodynamics. Pharmaceutical chemistry work is usually done in a lab setting.

(PDF) Pharmaceutical Chemistry Laboratory Manualphm.utoronto.ca/~ddubins/DL/PHC340Y_Lab_Manual.pdf · Pharmaceutical Chemistry Laboratory Manual ... Lab 17: Synthesis and Examination of

Student Name: _________________________________

Lab Group: _______ Locker #: _______

Pharmaceutical Chemistry Laboratory Manual

PHC 340Y 2017-2018

Laboratory Website: http://phm.utoronto.ca/pharmlab

http://phm.utoronto.ca/pharmlab

PHC 340Y Lab Manual 2017-2018

Pharmaceutical Chemistry Laboratory Manual

Pharmaceutics Principles and Evaluation

PHC 340Y 2017-18

Leslie Dan Faculty of Pharmacy University of Toronto

Lab Coordinator: Dr. David Dubins

[emailprotected]

mailto:[emailprotected]

This manual was written for use by students enrolled in the undergraduate Pharmacy Program at the Leslie Dan Faculty of Pharmacy, University of Toronto.

Permission is granted to copy the manual provided no charge is made beyond reasonable reimbursem*nt for duplication and handling costs, and provided that this notice is retained in all such copies.

This manual was compiled and edited by David Dubins and Adam Downie, Copyright © 2014, David Dubins, Copyright © 2013, David Dubins and Kaitlyn Willams, Copyright © 2012, David Dubins and Caitlin Westerhout, Copyright © 2011, David Dubins and Trisha Warren, Copyright © 2009, University of Toronto. The original laboratories for PHC 340Y were written by Barry Bowen, Ping Lee, and Robert B. Macgregor, Jr. Portions of this manual were adopted from

The oscillator circuit in this lab manual was generously redesigned by bioanalytical scientist and electronics enthusiast Andrew Cooper. The board layout and assembly was performed by David Dubins.

Special thanks to Matthew Marchment, Shankar Sethuraman, Alfred Chen, Sammy Zheng, Joanna Ma, Lutan Liu and Vinson Li for their efforts in the redesign of the CMC lab. Specific contributions included optimizing the experimental procedure to better detect phase inversion, ensuring a robust incoming voltage by introducing and modifying the 9V DC adaptors, developing and testing the metal electrodes, and revising the lab protocol.

Cover art was kindly provided by Misaki Kondo.

Contributions to the PHC 340Y Pharmaceutics Laboratory Manual were also made by James Rogers, University of Alberta.

This manual is supplemented by notices and information on Blackboard.

PHC 340Y Lab Manual 2017-2018

1 Preface

Table of Contents

Preface ..................................................................................................................................................... 6

Recommended Textbooks ..................................................................................................................... 7

Teaching Staff ............................................................................................................................................ 7 Role of Teaching Assistants ................................................................................................................... 8

Attendance ............................................................................................................................................ 8

Lateness Policy ........................................................................................................................................... 9 Laboratory and Lecture Schedule ............................................................................................................ 10 Locker Check-In / Check-Out ................................................................................................................... 12 Recording Data, Analysis, and Results ..................................................................................................... 14

Plagiarism and Falsification ................................................................................................................. 14

Clean-up Check-List ................................................................................................................................. 15 Assignment of Grades .............................................................................................................................. 15 Guidelines for Writing Pre-Labs, Worksheets and Individual Laboratory Reports .................................. 15 Laboratory Safety .................................................................................................................................... 18

Chemical Inventory .............................................................................................................................. 18

Labeling of Preparations ...................................................................................................................... 18

Chemical Disposal ................................................................................................................................ 19

Dress Code ........................................................................................................................................... 19

Dress Code Rationale ........................................................................................................................... 19

Working with Hazardous Chemicals .................................................................................................... 19

Emergency Response ........................................................................................................................... 20

In Case of Personal Injury .................................................................................................................... 20

In Case of Spills .................................................................................................................................... 21

In Case of Fire ...................................................................................................................................... 21

If the Fire Alarm Sounds ...................................................................................................................... 22

Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve ....................................... 23 Introduction ............................................................................................................................................. 23 Background .............................................................................................................................................. 23 Experiment Protocol ................................................................................................................................ 25

Part A. Preparing a Calibration Curve .................................................................................................. 25

Part B. Plotting Your Calibration Curve ................................................................................................ 27

Questions ................................................................................................................................................. 27

Lab 2: Preparation of pH Buffers ............................................................................................................ 28 Introduction ............................................................................................................................................. 28 Background .............................................................................................................................................. 28

Definition of pH and pKa ...................................................................................................................... 29

Buffer Capacity .................................................................................................................................... 34

Experiment Protocol ................................................................................................................................ 36 Part A. Preparing Sorensen’s Buffer .................................................................................................... 36

Part B. Preparing McIlvaines’s Buffer .................................................................................................. 37

PHC 340Y Lab Manual 2017-2018

2 Preface

Questions ................................................................................................................................................. 38

Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid ..................................... 39 Introduction ............................................................................................................................................. 39 Background .............................................................................................................................................. 39 Experiment Protocol ................................................................................................................................ 43

Part A. UV Absorbance Standard Curve of Sodium Salicylate ............................................................. 44

Part B. Determination of the Partition Coefficient .............................................................................. 45

Part C. Direct Measurement of the Partition Coefficient .................................................................... 46

Questions ................................................................................................................................................. 47

Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa ...................................... 48 Introduction ............................................................................................................................................. 48 Background .............................................................................................................................................. 49

pKa and Intrinsic Solubility ................................................................................................................... 49

Calculation of pHp ................................................................................................................................ 51

Polymorphism ...................................................................................................................................... 55

Experiment Protocol ................................................................................................................................ 56 Part A. Intrinsic Solubility Determination ............................................................................................ 56

Part B. Preparing Different Salts of Sulfathiazole ................................................................................ 57

Part C. Preparing Different Polymorphs of Sulfathiazole .................................................................... 57

Part D. pKa Determination ................................................................................................................... 58

Part E. Melting Point Determination ................................................................................................... 59

Part F. Macroscopic Evaluation ........................................................................................................... 60

Questions ................................................................................................................................................. 60

Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification ......................................................................................................................................... 61

Introduction ............................................................................................................................................. 61 Background .............................................................................................................................................. 62 Experiment Protocol ................................................................................................................................ 63

Part A. Co-solvency .............................................................................................................................. 63

Part B. Salt Selection ............................................................................................................................ 64

Part C. Polymorph Identification ......................................................................................................... 64

Questions ................................................................................................................................................. 64

Lab 6: Thermodynamics of Mixing – Enthalpy and Volume .................................................................... 65 Introduction ............................................................................................................................................. 65 Background .............................................................................................................................................. 65

Specific Heat Capacity ......................................................................................................................... 65

Partial Molar Quantities ...................................................................................................................... 68

Experiment Protocol ................................................................................................................................ 69 Part A. Calibration of the Calorimeter ................................................................................................. 70

Part B. Specific Heat Capacity of Copper Metal ................................................................................... 71

Part C. Heat of Reaction and Heat of Hydration .................................................................................. 71

Part D. Measurement of Molar Enthalpy of Reaction ......................................................................... 71

Part E. Illustration of Partial Molar Volume ......................................................................................... 71

Lab 7: Examination of Viscosity and Suspending Agents ........................................................................ 73

PHC 340Y Lab Manual 2017-2018

3 Preface

Introduction ............................................................................................................................................. 73 Background .............................................................................................................................................. 74 Experiment Protocol ................................................................................................................................ 79

Part A. Characteristics of a Polymeric Solution: Intrinsic Viscosity ...................................................... 79

Part B. Characteristics of a Polymeric Solution: Fluid Type ................................................................. 80

Part C. Measurement of the Sedimentation Rate of an Ion Exchange Resin (Glass Beads) ................ 80

Questions ................................................................................................................................................. 81

Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis .................................................................................... 82 Introduction ............................................................................................................................................. 82 Background .............................................................................................................................................. 83

Half-Life and Shelf-Life ......................................................................................................................... 85

Temperature dependency of Kinetics: The Arrhenius Equation.......................................................... 85

Kinetics of ASA Hydrolysis ................................................................................................................... 86

Calculating the Amount of ASA as a Function of Time ........................................................................ 87

Experiment Protocol ................................................................................................................................ 88 Part A. Acetylsalicylic Acid Hydrolysis – Effect of Temperature .......................................................... 89

Part B. Acetylsalicylic Acid Hydrolysis – Effect of Concentration ........................................................ 90

Part C. Acetylsalicylic Acid Hydrolysis – Effect of a Suspension........................................................... 90

Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement ........................................... 93 Introduction ............................................................................................................................................. 93 Background .............................................................................................................................................. 93 Experiment Protocol ................................................................................................................................ 99

Part A. Standard Curve: Salicylate ....................................................................................................... 99

Part B. The Diffusion Experiment ...................................................................................................... 100

Questions ............................................................................................................................................... 101

Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases ......................... 103 Introduction ........................................................................................................................................... 103 Background ............................................................................................................................................ 104 Experiment Protocol .............................................................................................................................. 108

Part A. UV Absorbance Standard Curve of Salicylic Acid ................................................................... 108

Part B. Ointment Base Preparation ................................................................................................... 110

1. Hydrocarbon Base .............................................................................................................................. 111 2. Absorption Base ................................................................................................................................. 112 3. Emulsion Bases W/O Type ................................................................................................................. 113 4a. Emulsion Bases O/W Type ............................................................................................................... 114 4b. Emulsion Bases O/W Type ............................................................................................................... 115 5. Hydrophillic/Water Soluble Bases ..................................................................................................... 115 6. Poloxamer Gel/Cream ....................................................................................................................... 116

Part C. Salicylic Acid Base Compounding and Drug Release .............................................................. 117

Part D. Using an Ointment Mill .......................................................................................................... 119

Results & Questions ............................................................................................................................... 119

Lab 11: Tonicity and Pharmaceutics ..................................................................................................... 121 Introduction ........................................................................................................................................... 121 Background ............................................................................................................................................ 122 Experiment Protocol .............................................................................................................................. 126

Part A. Determination of the Tonicity of Sodium Chloride Solutions ................................................ 126

PHC 340Y Lab Manual 2017-2018

4 Preface

Part B. Determination of the Tonicity of Atropine Sulfate Solutions ................................................ 127

Part C. Calculation and Preparation of an Isotonic Solution of Atropine Sulfate .............................. 128

Part D. Preparation of an Isotonic Phosphate Buffer ........................................................................ 128

Part E. Demonstration of the Action of a Hypotonic, Isotonic, and Hypertonic Sodium Chloride

Solution on Erythrocytes ................................................................................................................... 128

Questions ............................................................................................................................................... 129

Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water ..................................... 130 Introduction ........................................................................................................................................... 130 Background ............................................................................................................................................ 131 Experiment Protocol .............................................................................................................................. 137

Part A. Preparing the Solutions ......................................................................................................... 137

Part B. Phase Inversion ...................................................................................................................... 142

Questions ............................................................................................................................................... 144

Lab 13: Optimization of Powder Flow and Particle Size Determination ................................................ 146 Introduction ........................................................................................................................................... 146 Background ............................................................................................................................................ 147 Experiment Protocol .............................................................................................................................. 152

Part A. Compounding Powder Blends................................................................................................ 152

Part B. Determining Tapped Density ................................................................................................. 153

Part C. Determining the Angle of Repose .......................................................................................... 154

Part D. Determining Powder Flowability ........................................................................................... 154

Part E. Sieve Analysis ......................................................................................................................... 157

Questions ............................................................................................................................................... 158

Lab 14: Pharmaceutical Granulations ................................................................................................... 159 Introduction ........................................................................................................................................... 159 Background ............................................................................................................................................ 159 Experiment Protocol .............................................................................................................................. 161

Part A. Preparing a Standard Curve for Acetaminophen ................................................................... 161

Part B. Preparing the Powder Blends and Granulating ...................................................................... 162

Part C. Milling and Sizing ................................................................................................................... 163

Questions ............................................................................................................................................... 163

Lab 15: Tableting, Capsuling, and Dissolution Testing .......................................................................... 165 Introduction ........................................................................................................................................... 165 Background ............................................................................................................................................ 166

Tableting Methods............................................................................................................................. 168

Tablet Properties ............................................................................................................................... 168

Experiment Protocol .............................................................................................................................. 175 Lab Period 1: .......................................................................................................................................... 176

Part A. Tableting ................................................................................................................................ 176

Part B. Stability (Shelf Life) ................................................................................................................ 177

Demonstration: Tablet Coating ......................................................................................................... 178

Lab Period 2: .......................................................................................................................................... 180 Part C. Tablet Dissolution .................................................................................................................. 180

Part D. Formulating Capsules ............................................................................................................ 180

Part E. Content Uniformity: Tablets and Capsules............................................................................. 181

PHC 340Y Lab Manual 2017-2018

5 Preface

Lab Period 3: .......................................................................................................................................... 181 Part F. Capsule Dissolution ................................................................................................................ 181

Part G. Detection of Degradation Products / Decomposition: Thin Layer Chromatography ............ 182

Summary of Formulation Testing ...................................................................................................... 184

Questions ............................................................................................................................................... 184

Lab 17: Synthesis and Examination of Colloids ..................................................................................... 186 Introduction ........................................................................................................................................... 186 Background ............................................................................................................................................ 186 Experiment Protocol .............................................................................................................................. 187

Part A. Yttrium Citrate Colloid ........................................................................................................... 188

Part B. Rhenium Heptasulphide Colloid, Method 1 ........................................................................... 189

Part C. Rhenium Heptasulphide Colloid, Method 2 (Performed as a Demonstration only) .............. 189

Part D. Analysis of Colloids ................................................................................................................ 190

Questions ............................................................................................................................................... 191

Lab 18: Formulating Using Molds ......................................................................................................... 192 Introduction ........................................................................................................................................... 192 Background ............................................................................................................................................ 192 Experiment Protocol .............................................................................................................................. 199

Part A. Formulating 325 mg Acetaminophen Suppositories (Calibrated Batch Volume Method) .... 200

Part B. Formulating 20 mg Benzocaine Lollipops (Mass of Drug Negligible) ..................................... 202

Part C. Formulating 20 mg Hydrocortisone Troches (Displacement Factor Method) ....................... 204

Part D. Double Casting Method: 70 mg Hydrocortisone/150 mg Lidocaine Lip Balm ....................... 206

Questions ............................................................................................................................................... 208

APPENDIX ............................................................................................................................................ 209 U.S. Standard Sieve Sizes and Lab Sieve Inventory ................................................................................ 209 Quadro Comil Meshes ........................................................................................................................... 210 Methocel and Avicel Grades .................................................................................................................. 211 Avicel Grade Usage Chart ...................................................................................................................... 213 Capsule Properties ................................................................................................................................. 214 Working Ranges of Typical Granulating Fluids....................................................................................... 215 Viscosities of Typical Fluids .................................................................................................................... 215 Powder Flowability Indices .................................................................................................................... 215 Average HLB Values of Some Surface Active Agents ............................................................................. 216 General Physical Properties of Spans and Tweens ................................................................................ 218 HLB Requirement for Some Common Oil Components ......................................................................... 220 Buffer Solution Preparation: Polyprotonic Acids and Bases .................................................................. 221 Dissociation Constants of Acids in Aqueous Solutions at 25°C .............................................................. 222 Dissociation Constants of Bases in Aqueous Solutions at 25°C ............................................................. 222 Sorensen Phosphate Buffers .................................................................................................................. 223 Fundamental Lab Calculations ............................................................................................................... 223

Preparing a Known Molar Concentration .......................................................................................... 223

Weight-Volume Percent (%w/v) ........................................................................................................ 224

Dilution Equation ............................................................................................................................... 224

How to Use a Syringe Filter ............................................................................................................... 226

Capsule Filling: Quality Control.......................................................................................................... 227

YOUR NOTES .......................................................................................................................................... 229

PHC 340Y Lab Manual 2017-2018

6 Preface

Preface There are a lot of rules and guidelines that accompany working in a laboratory, as there is a lot of potential for you harming equipment, or far worse, the equipment harming you. Rising above the details, there are three basic tenets that will permeate through each laboratory:

1) Be aware of the specific hazards and protect yourself accordingly;

2) Think about the exercises as you are doing them, and learn the techniques and principles behind them;

3) Have fun! A lab is a refreshing change from the classroom, where you get to try things out, rather than just being told how they work.

Concepts in these labs are used in pharmaceutical industry, in pharmacies, and in research, particularly with respect to drug formulation, manufacture, and compounding. The protocols outlined in the labs provide suggestions on how to observe the phenomena of interest. However, there is more than one way to accomplish something, and there is certainly more than one way to measure something. In many cases, common sense will play an important part of your lab work. For instance, is it more accurate to measure out 5 mL of de-ionized water in a 10 mL graduated cylinder, or a 100 mL graduated cylinder?

Subtle methods in the labs may be changed by your instructor, TA, or even by you, depending on the equipment and supplies available to you on your lab day. There is room for creativity.

If you find a specific section, step, or explanation in this manual vague or difficult to follow, ask your TA or instructor for help. Please let us know, so we can improve the manual for future editions.

The following icons are used in the margins throughout this manual:

Useful tip on an experimental method. Read carefully.

Important discussion point that is particularly useful in understanding the exercise.

Important safety tip.

Time-critical experimental step.

PHC 340Y Lab Manual 2017-2018

7 Introduction

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Introduction

General Information

Check-in for the laboratory will be on September 15, 2017, during the first laboratory session. During the check-in, you will be given your locker key and should make sure all the equipment in your locker is complete and clean.

To prepare for a lab, read the part of the lab manual pertaining to that lab exercise, understand the rationale of the exercise, watch any associated videos on the laboratory website, perform any calculations that may be necessary to prepare for the lab, be aware of any potential hazards, review the questions, and go to bed early the night before. The laboratories start on time. There will be various “surprise” pre-lab quizzes during the pre-lab tutorials. They will consist of five or six short questions related to the experiments being performed during that session. Students who are late are not eligible to write the quiz.

Recommended Textbooks

There are no required textbooks for the PHC340 laboratory. This manual will serve as the primary reference to the laboratory. The following textbooks are recommended to clarify concepts or to serve as useful general references:

1. Sinko, Patrick J. Martin’s Physical Pharmacy and Pharmaceutical Sciences. Lippincott Williams & Wilkins; 6 edition (Feb 21, 2010)

2. Troy, David B. Remington – The Science and Practice of Pharmacy. Lippincott Williams & Wilkins; 21 edition (May 19, 2005)

3. Aulton, Michael E. Aulton’s Pharmaceutics: The Design and Manufacture of Medicines. A Churchill Livingstone Title; 3 edition (Nov 1, 2007)

4. Allen, Loyd V. Jr. et al. Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Lippincott Williams & Wilkins; 9 edition (Jan 7, 2010)

5. Rowe, Raymond C et al. Handbook of Pharmaceutical Excipients. Pharmaceutical Press; 6th edition (2009). Available online, U of T Library permalink: http://simplelink.library.utoronto.ca/url.cfm/141954 (UtorID login required)

Teaching Staff

The following people will be teaching, helping, and evaluating your work in the lab:

PHM340Y Laboratory Coordinator E-mail Address

David Dubins [emailprotected]

PHM340Y Teaching Assistants E-mail Address

Noor Al-Saden [emailprotected]

Giovanna Schver [emailprotected]

http://simplelink.library.utoronto.ca/url.cfm/141954

mailto:[emailprotected]

PHC 340Y Lab Manual 2017-2018

8 Introduction

Role of Teaching Assistants

One Teaching Assistant (TA) will be assigned to each laboratory period. The following are the roles and duties of the TA:

Before the lab:

Ensure the availability of chemicals and supplies, and inform the Instructor if orders are required in advance

Work with the instructor to ensure equipment in their assigned section is set up, functional and serviced

Prepare buffers, reagents, and indicators in advance

Arrive before the lab in order to warm up any relevant equipment and appropriately set up the lab

During the lab:

Take attendance, checking student TCards

Ensure laboratory safety

Notify the Instructor of any injuries or hazards in the lab

Handling of the disposal of hazardous chemicals

Provide pre-laboratory lectures in an interactive format

Supervising students regarding procedure, process, technique and safety elements of laboratory session

Coordinate equitable access to equipment

Collect student attendance via sign-in sheets

Supervise the progress of student work - by asking appropriate questions, not only by providing answers

Provide directions and clarify instructions

Ensuring the cleanliness of the lab, and coordinating laboratory clean-up

Ensure equipment is clean and shut down at the end of the lab (especially spectrophotometers)

After the lab:

Assisting in lab check-in and check-out

Evaluate submitted laboratory reports, quizzes, products and work plans

Recording and entering marks on the Blackboard system

Attending and supervising student tours

Attendance

Attendance in labs is mandatory. Attendance in each lab, and the lab tour, will be recorded. If you miss a lab or lab tour due to medical, personal, family, or other unavoidable reasons, you must provide supportive documentation (e.g. a doctor’s note) to the course Instructor for consideration of accommodation. The U of T Verification of Illness or Injury form is available online at:

http://www.illnessverification.utoronto.ca/

If accommodation is granted, you will be asked to complete a make-up assignment on the same topic of the missed course material. Otherwise, if you miss a single laboratory session, you will obtain a zero for that laboratory. If you miss one laboratory session of a laboratory that spans

http://www.illnessverification.utoronto.ca/

PHC 340Y Lab Manual 2017-2018

9 Introduction

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Raw Score _____

-1% Cleanliness

-1% Timeliness Pre-Lab Penalty (max 5%) _____ Late Penalty (10%/day) _____

more than one session, your final mark will be multiplied by the ratio of the number of sessions you attended, provided that you participate in writing the final report.

Lateness Policy

You may submit lab reports via email, or by hard copy. Labs are generally due one week from performing the lab, by the beginning of class. For late submissions, there will be an academic penalty imposed of 10% per day, in accordance with departmental policies. Submissions will not be accepted beyond 1 week from the original due date.

Each lab will also be subject to a cleanliness and timeliness penalty. Cleanliness penalties will be issued if a lab area or lab scale is left untidy. Timeliness penalties will be issued if you remain inside the lab more than 10 minutes past the end of the scheduled lab. Budget your time in the lab to allow for sufficient clean-up once you are finished.

Lab worksheets, when made available, will have the following box beside the final score to indicate if a penalty has been issued:

PHC 340Y Lab Manual 2017-2018

10 Introduction

Laboratory and Lecture Schedule

All pre-labs and labs will take place in PB860.

Fall Term 2017

Labs (PB860)

Thursday 9am-1pm

Lectures (PB255) Monday

3pm-4pm

Lecture 1 – Acid/Base Equilibria (Keith Pardee) 8am-9am, 07-Sep-17

Lab 1* – Safety Lecture, Locker Check-in, Examination of UV Spectroscopy and Preparation of a Standard Curve

07-Sep-17

Lecture 2 – Phase Partitioning (Keith Pardee) 11-Sep-17

Lab 2 – Preparation of pH Buffers 14-Sep-17

Lecture 3 – Mixing (Keith Pardee) 18-Sep-17

Lab 3‡ – Effect of pH on the Partition Coefficient of a Slightly Soluble

Weak Acid 21-Sep-17

Lecture 4 – Polymorph and Salt (Ping Lee) 25-Sep-17

Lab 4* - Characterization of Drug Candidates (I) – Measuring Solubility and pKa

28-Sep-17

Lab 5* – Characterization of Drug Candidates (II) – Co-solvency, Salt Selection and Polymorph Identification

05-Oct-17

Lab 6* – Thermodynamics of Mixing – Enthalpy and Volume Workshop: Writing Formal Lab Reports for PHC 340 (Heather Sanguins)

12-Oct-17

Lecture 5 – Rheology (Keith Pardee) 16-Oct-17

Lab 7* – Examination of Viscosity and Suspending Agents 19-Oct-17

Lecture 6 – Chemical Kinetics & Stability (Ping Lee) 23-Oct-17

Lab 8‡ – Kinetics of Acetylsalicylic Acid Hydrolysis 26-Oct-17

Lecture 7 – Diffusion and Membrane Transport 1 (Ping Lee) 30-Oct-17

Lab 9 – Diffusion and Membrane Transport 1: Permeation Measurement [Exercise 2: Quassignment for Lab 11]

02-Nov-17

Lecture 9 – Colligative Properties (Keith Pardee) (intentionally out of order)

13-Nov-17

Lab 11* – Tonicity and Pharmaceutics (intentionally out of order)

16-Nov-17

Lecture 8 – Diffusion and Membrane Transport 2 (Ping Lee) 20-Nov-17

Lab 10‡ – Diffusion and Membrane Transport 2: Drug Release from

Ointment Bases 23-Nov-17

Lecture 10 – Molecules at Interfaces (Keith Pardee) 27-Nov-17

Lab 12* – Estimation of Critical Micelle Concentration (CMC) of a Surfactant in Water

30-Nov-17

Lecture 11 – Particle Size and Powder Flow (Ping Lee) 04-Dec-17

Winter Term 2018 Labs

Thursday 1pm-5pm

Lectures Wednesday

11a-12p

Lab 13* – Optimization of Powder Flow and Particle Size Determination 04-Jan-18

Lecture 12 – Pharmaceutical Granulation (Ping Lee) 10-Jan-18

Lab 14 – Pharmaceutical Granulations, Part 1 11-Jan-18

PHC340 Midterm 17-Jan-18

Lab 14‡ – Pharmaceutical Granulations, Part 2 18-Jan-18

Lecture 13 – Tabeting and Dissolution Testing (Ping Lee) 24-Jan-18

Lab 15 – Tableting and Dissolution Testing, Part 1 25-Jan-18

Lecture 14 – Measurement, Part 1 (Keith Pardee) 31-Jan-18

Lab 15 – Tableting and Dissolution Testing, Part 2 01-Feb-18

PHC 340Y Lab Manual 2017-2018

11 Introduction

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Lecture 15 – Measurement, Part 2 (Keith Pardee) 07-Feb-18

Lab 15‡ – Tableting and Dissolution Testing, Part 3 08-Feb-18

Lecture 16 – Synthetic Biology and Human Health, Part 1 (Keith Pardee) 14-Feb-18

Lab 16 – Mystery Laboratory 15-Feb-18

Lecture 17 – Synthetic Biology and Human Health, Part 2 (Keith Pardee) 28-Feb-18

Reading Week Feb 20-23

Lab 17‡ – Synthesis and Examination of Colloids 01-Mar-18

Lecture 18 – Mold Calculations (David Dubins) 07-Mar-18

Lab 18* – Formulating Using Molds 08-Mar-18

Lab 19 – Advanced Formulations Project, Part 1 15-Mar-18

Lecture 19 – Ethics & Academic Integrity (Alison Thompson) 21-Mar-18

Industrial Tour (to be confirmed) 22-Mar-18

Lecture 20– Ethics & Academic Integrity (Alison Thompson) 28-Mar-18

Lab 19‡ – Advanced Formulations Project, Part 2. Lab Check-Out. 29-Mar-18

PHC340 Final Exam During final exam period ‡ Lab Report to be completed for evaluation

* Lab Worksheet to be completed for evaluation

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12 Introduction

Locker Check-In / Check-Out

Check-In: September 15th, 2017 Your Locker #: ____________

You will be assigned your own locker. The contents of your locker have been arranged by students of previous years. It is your privilege to use the locker and your responsibility to maintain the locker. Today, make sure you have all the glassware according to the “Content of your Locker” list. You may also want to clean the glassware. Replacement of damaged equipment can be obtained from the back shelves or from your Teaching Assistants (TAs).

Take time to review the laboratory safety section of this manual and locate the following safety equipment in the laboratory. Indicate the location in the space provided below:

Safety Equipment Location

Fire Extinguishers

Fire Alarm

Eye Wash Fountains

Safety Shower

First Aid Box

When your group has completed the locker check-in, notify your teaching assistant and he/she will ask you a few safety questions.

Locker key issued _________________________ (student signature)

Locker Check-Out: Marth 30th, 2018

A fee of $10.00 will be charged for locker key replacement. You are encouraged to attach the key to a secure key ring or case.

Keep this and the following page in your laboratory manual.

Lab Check-Out Procedure Clean your lab bench and any dirty glassware;

Throw out any remaining formulations or garbage;

Empty your locker water bottle;

Verify that your locker contents are complete;

Return any extra glassware to the laboratory back shelves;

Get a TA or Instructor to verify the above, and sign your check-out list (next page);

Be assigned a special area in the lab to clean;

Lock your locker, and return your lab key when the above is completed.

PHC 340Y Lab Manual 2017-2018

13 Introduction

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Locker Contents 2017-2018 Student Name Locker # Date

Name of Apparatus/Item Qty In Out 50 mL Volumetric Flask 2 100 mL Volumetric Flask 2 200 mL or 250 mL Volumetric Flask 3 10 mL Graduated Cylinder 1 25 mL Graduated Cylinder 1 100 mL Graduated Cylinder 2 50 mL Erlenmeyer Flask 2 125 mL Erlenmeyer Flask 2 250 mL Erlenmeyer Flask 2 500 mL Erlenmeyer Flask 2 5 cm Glass Funnel 1 7.5 cm Glass Funnel 1 10 cm Glass Funnel 1 Test Tube Rack 1 1 mL Bulb Pipette 1 5 mL Bulb Pipette 1 10 mL Bulb Pipette 1 20 mL or 25 mL Bulb Pipette 1 1 mL Graduated Pipette 1 10 mL Graduated Pipette 1 Thermometer (°C) 1 Watch Glass small 1 Watch Glass large 1 8” Glass Stirring Rod 1 50 mL Beaker 2 150 mL Beaker 2 250 mL Beaker 2 400 mL Beaker 2 600 mL Beaker 2 Glass Slab 1 3” Ceramic Evaporating Dish 1 6” Ceramic Evaporating Dish 1 Plastic Wash Bottle 1 Ceramic Mortar & Pestle Set (Glass set additional in some cases) 1 Funnel Clamp and Holders 1 Sharpie Lab Marker 1

TA Signature - Lab Check-In TA Signature - Lab Check-Out

Volumetric Flask

Graduated Cylinder

Erlenmeyer Flask

Bulb Pipette

Grad. Pipette

Watch Glass

Beaker

Evaporating Dish

Mortar & Pestle

Funnel Clamp

PHC 340Y Lab Manual 2017-2018

14 Introduction

Recording Data, Analysis, and Results

In this laboratory, we are attempting to introduce laboratory practices that are employed in research and development labs in the pharmaceutical industry. Practices such as daily initialing of laboratory results and the use of bound books are used to increase security and in some cases to document intellectual property.

Hard cover bound laboratory notebooks will be used to record your data. At the beginning of selected laboratories, the TA will give you a laboratory worksheet which you will use to record your data, present your results and interpretation for grading. The worksheets will also contain specific questions to answer about the labs. Other times you will record data in your lab book.

During the laboratory, you may work in groups of two, and sometimes in larger groups, for the collection of data. Any data that is collected during the lab period must be recorded in each member of the group’s data booklet. The analysis of the data, presentation and calculations are to be done individually, and recorded in the appropriate section in the data booklet.

All lab books are to be initialed by both the student and an instructor at the end of each laboratory session. It is your responsibility to make sure that your book is initialed. Books will be initialed after satisfactory laboratory clean-up has been completed.

A lab has a lot of potential for entropy (read: mess). Please keep your lab area tidy.

The lab reports will be graded according to the Report Format which is outlined in this introduction. In some cases, grades may also be assigned to the quality of the product, and product label that you made during the lab. Lab reports will be not be returned to you until all students in the class have completed the same exercise.

In addition to the lab reports and quizzes, there will be a written exam in December as part of the PHM340 Mid-term exam. Questions related to the work in the Winter Term will be included in the April final exam.

Try to work cooperatively with others in your group. If there are unresolved conflicts, approach your TA or the lab coordinator to seek a solution.

Plagiarism and Falsification

At some point in your laboratory, you might look at your results and think,

“OH NO! This can’t be right!”

You will be nervous. You will wonder what happened. What went wrong? Worse off, you might be tempted to misreport the results for that ONE point that should have fallen on the line.

However, you are reminded to always report what you observed, rather than what you would have liked to observe. Provided you made the correct calculations and performed your exercises meticulously and carefully, you will not lose marks for less than perfect looking observations. Real data rarely look perfect. Things don’t always work. If they did, there would be no need for formulation scientists.

If you encounter suspicious looking data, identify your concerns in your analysis, and explain where you think things may have gone wrong (sources of error). If your entire data set is concerning you, seek the assistance of your T.A. or instructor. There could be a malfunction in the equipment, a problem with the method, or a systematic error in your calculations. If you have time, you can repeat the outlying measurements to refute or confirm their validity.

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DO NOT PLAGIARISE OR FALSIFY YOUR DATA. Doing so is an offence under the University of Toronto Governing Council’s Code of Behaviour on Academic Matters.

Clean-up Check-List

Your experiment is done. Are you all ready to go? Here are some helpful tips on leaving the lab clean for the next group of students:

I cleaned all lab equipment (especially balances!), so other students can use them.

I rinsed out my pipettes and burettes with water, so crystallization won’t gum up the tips.

I washed and shook out all my glassware, and put it back in my locker so it’s clean for my next lab.

I wiped my work area, lab bench, and bench top (including the balances I used).

I properly labeled and handed in my preparation (if there is one to hand in).

I properly disposed of all chemicals:

solid and semi-solid inert waste in the garbage,

liquid inert waste down the sinks

hazardous chemicals in appropriately labeled waste bottles in the fume hoods

I double-checked the fume hood. It’s clean, and I didn’t leave anything in it.

Assignment of Grades

Laboratory Reports*, Exercises 65 % Quizzes and Problem Sets (weighted equally) 5 % Mid-term and Final exam (weighted equally) 30 % Total 100 %

*Lab reports are weighted in proportion to the number of lab periods.

Guidelines for Writing Pre-Labs, Worksheets and Individual Laboratory Reports

Pre-Labs

Prior to the lab, regardless of whether a worksheet or formal lab report is assigned, you will be expected to prepare a pre-lab in your lab notebook, which should include the following sections:

Purpose: Why are you doing this lab? What scientific questions will be addressed? Procedures: In flow-chart form, organize your activities in the lab. This will help you

prepare for complicated procedures, and allow you to be more efficient in the lab.

Pre-labs will be checked at the beginning of the lab, and will be worth 5% of the lab report or worksheet mark. Preparing a proper pre-lab will help you succeed in surprise quizzes.

Individual Lab Worksheets

For selected labs, worksheets will be handed out in the beginning and will be made available for download from Blackboard. For these labs, filling out the worksheets and answering the worksheet questions is all that is required for the lab. For these labs, the mark breakdown will be indicated on the worksheets.

PHC 340Y Lab Manual 2017-2018

16 Introduction

NOTE: Where applicable, your submitted, properly labeled product will constitute a proportion of the “Presentation, neatness” component.

Other laboratories will involve creating a formal lab report. The following is a guide on what is expected for these reports. As each lab is individual, the marking scheme may vary slightly for each lab.

Rationale of Laboratory Reports

The purpose of writing a scientific report is to communicate your findings with the outside world. Enough detail should be conveyed so that someone who did not do the experiment could repeat it, and be able to fairly compare their results with yours. Writing laboratory reports (and technical writing in general) is an extremely useful and valuable skill to develop. Avoid providing one word answers and bullet points. Use sentence form, and summarize where appropriate. The ability to condense the purpose, observations, and results into an abstract will help the reader connect with the material, and will put your results in perspective for the reader. This process will help prepare you for writing scientific publications.

Be consistent with grammar. For events that happened in the lab, use the past tense for reports, and the passive voice.

e.g.: “1 mg of the free acid of sulfathiazole was incubated at 25 C in 10 mL of phosphate buffer for 1 hour, with agitation every 15 minutes.”

For scientific principles, use the present tense.

e.g.: “Ethanol is a co-solvent, and disrupts the hydrogen bonding between water molecules and the surface of the drug molecule.”

Details on Writing a Formal Laboratory Report

1. Pre-Lab (5%) Your pre-lab mark will be evaluated at the beginning of each laboratory.

2. Title Page (1%)

Please include lab number and title, student name(s), date submitted, and course code.

3. Abstract (10%)

No more than 200 words, an abstract is a mini-version of the entire lab report. It provides a brief introduction, purpose, a summary of results (not the raw data itself but parameters estimated), conclusions, and the relevance of the conclusions to the field of study. It is usually the last section that you will write, although it comes first in the report.

4. Introduction (5%)

This section should be 1-2 paragraphs long, and include the purpose of the experiment and a brief overview.

What is the main purpose of the lab? Which scientific principles are being investigated? What is the value of the results to the field of study? A good introduction will spark the interest of the reader and explain the purpose of the work.

5. Experimental (10%)

This section should be no more than 2 pages long, but depending on the

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17 Introduction

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experiment, may only be a few paragraphs. Do not copy and paste the methods section from the lab manual – this is a protocol. The purpose of the methods section is to summarize what you did with sufficient detail for someone to repeat the experiment, without getting into step-by-step instructions.

Provide details of the chemicals you used. Key equipment (e.g. a UV spectrophotometer) should be mentioned; however, glassware (e.g. 100 mL graduated cylinder) should not unless it was integral to the method (e.g. tapped density).

e.g.: “A standard curve of salicylic acid was prepared by diluting a standard solution of 0.2 M sodium salicylate at ratios of 1:50, 1:100, 1:200, 1:250, and 1:500. The assay procedure involved adding 1 mL of sample with 5 mL of de-ionized water and 2 drops of ferric chloride TS. Absorbance was measured at 525 nm in a UV spectrophotometer.”

Document what you actually did, not what you were supposed to do. If there was a change or deviation from the lab manual, describe it. Explain what you did in chronological order (the order that you did things in the lab).

6. Results (30%)

The length of your results section will depend on the experiment.

All of your data and observations go into this section, in table form. Attach any graphs printed out in the lab. This should be the easiest section to write.

Provide sample calculations for key elements of the lab: dilutions, standard curve use, etc.

Make sure you:

Properly label all graph axes;

Always report the units with each measurement;

Report your parameters with the appropriate number of significant digits (e.g if the pH meter reads 2 decimals, don’t report a pKa of 6.39281);

State final estimated parameters in sentence form briefly.

e.g.: “The pKa of sulfathiazole was estimated to be 5.98.”

7. Discussion (35%)

The discussion section will likely be the longest section, and should be no less than 2 pages long. It is your chance to demonstrate your understanding of the lab. For the majority of labs, the scientific principles are discussed in the Background section of each lab in this manual. They will lay the foundation of your discussion, but it is up to you to make the link between the scientific principles, and the data you collected in the lab.

Answer any discussion questions at the end of the lab protocol (10%)

Summarize the key scientific idea(s) behind the lab. If there was a key equation (e.g. Hendersson-Hasselbalch), report it here and describe its significance.

Did the results confirm or refute the scientific principles involved? Discuss the precision of your data (e.g. how good the r2 was of a fitted linear regression). Were the results obtained what you expected? Sometimes in the lab you may observe a trend opposite to what you were expecting. It is up to you to either re-evaluate your understanding of the phenomena, or try to identify the sources of error. Some reasons may include:

Limitations on the sensitivity of the instruments (noise)

Improperly performed calculations before or during the lab

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18 Introduction

Deviations from the lab protocol

Errors in the lab protocol

Limitations of the method used to evaluate the phenomena of study

Equipment malfunction or improper use of the equipment

If the error was a result of experimental design, suggest how the design could be improved.

If relevant, put your results in the context of literature values. Were they in agreement? e.g.: The pKa of sulfathiazole was estimated to be 5.98. This is not in good agreement with a published value of 7.14 (reference 1).

You may also discuss other related theories.

8. Conclusions (4%)

Conclusions are relatively short compared to the discussion. They are typically 1-2 paragraphs, and serve as the bottom line of the lab. In sentence form, report the final estimated values of parameters, and summarize the results/discussions with a closing thought. Recommendations for future work or how the lab could change may also be included here.

9. References

Include literature references you referred to in this section. If you did not refer to the references in the laboratory manual, you do not need to include them here. e.g.:

(1) Fioritto AF et al., Int J Pharmaceutics (2007);330:105-113.

10. Appendices

You may include extra calculations, additional information, and supplementary analyses attached as appendices.

Make sure you staple your lab report together, and that you present your work neatly. At your option, you may submit the report in a folder.

Laboratory Safety

Chemical Inventory

A complete chemical inventory for PB 860 is located through the lab website:

http://pb860.pbworks.com/w/page/41084070/PB860-Chemical-Inventory

In consideration for others, be frugal with chemicals and buffers – take only what you need.

Return the balance of chemicals to the TA’s cart or the Preparation Room (Room 865) when you are finished with them.

Replace the caps of chemicals when you are finished weighing them.

Use the fume hood when handling flammable or volatile solvents.

Avoid leaving unlabelled weighing boats filled with white powder by the scales. Not only is this wasteful, but it is dangerous as well.

Labeling of Preparations

“What was in that beaker again? It looks like water…”

Nothing is more frustrating than spending an hour to make a product, and then

http://pb860.pbworks.com/w/page/41084070/PB860-Chemical-Inventory

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19 Introduction

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forgetting which beaker you poured it in. It will save you aggravation to get in the habit early of clearly labeling your preparations as you go along.

Chemical Disposal

There are large green buckets available for broken glassware in the lab. Please use them instead of the garbage, to respect the safety of the cleaning staff.

There will be designated waste jars for hazardous waste and organic solvents in the fume hoods for each lab. When appropriate, there will also be a designated container for sharps (e.g. needles).

Solid and semi-solid chemically inert waste (e.g. petrolatum) will gum up the drains, and are properly disposed of in the garbage.

If you are unsure how to properly dispose something, ask your TA or instructor.

ACIDS CORRODE PIPES, AND SHOULD BE DISPOSED OF IN WASTE BOTTLES ONLY.

Dress Code

For your protection, you are required to wear the following protective gear, at all times during the lab:

A lab coat

Safety Goggles

Closed-Toed Shoes (no sandals or open-toed shoes)

Clothing that covers your legs

The following special protective equipment is available for specific tasks, or on your request:

Latex (and non-allergenic neoprene) gloves

N95 Masks

Protective hair covers

Dress Code Rationale

If you have ever taken a laboratory course, you have likely already heard much of the following safety advice at some point. Common sense plays a large part in lab safety. However, it is useful to outline a few principles that pertain to the labs in this manual, so they are fresh in your mind.

Laboratory coats offer first line protection to your clothes and body against chemical burns. They work best when they are done up – an open lab coat will not properly protect you from a spill.

Closed-toed shoes protect your feet from chemical spills.

Safety glasses will help to shield your eyes from any chemical splashes, including boiling solutions.

Latex (and nitrile) gloves are available for use in the laboratory. In particular, hydrochloric acid (HCl), potassium hydroxide (KOH), and sodium hydroxide (NaOH) are extremely corrosive. Gloves should be worn if you are going to be handling these solutions. Gloves also offer protection if you have a known specific allergy or sensitivity to a certain chemical.

Working with Hazardous Chemicals

When in doubt, treat all chemicals as hazardous, until you are familiarized with their

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20 Introduction

properties. Consult the Material Safety Data Sheets (MSDS) or your TA for relevant information.

Whenever possible, or necessary, handling chemicals in a fume hood will protect you as well as those around you from toxic and flammable fumes.

Handle all volatile and flammable solvents in a fume hood.

Do not put a sealed container over any heat source, as it may explode.

If you are not sure how to use something, ask your TA.

Notify your TA if there is any broken glassware, so they can safely clean and dispose of any chemical or sharps hazards.

Notify your TA immediately if there is a mercury spill. They will have access to a mercury spill kit.

Be cautious when testing for odours. Never inhale a chemical directly. Fan the vapours towards your nose. Many vapours can cause irreparable damage.

Never ingest any excipients or products in the teaching laboratory.

Other safety references:

o Merck Index o Material Safety Data Sheets (MSDS), a part of the WHMIS (Workplace

Hazardous Material Information System) right-to-know system o Fisher Scientific Catalog o Sigma-Aldrich MSDS

Follow these guidelines to decrease the risks of working with chemicals:

Work with a minimum amount of chemicals necessary.

Read the warning labels and/or consult the MSDS before using a chemical.

When storing, using or disposing of chemicals, avoid accidental mixing of incompatible chemicals such as acids and bases, flammables and toxics, flammables and oxidizers, oxidizers and reducers.

Highly toxic and flammable chemicals must be stored in ventilated areas in unbreakable, chemically resistant containers.

Emergency Response

The University Emergency phone number is 416-978-2222.

In Case of Personal Injury

Inform the Teaching Assistant, or the Laboratory Coordinator of any injury acquired during a lab, no matter how slight it may appear.

An open or even partially healed cut is dangerous, since it allows easier penetration of chemicals. Cover any exposed areas with a bandage when working in the laboratory. Protective latex gloves are available from your TA.

In case of chemical eye injury, hold the eye open in the eye-wash, even if painful, and wash the eye for 15-20 minutes.

In case of chemical body burns, use cold water to wash chemicals from the skin immediately, and thoroughly. Hot water may increase the absorbency of the chemical.

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21 Introduction

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In Case of Spills

Chemicals spilled in the laboratory must be cleaned up immediately to reduce and eliminate hazards. The Chemical Spill Cart is located in the laboratory outside the entrance of Room 865.

In the event of a localized, minor spill, use the following procedure:

In the event of a major spill that exceeds the clean-up capabilities of the laboratory, the following procedure is to be followed:

In Case of Fire

If the fire is contained in beakers or flasks, smother the fire simply by covering the vessels so that no oxygen can enter.

If electrical equipment is on fire, unplug it quickly or cut the power if possible.

If your clothing is on fire, do not run. Stop, drop, and roll. If the clothing of someone next to you is on fire, help him to the floor and use your lab coat or fire blanket, or whatever is available to smother the fire. Once the fire is extinguished, help the person away from the general fire area.

If the fire is small and contained, a qualified person should attempt to use a fire extinguisher to eliminate the fire. Many fire extinguishers handle multiple types of fires. There are 4 major classes:

Responding to a Major Spill

Notify everyone to evacuate the area immediately.

Contact the University of Toronto Emergency Number 416-978-2222 and state the location of spill, extent of the spill, and the chemical involved.

Or, call 911.

Wait in a safe area until the response team arrives.

Responding to a Minor Spill

Report all spills to the TA. Notify other students who are working in the area.

Confine the spill to a small area. Do not allow the spill to spread.

If the material involved is flammable, turn off any ignition sources/electrical equipment present.

Ventilation should be established to dispel vapour, if necessary, and if safe to do so.

Absorb and neutralize the spilled liquid chemical. For example, strong acids should first be neutralized with sodium bicarbonate, then washed with water. It is always advisable to add acid into water when mixing, since water has a much larger heat capacity and will therefore be able to absorb any resulting heat much better. You can always remember the catch phrase: “Do as you aughta, add acid to watah”.

The TA or Instructor should handle a mercury spill. Spilled mercury is collected with a mercury collector. Sprinkle the affected area with sulfur powder. The sulfur-mercury powder is then swept up and discarded in the appropriate labeled container.

When cleaning up a spill, wear the proper protective equipment, such as gloves and goggles.

After the spilled chemicals have been removed, wash the area with warm, soapy water to remove any residue left behind.

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22 Introduction

Fire Extinguisher Class

Appropriate for: Examples:

Class A Ordinary combustibles (paper, wood, cardboard)

Class B Flammable/Combustible Liquids and Gasses

(gasoline, organic solvents)

Class C Electrical Equipment Computers, monitors, melting point apparatus

Class D Combustible metals Magnesium, titanium, potassium, sodium

Class K Grease fires Cooking Oils, fats

The fire extinguishers in Room 860 are rated for Classes A, B, and C. They are located by each exit, and outline the following procedure: (PASS)

Pull the pin out

Aim at the base of the fire

Squeeze the handle

Sweep the nozzle back and forth

If the fire is too large to be contained with a fire extinguisher, pull the fire alarm, and evacuate the building. Once out of harm’s way, call the University of Toronto Emergency 978-2222 or call 911. Specify the site and extent of the fire.

Wait outside the building, away from the main entrance so that you do not block the entrance when the fire personnel arrive.

If the Fire Alarm Sounds

Evacuate the building quickly, using the stairwells. The elevators will automatically go out of service. Do not try to use them.

Wait in the designated emergency area (the area between the Medical Sciences Building and the Leslie L. Dan Pharmacy Building),

Keep clear of the building.

Do not re-enter the building until authorized by a Fire Officer.

PHC 340Y Lab Manual 2017-2018

23 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve

Lab

Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve

Preparing for the Lab

Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:

UV/Vis Spectrophotometry - Determining Absorbance (http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv) Calculate the volume of stock required for each standard solution in the calibration curve.

Group Allocation You will be working in groups of 2 students

What You’ll Be Doing Part A: Prepare a calibration curve for hydrochlorothiazide Part B: Plot your calibration curve Demonstration: Using a spectrophotometer

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/calibration.xls

What You’re Handing In Lab 1 Worksheet (due at the beginning of the next lab)

Introduction

One of the fundamental tools to be used in any pharmaceutics laboratory is the analysis of the drug that is the subject of the experiment. In this introductory session a standard solution will be prepared and some of the principles related to the Beer-Lambert Law will be examined. The standard curve will be able to be used in a later session.

Background

Lambert’s Law

Lambert showed that each unit length of material through which light passes absorbs the same fraction of the incident or entering light and compares the relation between the incident light (Io) and the transmitted light (IT) for various thicknesses t.

loge

Where:

I is the intensity of light t is the thickness of the substance

the absorption coefficient

Conversion to log 10 results in the equation:

log.

2 3026

It Kt

Where K is the extinction coefficient generally defined as the reciprocal of the thickness (t in cm) required in order to reduce the intensity of the incident light to its original intensity.

http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

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24 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve

Beer’s Law

Despite what it sounds like, Beer’s Law does not describe the relationship between number of beers consumed and physical attraction. Beer examined the relationship between absorption and the concentration of coloured solutions.

The equation is similar:

(1)

ckI

Ilog 1

010

If this is performed in a cell with a uniform thickness then a measure of the length l may be added:

(2)

lckI

Ilog 1

10 or AI

Ilog

The value of k1 depends on how c is expressed. There are several proportionality factors. The

most common use in pharmacopoeias is the term , the extinction coefficient, which is equal to the absorbance of a 1% solution, at a path length of 1 cm:

(3) = A (1 %w/v, 1 cm)

× l is equal to the slope of the calibration curve (absorbance vs. concentration):

(4) A = × l × c

Where:

= extinction coefficient ((concentration units)-1cm-1) c = concentration (concentration units) l = path length (usually 1 cm)

There are many other names/conventions for A, such as E (extinction), and OD (optical density). They all mean the same thing. Usually a subscript is used to specify a specific wavelength. For instance, A260 (or E260, or OD260) would be used to denote the absorption of light at 260 nm. If we plot E against the concentration c then a straight line is obtained.

Figure 1. A Standard Curve for an Iron Solution

0 1 2 3 4 5 6 7 8 9

ml Standard Fe (0.02 mg Fe/ ml)

0.1

0.2

0.3

0.4

0.5

tin

cti

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25 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve

Lab

Beer’s law must always be tried for each substance being measured in order to see if there is a linear relation between E and the concentration of the drug in solution. In the above example it applies at least up to the 8th mL of sample.

There is an assumption in both cases that monochromatic light will be used. In addition, one must be sure that the wavelength of the light is not only the optimum wavelength for the analysis but also remains constant throughout the experiment.

In the following example, the drug displays a different E at several wavelengths. In this example, the instrument should be set at about 235 – 240 nm in order to not only give the highest E value, but also to place the wavelength in a location where slight shifts in the wavelength of the

light would not adversely affect the measurement. This plot is called a scan.

Figure 2. Graph Showing the Change in E (1%, 1cm) at Several Wavelengths

Areas of the curve where the change [E (1%, 1cm)] is large should never be used in drug analysis. On the curve in Figure 2, wavelengths of 205, 230, and 265 nm are sub-optimal.

Experiment Protocol

Chemicals Supplies Special Equipment Hydrochlorothiazide (5 mg/mL) in Sodium Hydroxide Solution (0.1 N) Sodium Hydroxide (40.0 g/mol)

Plastic transfer pipettes UV Cuvettes (Plastic) Parafilm

Helios UV/Vis Spectrophotometer Volumetric flasks

The following solutions are prepared or provided by the TA:

250 mL of Hydrochlorothiazide stock solution (5 mg/mL in 0.1 N NaOH)

The Helios spectrophotometers will be turned on prior to the laboratory. Each person will do their own measurements.

Part A. Preparing a Calibration Curve

1. Prepare 1 L of 0.1 N Sodium Hydroxide solution.

Note: do not leave sodium hydroxide pellets exposed to air. Close the cap of the bottle when not weighing pellets. Wear gloves when weighing and handling sodium hydroxide.

190 200 210 220 230 240 250 260 270 280 290

Wavelength

E( 1

1 c

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26 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve

2. Prepare dilutions of the hydrochlorothiazide stock solution in 0.1 N NaOH as follows, in triplicate:

1:10, 1:50, 1:100, 1:200, 1:250

To clarify, “in triplicate” means that you create each solution three times, rather than measure the absorbance of the same solution three times, to get an estimate of the error associated with creating the standard solutions. Measuring the standards in triplicate will allow you to report the average, standard deviation, and %RSD at each standard concentration. An efficient way to accomplish this is to have three different people run the same curve in parallel. The same spectrophotometer must be used.

3. Use the volumetric glassware, glass pipettes, and rubber pipette bulb for the dilution. Use Parafilm to close the top of the flask to allow mixing. Show details of your preparation and calculation.

4. Set the wavelength on your spectrophotometer to 270 nm. Place about 1.5 mL of the blank solution (0.1 N NaOH) supplied in a cuvette (fill the cuvette to the filling line) and determine zero absorbance. Blank the spectrophotometer.

5. Repeat the above steps with each of the five dilutions of the sample.

6. Measure the absorbance of the stock solution (remember 3 determinations). Calculate the average and standard deviation for each concentration.

*NOTE: Plastic UV cuvettes are tapered towards the bottom, to accommodate a smaller sample volume. The fill line is just above the clear part of the cuvette window. The “V” shaped arrow on the Plastic UV cuvette indicates the side of the cuvette that the UV beam will travel through the entire 1 cm path length (not widthwise, which is only 0.5 cm):

Fill line (fill to at least here)

Spectrophotometer beam travels this way

SPECTROSCOPY NOTES

Fill the cuvette to the etched line (approx ¾ full)

Make sure the cuvette is facing the correct way (the light path should go through the clear windows through the longest path length, not the ridged sides)

To avoid fingerprints, only handle the cuvettes by the ridged sides, not the clear windows.

Fill the cuvette slowly, and gently tap to release bubbles clinging to the sides of the cuvette

Gently wipe the clear windows with a Kimwipe prior to measuring

Make sure the sample door is closed before measuring absorbance

Make sure you use the same UV spectrophotometer for calibration and sample measurements.

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27 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve

Lab

Helios Spectrophotometer (PB 860) Varian Spectrophotometer (PB 819)

Part B. Plotting Your Calibration Curve

You will be preparing two calibration curves using calibration.xls (available in the Downloads section of the laboratory website):

One curve with all of your collected data;

One curve with the linear portion of the curve (excluding the higher concentrations).

Questions

1. Describe the shape of the curve that results from your data.

2. Does the best-fit curve go through zero? Is this necessary for Beer’s law to be valid?

3. Which of the linear fits in the two curves in Part B would you use to convert OD to concentration? Why?

4. What is the accuracy of your measurements? What is the precision?

5. Hydrochlorothiazide is a very weak acid. Why is 0.1 N NaOH used to help dissolve hydrochlorothiazide?

Beam direction Beam direction

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

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28 Lab 2: Preparation of pH Buffers

Lab 2: Preparation of pH Buffers

Preparing for the Lab

Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:

Measuring pH (http://phm.utoronto.ca/~ddubins/DL/pH.wmv)

UV/Vis Spectrophotometry - Determining Absorbance (http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv)

Calculate the volume of stock required for each standard solution in the calibration curve.

Group Allocation You will be working in groups of 2 students

What You’ll Be Doing

Part A: Preparing Phosphate (Sorensen’s) Buffer @ pH 7.4 Part B: Preparing McIlvane’s Buffer at 2 assigned pH values Demonstration: Using a pH meter Your buffers will be stored in the cold room on the 9

th floor, for

use in Lab 3.

Spreadsheets You Will Need Not applicable.

What You’re Handing In Not Applicable. Retain and store buffers prepared in this lab for use in Lab 3 (McIlvaine’s) and Lab 4 (Sorensen’s).

Introduction

Buffers are fundamental to wet chemistry, although the basic idea of buffers extends far beyond solutions. The primary idea behind a buffer is to dampen or minimize the effects of changes to or within the system so that the impact on the system is not as bad. There are buffers in electrical systems, irrigation systems, computers, and mechanics. Shock absorbers, for instance, prevent you from feeling bumps in the road when you are driving. Similarly, in wet chemistry, buffers can help reduce or minimize external stresses (changes in temperature or pressure), or chemical reactions from changing the overall pH of a solution. It is critical to select a buffer that is well suited to the system you are studying. Will the temperature of the system be changing? Will the pressure be changing? How does a change in either affect the pKa of the buffer? What is the pH value you would like to maintain? A buffer is most effective when the pH of the solution is in the vicinity of its pKa value (±1 pH unit). In this laboratory, you will learn how to calibrate and use a pH meter. You will be preparing two buffer systems: Sorensen’s Buffer and McIlvaine’s Buffer. You will be using these buffers in the following two labs.

References

1. Glasstone, Samuel. An Introduction to Electrochemistry. New York, NY USA (1942). p372.

2. http://www.chembuddy.com/?left=pH-calculation&right=pH-buffer-capacity

3. http://biotech.about.com/od/buffersandmedia/ht/phosphatebuffer.htm

4. http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer

5. http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html

Background

The mathematics behind buffer calculations for weak acids find their roots in the fundamental equation for a monoprotic acid dissociating. The following is a discussion behind the theory; however, it is important especially in the case of preparing the buffer to keep in mind that the

http://phm.utoronto.ca/~ddubins/DL/pH.wmv

http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv

http://www.chembuddy.com/?left=pH-calculation&right=pH-buffer-capacity

http://biotech.about.com/od/buffersandmedia/ht/phosphatebuffer.htm

http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer

http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html

PHC 340Y Lab Manual 2017-2018

29 Lab 2: Preparation of pH Buffers

Lab

theoretical (or even published) values of how much of each buffer component to add are just that – theoretical. The actual recipe required could be different, depending on the purity of components, errors in weighing and measurement, and even the quality of water used. There is a lot of theory involved, but at the end of the day, the calculated theoretical values serve only as a guide. Making a buffer is relatively quick and straightforward once you’ve tried it a few times. In order to understand buffers and how they work, a “crash course” in pH and pKa is offered in this section.

Definition of pH and pKa

An acid will dissociate in water to a conjugate base and proton. Consequently, acids are typically thought of as proton donors:

(1) [HA] [H+] + [A-] Acid Proton Conjugate Base

Ka is the equilibrium constant that determines the extent that the acid will dissociate in water:

(2) ]HA[

]A][H[K a

Recall that the pH of a solution in water is the negative log of the concentration of hydrogen ions, and is a more convenient way to express tiny concentrations. Similarly, the pKa is also the negative log of the equilibrium constant Ka:

(3) pH = -log[H+], or alternatively, 10-pH = [H+] (4) pKa = -log(Ka), or alternatively, 10-pKa = Ka

By substituting Equations (3) and (4) into Equation (2), we can derive the Henderson-Hasselbalch equation:

(5) [HA]

][A1010

pHpKa-

(6) pH

pKa

][A

[HA]

(7) pHpKa10][A

[HA]

According to the Henderson-Hasselbalch buffer relationship, pH, pKa, and the buffer component concentrations for a weak acid are related as follows:

(8) pHpKa10[base]

[acid]

Here, the ‘acid’ is the proton donor (HA), and the ‘base’ is the conjugate base (A-) in Equation (1). This is a very convenient form of the equation, because it allows us to see the following:

Key Concepts

if the pKa is greater than the pH, there will be more of the acid form in the solution.

If the pKa is equal to the pH, there will be an equal amount of acid and base in the

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30 Lab 2: Preparation of pH Buffers

solution.

If the pKa is less than the pH, there will be more of the base form in the solution.

A strong acid is defined as one that will dissociate completely. Consequently, the lower the pKa of the acid, the stronger the acid.

The same scheme can be re-written to describe the reaction of a base with water, to form its conjugate acid:

(9) [B] + H2O [B-H+] + [OH-] Base Water Conjugate Acid Hydroxide Ion

Bases are thought of as proton acceptors. A similar derivation can be made for the Henderson-Hasselbalch equation of a weak base; however, the equilibrium constants for bases are now more commonly reported using Ka, which allows Equation (8) to be used for bases as well. We can start with the Equilibrium expression for Equation (9), and then substitute the following identities in order to obtain Equation (8).

(10) pOH = -log[OH-]; pOH = 14 – pH; (11) pKb = -log[Kb]; pKa = 14 – pKb

Try it out for yourself. This saves us having to remember two sets of Hendersson-Hasselbalch equations. If the pKa of a base is greater than the pH, there will be more conjugate acid. It need only be remembered that [B-H+] is the concentration of conjugate acid and [B] is the concentration of base. The higher the pKa of a base, the stronger the base.

By using the appropriate experimental conditions, the pKa of a drug may be measured directly with a pH meter.

Pairing a Weak Acid with its Salt: Sorensen’s Buffer

Many buffer systems are weak acids paired with their respective salts. One example is citric acid paired with sodium citrate. The reason that two solutions are made at the beginning – a solution of the acid of the buffering agent, and another solution of its salt – is so that we may titrate one with the other to attain the exact pH we are looking for. It is assumed that when the salt of a buffer is dissolved in water, it will dissociate completely and go into solution in the ionic form.

It is important to note however that a buffering system can be as simple as a weak acid added to de-ionized water, with the pH of solution adjusted close to the pKa using either NaOH or HCl. Buffering systems are not limited to weak acids, they can also be weak bases (e.g. ammonia + ammonium chloride). Weak bases may be used for solutions where the pH desired is above 7. We will focus our discussion on weak acid buffers paired with salts of their conjugate bases.

Since it was already stated a buffer is most effective within 1 pH unit of its pKa, you would think that a given buffer would only be useful in the vicinity of one pH value. However, many buffers have more than one acidic group attached, which vary in affinity to their respective protons. Sorensen’s Buffer (phosphate buffer) has three acidic groups, each with different pKa values (see right panel).

O OHP

Phosphoric Acid

pKa3 = 12.32

pKa2 = 6.86

pKa1 = 2.15

PHC 340Y Lab Manual 2017-2018

31 Lab 2: Preparation of pH Buffers

Lab

This makes Sorensen’s buffer useful in the pH ranges 1.15 – 3.15, 5.86 – 7.86, and 11.32 – 13.32. The second pKa is close to 7, and so Sorensen’s buffer is typically used for buffer systems at pH 7. Since we would like to make use of the second pKa of phosphate, we might as well choose the weak acid and corresponding salt of the conjugate base of the second acidic group:

Na+

O OH

O-

Sodium Phosphate Monobasicweak acid

Na+

O O-

O-

Na+

Sodium Phosphate Dibasicsalt of conjugate base

Even though the sodium phosphate monobasic is a salt (and here is the potentially confusing part), the second hydroxyl group is still acidic, can drop its proton:

(12)

Na+

O OH

O-

Na+

O O-

O-

P + H+

sodium phosphate monobasic(acid)

sodium phosphate dibasic(conjugate base)

The monobasic acid dissociates into its conjugate base, and thus becomes dibasic. (It’s called “basic” since the charged –O- form is the acid’s conjugate base. Confused yet?). It will do this depending on the pH of the solution, according to the Henderson-Hasselbalch equation.

In contrast, when you add the salt form of the dibasic phosphate, the proton on the second hydroxide group is already gone. The molecule is being added as a conjugate base, rather than as an acid. For this very reason, you can sprinkle the sodium salt of the conjugate base of hydrochloric acid (NaCl) on your fries and be none the wiser, however HCl would have a very different effect.

The conjugate base is still free to revert back to its acid form:

(13)

Na+

O O-

O-

Na+

Sodium Phosphate Dibasic(salt of conjugate base)

+ OH2

Na+

O OH

O-

Na+

+ OH-

Sodium Phosphate Monobasic(weak acid)

PHC 340Y Lab Manual 2017-2018

32 Lab 2: Preparation of pH Buffers

However, to a first approximation, we treat the system as if the salt completely dissociates and stays in the ionic form.

A buffer is usually prepared in concentrations ranging from 0.1 – 10 M. The way that a buffer works, is that provided there are both forms (acid and conjugate base) of the buffer present (i.e. the pH is around the pKa), then if another acid dissociates to add a proton to solution, the proton will be absorbed by the buffer’s conjugate base instead of lowering the pH. If a base is added to the solution, it will result in a hydroxide ion, which will in turn react with the buffer’s weak acid instead of raising the pH. In this way, the balance of hydrogen ions is protected, and changes in pH are much smaller than they would have been in the absence of buffer.

Calculating the Amount of Acid and Salt of Conjugate Base Required

There are essentially four decisions to make when selecting a buffer:

1) What is the desired pH of the solution? 2) What type of buffer system will you choose? 3) Which pKa will you be making use of? 4) What buffer concentration will you need?

Let’s go through the exercise with Sorensen’s Buffer. We decide we would like to maintain the pH of solution at 7.4, which makes Sorensen’s an attractive choice. We decide on a buffer concentration at 0.1 M (more on that later).

To calculate the amount of salt and acid required, we return to the Henderson-Hasselbalch equation - Equation (5):

(14)

pHpKa10[base]

[acid]

In this case, the ‘acid’ is the un-ionized sodium phosphate monobasic, and the ‘base’ is the salt of the ionized form (sodium phosphate dibasic).

If we would like to make a 0.1 M Sorensen’s buffer at pH 7.4, we would substitute the desired pH, and the relevant pKa into the Henderson-Hasselbalch equation:

(15) 0.28841010][A

[HA]

[base]

[acid] 0.544.76.86

Simplifying (15):

(16) [HA] = 0.2884 [A-]

Since we want the buffer to be 0.1 M, we also have a mass balance to think about:

(17) [HA] + [A-] = 0.1 M

Substituting equation (16) into (17), we can solve for [A-], the concentration of conjugate base we will be adding in the salt form:

(18) 0.2884[A-] + [A-] = 0.1 M

(19) 1.2884[A-] = 0.1 M

(20) [A-] = 0.0776 M

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33 Lab 2: Preparation of pH Buffers

Lab

Substituting equation (20) into (17), we can solve for [HA], the concentration of acid that will be added in the un-ionized acid form:

(21) [HA] + 0.0776 = 0.1 M (22) [HA] = 0.0224 M

So now we have to do the important part: we have to translate a theoretical calculation into reality. We need to create two solutions and mix them together, such that the final concentration of sodium phosphate monobasic is 0.0224 M, and the concentration of dibasic is 0.0776 M. If we would like to start with two stock concentrations, 250 mL each, at a concentration of 0.2 M:

Sodium Phosphate Monobasic (NaH2PO4*H20)

g 900.6m

L 0.250mol

g 99.371

mol 0.2m

VMWCm

Sodium Phosphate Dibasic (Na2HPO4*7H20)

g 13.404m

L 0.250mol

g 268.07

mol 0.2m

VMWCm

So, 6.900 g of monobasic is added to a 250 mL volumetric flask and diluted with water to the mark, and 13.404 g of dibasic is added to another 250 mL flask and diluted with water to the mark. Now we have to find out how much of both we require to end up with final concentrations as calculated above. Suppose we would like to make 250 mL of the final buffer:

Sodium Phosphate Monobasic

mL 28V

mol0.2

mL 250L

mol 0.0224

VCV

VCVC

221

2211

Sodium Phosphate Dibasic

mL 97V

mol0.2

mL 250L

mol 0.0776

VCV

VCVC

221

2211

In theory, adding 28 mL monobasic plus 97 mL dibasic will give us a 0.2 M phosphate solution with the right proportions for pH 7.4. For a 0.1 M solution, we would then dilute this by a factor of 2 (adding an equal volume of water).

However, in reality if you were to add these two volumes together, you would not attain a pH of 7.4, exactly. In a lab environment, it is important to remember that theory does not always translate directly and literally to reality. This typically causes confusion for a new graduate student. For example, inevitably the graduate student calibrates the pH meter for the first time, and measures the pH of de-ionized water, finding that it is around pH 5. Clearly there is something wrong with the pH meter? Water is supposed to be pH 7, right? In reality, carbon dioxide from the air dissolves into the water creating carbonic acid, thus lowering the pH. In reality, the standard buffers the graduate student is using to calibrate the pH meter may have expired 6 years ago, and are potentially growing fungus.

In practice, what is done is that the larger of the two volumes (in this case, the 97 mL of dibasic) is added to a beaker, and the smaller of the two (in this case the monobasic) is loaded into a

PHC 340Y Lab Manual 2017-2018

34 Lab 2: Preparation of pH Buffers

burette. The pH electrode is inserted into the beaker, and solution is titrated until the desired pH is attained. Once it is attained, for this particular protocol, an equal volume of water (volume in beaker + volume of solution titrated) is added to bring the concentration of buffer from 0.2 M to 0.1 M.

Question: Why can’t you just prepare 0.1 M of each solution, and add them together? Wouldn’t that give you a 0.1 M Sorensen’s buffer?

Answer: You can. The reason we start with 0.2 M stock solutions in this case is out of convenience, because the burette can only hold 50.0 mL. A 250 mL solution of 0.1 M sodium phosphate monobasic would require 56 mL for the final mix, which wouldn’t all fit in one burette.

What makes these calculations lengthy is compensated by the simplicity of published tables in the literature of volumes of each solution to attain the desired pH. In practice, you need not perform the calculations routinely, you can use the tables as a starting point and titrate to your desired pH. However, studying the theory behind these calculations will make all the difference in your understanding of how a buffer works.

Buffer Capacity

One question that might arise is how well will the buffer protect the pH from changing? This will depend on the pKa of the buffer, pH of the solution (the closer to the pKa the pH is, the more effective the buffer will be), and on the concentration of buffering agent used.

One definition of buffer capacity is the amount of (external) acid or base required to change the pH of 1 L of the solution by 1 pH unit. The higher the buffer capacity, the larger this number will be.

A formula to calculate buffer capacity is presented here:

(23)

abufw

])[H(K

][HKC][H

][H

K 2.303β

dpH

Where,

: buffer capacity (mol/(L*pH unit)). n: number of moles of acid or base added (assumed to be monoprotic) Kw: The equilibrium constant of water (1.00×10-14 at 25 °C) Cbuff: the concentration of buffering agent

The summation sign in Equation (23) means that you can enter more than one Ka of your buffering agent if it has multiple acidic groups, or the equation can be used for buffers with multiple components. It is beyond the scope of this discussion to derive Equation (22). However, we may use it to calculate the buffer capacity of our Sorensen’s buffer. At a pH 7.4:

PHC 340Y Lab Manual 2017-2018

35 Lab 2: Preparation of pH Buffers

Lab

100

120

2 3 4 5 6 7 8

ffe

r C

apac

ity

l/(L

Buffer Capacity of McIlvaine's Buffer (0.2 M Sodium phosphate dibasic + 0.1 M Citric Acid)

pH)*mol/(L 0.04β

)103.981110(1.3804

103.9811101.3804M 0.1103.9811

103.9811

1012.303β

])[H(K

][HKC][H

][H

K2.303β

101.38041010K

103.98111010][H

287

878

14-

abufw

76.86K

87.4pH

In other words, if 0.04 moles of hydrochloric acid was added to 1 L of 0.1 M Sorensen’s buffer at pH 7.4, the pH would be expected to drop by 1 pH unit, to pH 6.4. Compare this with the expected pH change if you were to add 0.04 moles of HCl to 1 L of de-ionized water:

pH = -log[H+] = -log[0.04] = 1.3979

So the Sorensen’s buffer turned a pH that should have been 1.3979 into a pH of 6.4. Not too shabby!

As stated before, a buffer is most effective when the pKa is within one unit of the solution’s desired pH. In closing this discussion, we can look at the buffer capacity for our 0.1 M Sorensen’s Buffer as a function of pH:

We can see a peak (left panel, above) at the second pKa of phosphoric acid (6.88), and the buffer capacity decreases as the pH falls farther away from the pKa. At 1 pH unit away (pH 6 or pH 8) the buffer capacity is reduced to less than half of what it was at the pKa.

Some buffering systems make use of more than one buffering agent. For instance, McIlvaine’s buffer contains both phosphoric acid (pKa values: 2.15, 6.86, and 12.32), and citric acid (pKa values: 3.13, 4.76, and 6.40). Within the range pH 2 – 8, you are never farther than 1 pH unit away from a pKa. The buffer capacity graph for McIlvaine’s buffer is more complicated (right panel, above).

We can use buffer capacity to back-calculate what concentration of buffer we require (Cbuf), depending on what changes in pH we would like the system to tolerate. This will affect Equation (11), the mass balance of buffering agent. In practice, a concentration of 0.1 M is usually used.

Did you know?

The mathematics of acid/base chemistry are exactly the same as that for any other equilibrium reaction, e.g. drug/receptor interactions. So by learning the mathematics of buffers, you have just learned how to calculate binding constants of drugs with their targets.

PHC 340Y Lab Manual 2017-2018

36 Lab 2: Preparation of pH Buffers

Remember that the Ka values for weak acids and bases are dependent on the temperature and pressure of the solution. Many experiments make use of this, and measure the equilibrium constants at different temperatures or pressures to examine other interesting properties of the systems studied.

In practice, Sorensen’s buffer is simply referred to as “phosphate buffer”.

Key Concepts:

Many buffering systems are weak acids paired with the salt of their conjugate bases, or weak acids titrated to the viscinity of their pKa with NaOH or HCl.

A buffering agent is useful within 1 pH unit of its pKa.

Understanding the mathematics of buffering systems is important, but ultimately, your pH meter decides how much of each agent to add.

Experiment Protocol

Chemicals Supplies Special Equipment Sodium Phosphate Monobasic (verify MW on the bottle used) Sodium Phosphate Dibasic (verify MW on the bottle used) Citric Acid (MW 210.14 g/mol) pH Standardizing Buffers

N/A pH Meter Mixing plate and magnetic stir bar 100 mL graduated cylinder 250 mL volumetric flask 140 mL beaker 50 mL burette, burette clamp, retort stand

The following solutions are prepared or provided by the TA:

pH Meter Standardizing Buffers (pH 4, 7)

Part A. Preparing Sorensen’s Buffer

Prepare 250 mL of a 0.2 M solution of sodium phosphate monobasic.

Prepare 250 mL of a 0.2 M solution of sodium phosphate dibasic.

Load a burette with 50 mL of the 0.2 M sodium phosphate monobasic solution.

Set up the burette on a retort stand with a burette clamp.

Measure out 97 mL of sodium phosphate dibasic using a 100 mL graduated cylinder. Pour into a 140 mL beaker.

Calibrate your pH meter with the standard solutions provided.

Set the 140 mL beaker on top of a mixing plate under the burette, and insert the pH electrode.

Make sure the tip of the pH electrode is submerged, and does not touch the bottom of the beaker.

Place a magnetic stir bar in the 140 mL beaker, and set mixing to a low speed. Do not allow the stir bar to hit the pH meter, or a vortex to appear.

Slowly titrate the sodium phosphate dibasic solution with the sodium phosphate monobasic solution, until you attain a pH of 7.40.

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37 Lab 2: Preparation of pH Buffers

Lab

Record the volume of sodium phosphate monobasic added.

Transfer the titrated mixture to a 250 mL beaker. Add an equal volume of water to create a 0.1 M Sorensen’s Buffer.

Seal your buffer with parafilm, label it appropriately, and store it for Lab 4.

Part B. Preparing McIlvaines’s Buffer

McIlvaine’s Buffer is a mixture of phosphoric and citric acids. It is useful in the range pH 2.2 – pH 8. The two solutions to be prepared are sodium phosphate dibasic, and citric acid. In this exercise, you will be assigned two of the following pH values: 2.5, 3, 3.5, 4, and 4.5. You may be assigned different pH buffers by group. The following is a published chart on volume (in mL) of each solution to combine for the expected pH, through the useful range of McIlvaine’s Buffer. It makes 20 mL of buffer:

Source: http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer

In a 250 mL flask, prepare 0.2 M sodium phosphate dibasic solution.

NOTE: You may have enough stock left over from making the Sorensen’s Buffer.

In a 250 mL volumetric flask, prepare 0.1 M citric acid solution.

Load a burette with 50 mL of the 0.2 M sodium phosphate dibasic solution.

Set up the burette on a retort stand with a burette clamp.

Measure out 100 mL of citric acid using a 100 mL graduated cylinder. Pour into a 250 mL

http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer

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38 Lab 2: Preparation of pH Buffers

beaker.

Set the 250 mL beaker on top of a mixing plate under the burette, and insert the pH electrode.

Place a magnetic stir bar in the 250 mL beaker, and set mixing to a low speed. Do not allow the stir bar to hit the pH meter, or a vortex to appear.

Slowly titrate the citric acid solution with the sodium phosphate dibasic solution, until you attain the desired pH.

NOTE: Depending on the pH selected, you may have to re-fill the burette with the sodium phosphate dibasic solution.

Record the total volume of sodium phosphate dibasic added.

Seal your buffer with parafilm, label it appropriately.

The TA will store your buffers in the cold room on the 9th floor, for Lab 3. In general, many aqueous buffers will grow bacteria at room temperature, and are best stored at cold temperatures (e.g. at 5 °C in media bottles, or -20 °C in small aliquots, depending on the buffer).

Questions

1. Explain using the Henderson-Hasselbalch equation why the pH of the Sorensen’s buffer shouldn’t change when you add an equal volume of water for the final step.

2. The pKa of salicylic acid is 2.97. What form (ionized or un-ionized) will it predominantly be:

a. In the stomach, at pH 2? b. In the gut, at pH 7? c. If the ionized form (salicylate) cannot pass through cell membranes, where

would you expect the drug to be absorbed?

3. Borate buffer is used in gel electrophoresis. It is a combination of Boric Acid (MW 61.83 g/mol), titrated with NaOH to the desired pH.

Boric Acid

B + OH2

Borate

+ H+

pKa = 9.24

OHB

a. What is the useful pH range of borate buffer?

b. Create a protocol for preparing 500 mL of 0.1 M Borate buffer, at pH 8, assuming you already have a solution of 0.2 M NaOH. How will you prepare your stock solutions? How much of each will you require?

4. Calculate the buffer capacity of 0.1 M Borate buffer: a. At pH 8. b. At pH 5. c. Does your answer for (a) double at pH 8 for a Cbuff of 0.2 M?

PHC 340Y Lab Manual 2017-2018

39 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

Lab

Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

Preparing for the Lab

Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:

Measuring pH (http://phm.utoronto.ca/~ddubins/DL/pH.wmv)

Group Allocation You will be working in groups of 2 students (same groups as Lab 2)

What You’ll Be Doing

Part A: Prepare a standard curve for salicylate Part B: Salicylate partition experiment at 2 pH values (McIlvaine’s buffers from Lab 2) Part C: Salicylate partition experiment under acidic conditions Demonstration: Proper use of syringe filters

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/calibration.xls

What You’re Handing In Individual formal lab report, due at the beginning of the next lab (see Guidelines for Writing Individual Laboratory Reports for details)

Introduction

The partition coefficient of a drug is critical to the absorption of the drug, as it is often primarily the un-ionized form of the drug which crosses the intestinal lumen in the gut and ultimately drug cell membranes. It is also a key parameter in the solubility of the drug in a given media. A drug with a high oil/water partition coefficient will preferentially partition in the oil component and be less soluble in aqueous media.

There is another player at work here; the pKa of a drug will determine what proportion of the drug is in the ionized (charged) or un-ionized form. In knowing the pKa and partition coefficient of a drug, we can predict what proportion of the drug will be ionized at what pH, and also understand how well the drug will partition into oily media, which provides a model for the trip it must make to cross hydrophobic cell membranes. Chemical equilibria are rarely a one way street where there is all or nothing of one form. These equilibria are delicate and may shift substantially depending on the pH and hydrophilicity of the media they are dissolved in.

References

1. Garrett, E. and Woods, O.R., J. Pharm. Sci., 42, 736 (1953) 2. Handbook of Chemistry and Physics, 68th ed., Chemical Rubber Publishing Co., Cleveland, Ohio

1987, D 145 3. Fung, H.L. and W.D. Conway, Amer. J. Pharm. Ed. 38, 523 (1974)

Background

Familiarity with and comprehension of the effect of pH on the partition coefficient of weakly acidic and basic drugs are essential. The role of pH in gastrointestinal absorption and in the establishment of preservative requirements in oil-water systems are just two examples of the interesting aspects of this physical-chemical principle.

A theoretical understanding of this problem is often made difficult by the presence of complex equilibria, such as dimerization of the acid in the oil phase.

This experiment demonstrates the theoretical principles of the equilibra involved in the simple partitioning of a weak acid between aqueous and oil phases. The derivation of the equation

http://phm.utoronto.ca/~ddubins/DL/pH.wmv

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

PHC 340Y Lab Manual 2017-2018

40 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

used for plotting the data and for calculating the equilibrium constants can be easily understood.

Consider the distribution of a weak acid, HA, between an oil phase and an aqueous phase. Only the un-ionized form of the acid will partition into the oil phase. Provided there is no dimerization of the acid in the oil phase, the equilibrium is:

(1) oilo/w

W HAK

A special equilibrium constant, Ko/w, is used to describe the equilibrium between the concentration of the un-ionized form of the acid in oil ([HA]oil) versus the un-ionized form of the acid in water ([HA]w). Thus, the definition of the partition coefficient, Ko/w, is the ratio of the concentrations of un-ionized acid in oil versus water, at equilibrium:

(2) w

oil o/w

[HA]

[HA]K

NOTE: Some texts will use Po/w instead of Ko/w to denote partition coefficient. The term “true” partition coefficient in this lab also refers to Ko/w.

The second half of the story is the acid dissociating in the water phase to become its conjugate base:

(3) w-aW AH

KHA

acid

Please note that in this laboratory, we are starting with sodium salicylate solution, which is the salt of salicylate, the conjugate base of salicylic acid. The salt will dissociate completely as described in Equation (9) in Lab 2, to become salicylate and sodium ions:

(4) w-ANaANa

The reason we are using the salt is that it is much easier to get the salt of an acid into solution at higher concentrations, since it starts out in the ionized form. Recall that the ionized form is more hydrophilic, as it forms electrostatic interactions with water.

The equilibrium constant Ka governs how much the drug will be in the ionized form (A-)w versus the ionized form (HA)w in water. Thus, the definition of Ka is:

(5) w

a[HA]

][A][HK

The Ka is known as the acid dissociation constant, and is often expressed in log units for convenience sake:

(6) pKa = -log(Ka)

Putting these equations into context, the equilibrium scheme would look like this:

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41 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

Lab

In this laboratory, we are measuring the concentration of drug at equilibrium in the water phase. However, this does not directly give us the [HA]w, as the indicator reacts with both un-ionized and ionized forms of the drug in aqueous media. Instead, we measure Cw, the total molar concentration of the drug in the water phase:

(7) Cw = [HA]w + [A-]w

There is one more definition we need before we can proceed. The apparent partition coefficient (also known sometimes as the “distribution coefficient”, or experimentally observed partition coefficient) is defined as the ratio of total drug dissolved in the oil phase to the total drug (ionized + un-ionized) dissolved in the aqueous phase:

(8) w

oil

oilo/w

[HA]

][A[HA]

[HA]K'

The subtle difference between the apparent partition coefficient K’o/w, and the partition

coefficient, Ko/w, is that the K’o/w will change depending on the amount of ionized drug in solution

(and thus will change depending on the pH), whereas the true partition coefficient will not depend at all on pH. It is an intrinsic property of the un-ionized form of the drug.

Combining the boxed Equations (2), (5), and (8), we can derive a relationship between K’o/w and [H+]w. First we need to re-arrange Equation (2):

(9) wo/woil [HA]K[HA]

Substitute (9) into (8):

(10)

wo/wo/w

][A[HA]

[HA]KK'

Water

Oil

Partition

Ko/w

(HA)w

(A-)

w +(H

(HA)o

Dissociation

PHC 340Y Lab Manual 2017-2018

42 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

Simplify:

(11)

o/w

[HA]

][A1

KK'

Re-arrange Equation (5) to solve for [HA]w:

(12)

][A][H[HA]

Now substitute Equation (12) into (11):

(13)

aww

o/w

/K][A][H

][A1

KK'

Simplify:

(14)

o/w

][H

KK'

By taking the inverse of both sides of Equation (14) and simplifying, we obtain an equation that should produce a straight line when 1/K’o/w is plotted against 1/[H+]w:

(15)

o/ww

o/wo/w K][H

Equation (15) is a linear equation in the form y = mx + b. The slope of the line plotted is equal to Ka/Ko/w, and the intercept is equal to 1/Ko/w. Consequently, by performing this simple experiment, you can solve for both the partition coefficient of the drug, and the dissociation constant at the same time!

How do these equations apply to the experiment?

In this experiment, we can determine the apparent partition coefficient at each pH, using

Equation (8): w

oilo/w

[HA]K' . We measure Cw experimentally, using our ferric chloride assay on

the aqueous phase, and converting the absorbance into a concentration using the standard curve. We can’t measure [HA]oil directly. However, we know the total concentration of drug in the water phase at the beginning of the experiment (Cw,initial). The amount of drug entering the oil will be the initial amount of drug minus the amount left in water at equilibrium:

(16) noil = nw,initial – nw,equilibrium (17) noil = (Cw,initial × Vw) – (Cw × Vw)

(18) [HA]oil = noil / Voil

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43 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

Lab

So now we can plug Cw and [HA]oil into Equation (8) to solve for K’o/w at each pH. Since we measure the pH of the solution, we can calculate [H+]w (recall that pH = -log[H+]).

Once we have the apparent partition coefficient at each pH, we can plot 1/K’o/w versus 1/[H+], and then use the slope and intercept to calculate the true partition coefficient, Ko/w, and Ka for salicylic acid using the form of Equation (15).

Garrett and Woods (reference 1) used a similar approach for the study of the partitioning of benzoic acid between an aqueous phase and sesame oil. The derivation given in this report is more direct than that given by Garrett and Woods. Furthermore, Garrett and Wood's method required that the acid dissociation constant be known at the temperature and ionic strength in which the experiment is performed. This method does not require such information, and, in fact, the value for the acid dissociation constant obtained by the student can serve as a check on his/her experimental accuracy.

Experiment Protocol

Chemicals Supplies Special Equipment Sodium Salicylate (MW 160.11 g/mol) Sesame Oil Purified Five McIlvaine's standard buffer solutions pH 3.5 – 4.5 (from Lab 2) Hydrochloric Acid (2.0 N) Ferric Chloride TS

Plastic Cuvettes (at least 2) Scintillation Vials 30 mL Syringe & 0.45 µm Syringe filter Parafilm Test Tubes, test tube rack Class 2B lab goggles Face Shield Plastic Droppers

Spectronic 20 spectrophotometer or Helios UV/Vis Spectrophotometer pH Meter Mechanical Agitator 100 mL graduated cylinder 25 mL graduated cylinder 10 mL graduated cylinder

The following solutions are prepared or provided by the TA:

Hydrochloric Acid (2.0 N)

Ferric Chloride TS (9% w/w FeCl3 in water)

Sodium Salicylate (0.2 M)

NOTE: Ferric Chloride TS needs to be prepared freshly for this lab.

Prior to the lab:

To prepare the sesame oil, add the anticipated volume you will need for the lab with an equal amount of de-ionized water in an Erlenmeyer flask. Shake the solution vigorously. This will extract water soluble materials and impurities which might interfere with the analysis.

Preparation of the Indicator Ferric Chloride TS USP (9% w/w FeCl3 in water)

(your TA will prepare this solution)

IN A FUME HOOD, weigh out 9.89 g of ferric chloride and dissolve in 100 g of de-ionized water. Ferric chloride releases hydrogen chloride gas in contact with water or moist air. Caution should also be made as the dissolution of ferric chloride in water is exothermic.

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44 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

NOTE: If the ferric chloride doesn't dissolve completely, the solution should be filtered before using.

You will be performing this experiment using two of the McIlvaine’s Buffer solutions you made in Lab 2 (pH 2.5, 3.0, 3.5, 4.0, and 4.5).

Part A. UV Absorbance Standard Curve of Sodium Salicylate

The first phase of the experiment involves the procedure for the quantitative estimation of sodium salicylate in the aqueous phase. The assay of sodium salicylate is based on the estimation of the intensity of the purple colour produced by the addition of ferric chloride solution to the sample.

Your TA will prepare a 0.2 M sodium salicylate stock solution. You will need to calculate the volume of stock required for each of the standard curve concentrations.

Prepare 100 mL of each of the following sodium salicylate concentrations in de-ionized water, in triplicate:

0.5, 1, 1.5, 2, and 2.5 mM using volumetric glassware.

Calculate and report the volume of stock required to make each dilution.

NOTE: Use a 1 mL graduated pipette to dispense the appropriate amount of stock in each 100 mL volumetric flask to create each dilution.

Assay Procedure: Salicylic Acid

This assay procedure consists of combining the following in a test tube:

1 mL of the solution to be tested

5 mL of de-ionized water

Exactly 2 drops of ferric chloride (use a plastic dropper)

Cover with Parafilm® ensuring a tight seal.

Invert the mixture until uniform.

Perform the assay procedure for each concentration of your standard curve.

The blank consists of 6 mL of water and exactly 2 drops of ferric chloride solution.

NOTE: Allow 10-15 minutes for the colour to completely develop before measuring.

Set the wavelength of the spectrophotometer to 525 nm (visible light).

Zero the absorbance of the spectrophotometer using the blank solution.

Measure the absorbance of each concentration of the standard curve and record the absorbance.

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45 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

Lab

Using calibration.xls (available on the laboratory website), plot a graph of absorbance vs. known concentration of sodium salicylate. This is your standard (or calibration) curve.

Part B. Determination of the Partition Coefficient

NOTE: Perform Parts B and C at the same time. This will save you a lot of time. The next phase of the experiment involves the determination of the partition coefficient as a function of pH. Five aqueous solutions of sodium salicylate at various pH values are prepared in the following manner: (NOTE: your group will be assigned two pH values, and class data will be pooled for analysis.)

Pipette 10 mL of 0.2 M sodium salicylate into one 100 mL volumetric flask for each assigned buffer.

Dilute the solutions to the 100 mL mark with the assigned buffer. Label appropriately.

Due to time constraints, Part B is not performed in triplicate. You will be combining data with other lab groups to obtain an estimate of error.

Scintillation Vial Method

There are different methods used for the oil/water partition experiment. Typically, separatory funnels are used. The method used in this lab will be the Scintillation Vial method, as it requires much lower sample volumes.

Pour exactly 6 mL of each assigned pH solution into their own scintillation vials, using a 10 mL graduated cylinder.

Pour exactly 5 mL of sesame oil using a 10 mL graduated cylinder.

Shake the scintillation vial gently, and intermittently for about 30 minutes. A mechanical

SPECTROSCOPY NOTES

Fill the cuvette to the etched line (approx ¾ full)

Make sure the cuvette is facing the correct way (the light path should go through the clear windows, not the ridged sides)

To avoid fingerprints, only handle the cuvettes by the ridged sides, not the clear windows.

Fill the cuvette slowly, and gently tap to release bubbles clinging to the sides of the cuvette

Gently wipe the clear windows with a Kimwipe prior to measuring

Make sure the sample door is closed before measuring absorbance

Make sure you use the same UV spectrophotometer for calibration and sample measurements.

The same cuvette may be used to measure different solutions. Measure from least to most concentrated, and rinse with solution to be measured.

If a series of solutions is to be measured simultaneously, you only need to blank the spectrophotometer once, before measuring the series.

Ideally, measured absorbance values should fall between your lowest and hightest standard concentrations. If an absorbance value is too high (e.g. 3.5 OD), dilute it by a factor to obtain a more reliable measure, then multiply the result by that factor to calculate the concentration of the original sample.

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

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46 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

agitator may be used.

After the phases have completely separated, withdraw 4 mL of the aqueous phase using a Pasteur pipette.

Calibrate a pH meter and measure the pH of the aqueous phase.

Assay the aqueous phase using the same assay procedure described in Part A:

(1 mL sample + 5 mL de-ionized water + 2 drops ferric chloride TS).

Use the same blank solution prepared in Part A.

The total molar concentration of salicylate in the oil phase ([HA]oil) is then determined by calculating the amounts (moles) of sodium salicylate in a given volume of each phase using Equations (16) to (18). From the concentrations (molar) in each phase, the apparent partition coefficient (K’o/w) is then determined.

Part C. Direct Measurement of the Partition Coefficient

For this part of the lab, we have class 2B lab goggles and a face shield available to protect your eyes from acid splashes.

We can find the true partition coefficient by a more direct method. The true partition coefficient is equal to the apparent partition coefficient at pH values where all of the salicylate is essentially in the un-ionized form (>99%). To accomplish this:

Your TA will assign one volume to your group: 5 mL, 10 mL, or 20 mL sodium salicylate.

Add your assigned volume of 0.2 M sodium salicylate to a 100 mL volumetric flask.

Fill the volumetric flask approximately half way with de-ionized water.

Add 10 mL of 2.0 M HCl. Goggles and a face shield are required for this step. Obtain the face shield from your TA or instructor.

Dilute to 100 mL with de-ionized water.

Filter the entire solution with a 0.45 µm syringe filter and 30 mL syringe. You may need to use more than one syringe filter if it becomes clogged.

NOTE: Your TA or instructor will demonstrate how to use a syringe filter. Watch this demo prior to using syringe filters.

Assay the initial concentration (Cw,initial) of salicylic acid in the filtrate.

BLANK: Use de-ionized water for the blank, as HCl does not significantly absorb in our wavelength of interest.

Now, determine the partition coefficient of the filtered solution using the same procedure you followed for Part B (add 6 mL filtrate + 5 mL sesame oil, shake for 30 min, then assay (1 mL sample + 5 mL de-ionized water + 2 drops ferric chloride TS).

BLANK: use 6 mL deionized water + 2 drops of ferric chloride TS.

NOTE: You may need to dilute the sample further to get a meaningful value from the spectrophotometer (between 1.0 and 3.0 OD).

Since at low pH there will be no conjugate base, the apparent partition coefficient will be equal to the true partition coefficient, because:

Cw = [HA]w + [A-]w o/w

oilo/w K

[HA]

[HA]K'

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47 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

Lab

The [HA]oil can be calculated by subtraction:

[HA]oil = noil/Voil = (Cw,initialVw – Cw,finalVw) / Voil

Thus, Ko/w is determined directly.

How to Use a Syringe Filter:

Questions

1. Are there any variations of the true partition coefficient Ko/w with sodium salicylate concentration? Do you regard it as experimentally significant?

2. Why does salicylic acid precipitate out when the pH is reduced in Part C?

3. Why do we need to assay the initial concentration of salicylic acid in the aqueous phase in Part C, but not in Part B?

4. If we know the pH of the buffers we are using, then why do we need to measure the pH of the solution at equilibrium?

5. Compare your calculated pKa of salicylic acid with a literature value. Is it close to the literature value?

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48 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 3 students

What You’ll Be Doing

Part A: Determining the solubility of sulfathiazole Part B: Preparing two different salts of sulfathiazole Part C: Preparing two different polymorphs of sulfathiazole Part D: Determining the pKa of sulfathiazole Part E: Determining the melting point of sulfathiazole Part F: Macroscopic evaluation of sulfathiazole powder Demonstration: Using the Melting Point Apparatus

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/titration.xls

What You’re Handing In Lab 4 Worksheet (due at the beginning of the next lab)

Introduction

Characterization and pre-formulation is the process of characterizing the physical, chemical, and mechanical properties of a new drug substance, in order to develop stable, safe, and effective dosage forms. It is generally initiated after a compound shows sufficiently impressive results of biological screening. A new medicinal substance may have unsuitable physicochemical properties, such as instability or insolubility, that ultimately leads to poor bioavailability or efficacy in human clinical studies. To optimize the performance of drug products, it is necessary to have a complete understanding of the drug substance in hand.

Pre-formulation encompasses the study of parameters such as dissolution, polymorphic forms, crystal size and shape, pH profile of stability, and drug-excipient interactions. These may have profound effects on a drug’s physiological availability and physical and chemical stability.

In this laboratory exercise, a few pre-formulation parameters are investigated. Knowledge of the particle size, melting point, pKa and intrinsic solubility will assist in proper formulation of the final dosage form of a drug. Sulfathiazole has been chosen to illustrate these properties.

References

1. Gennaro AR, ed., Remington: The Science and Practice of Pharmacy, 20th

ed., U.S.A.: Mack Publishing Company, 2000, p. 700-720, 227-245, and 390-395

2. Lachman L, Lieberman H A and Kanig J L, ed., The Theory and Practice of Industrial Pharmacy, 2nd

ed., U.S.A.: Lea & Febiger, 1976, p. 1-31.

3. Bates RG, Paabo M and Robinson RA, J. Phys. Chem., 67, 1833 (1963). 4. Aulton ME, Pharmaceutics, the science of dosage form design, 2

nd ed., UK, Elsevier Science, 2002, p.

23-28 , 142-151 5. Apperley DC, et al, Sulfathiazole Polymorphism Studied by Magic-Angle Spinning NMR, J. Pharm

Sci., 88, No. 12, 1275-1280 (1999) 6. Ware, E.C. and D.R. Lu, An Automated Approach to Salt Selection for New Unique Trazodone Salts,

Pharmaceutical Research, 21, 177-184 (2004) 7. Kennepohl, D. et al. Chemistry 350 Organic Chemistry I Laboratory Manual 2002/2003. Athabasca

University (2003). 8. Operation and Service Manual: MPA160 and MPA161 DigiMelt Student Melting Point System.

Standford Research Systems, Revision 1.9 (September 2009).

http://phm.utoronto.ca/~ddubins/DL/titration.xls

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49 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Lab

Background

Since the pure drug entity is initially synthesized by the medicinal chemist in milligram quantities, it is important to note the general appearance, colour and odour of the compound. These characteristics provide a basis for comparison with future lots. Particle size and distribution may affect the dissolution rate, absorption rate and the content uniformity of the final product. Sedimentation and flocculation rates in suspensions are in part governed by the particle size. In addition, flow properties of powder formulation can also be affected by the particle size. There are a few common ways to measure particle size: microscopy, sieving, sedimentation, and dynamic light scattering.

If a solid substance exists in more than one crystalline form, the solid is said to exhibit polymorphism. The different crystal forms, which may have very different physical properties, can be distinguished by optical microscopy, X-ray powder diffraction, and differential scanning calorimetry.

The lipophilicity of a drug is usually indicated by its partition coefficient. In the biological context, partition coefficient usually refers to the equilibrium concentration ratio of a drug in the octanol phase (top) and the water phase (bottom). The ease with which a drug crosses a biological lipid membrane is related to the lipophilic nature of the drug involved.

pKa and Intrinsic Solubility

It is also important to know the aqueous solubility of a new drug substance. The solubility of a drug substance must be improved if it is less than the required concentration necessary for the recommended dose. This can be done by altering the pH of the delivery vehicle, using co-solvents (e.g. alcohols, propylene glycol, glycerin, sorbitol, and polyethylene glycols), using surfactants to help solubilize the drug, or re-crystallizing the drug into a different salt form (e.g. acetate, citrate, hydrochloride, or sulfate for anions; sodium or calcium salts for cations).

Since many chemical substances of pharmaceutical interest are weak acids and bases, it is important for the pharmacists to be aware of various fundamental physicochemical properties of such substances to fully appreciate their behaviours under the diverse conditions of storage, compounding, administration, and absorption. Knowledge of the pKa and intrinsic solubility (solubility of the non-ionized form of the drug) for a weakly acidic or basic drug will allow a better understanding and prediction of the performance and stability of a pertinent pharmaceutical preparation.

Knowing the pKa and intrinsic solubility of the drug in solution will help us to understand how solubility changes with pH. Typically, the pKa may be determined in aqueous solution using pH titration. However, how can the pKa of a drug be determined if the drug is sparingly soluble in water? One option is to determine the pKa of the drug at different concentrations of co-solvents. We can then find out what the pKa would be in water alone, by calculating the Y-intercept of a graph of pKa vs. % co-solvent. This is a valid approach provided the graph is linear. This method is an adaptation of the Yasuda-Sheldovsky Extrapolation. Computer spreadsheet programs, such as titration.xls found on the laboratory website, may be used to judge the linearity of the data, and calculate the Y intercept (pKa in water alone).

Parts C and D in this lab are designed to make use of the following relationship:

http://phm.utoronto.ca/~ddubins/DL/titration.xls

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50 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

0T pKpHS

SSlog

(for weak acids)

The following is a discussion of the derivation of this equation, and will help in answering the discussion questions.

Quick Review: The Henderson-Hasselbalch Equation

Recall from Lab 2 that an acid will dissociate in water to a conjugate base and proton. The acid dissociation equilibrium constant, Ka, is defined as the ratio of concentrations of products over reactants.

(1) [HA] [H+] + [A-] Acid Proton Conjugate Base

(2) ]HA[

]A][H[K a

In Lab 2, we substituted the identities pH = -log[H+] and pKa = -log(Ka) into Equation (2) to derive the Hendersson-Hasselbalch equation:

(3) pHpKa10[base]

[acid]

In the case of a weak acid, the [acid] term is the concentration of undissociated, uncharged acid (HA), and the [base] term is the ionic conjugate base (A-) in Equation (1).

The Henderson-Hasselbalch equation is also valid for the reaction of a weak base:

(4) [B] + H2O [B-H+] + [OH-] Base Water Conjugate Acid Hydroxide Ion

In the case of a weak base, provided the Ka for the base is used, the [base] in Equation (3) is the concentration of base, and the [acid] is the concentration of conjugate acid.

It is useful to re-iterate the following key ideas:

In addition:

the lower the pKa of the acid, the stronger the acid.

the higher the pKa of the base, the stronger the base.

By using the appropriate experimental conditions, the pKa of a drug may be determined with a pH meter. A titration with either strong acid or strong base should yield a flat region as the pH is adjusted. The centre of this region is where the drug is acting like a buffer – because there are both acid and basic forms of the drug present. Baselines can be fit to determine the midpoint of the flat region, or alternately, a derivative of this curve (dV/dpH) will provide a maximum at the pKa.

If the pKa is greater than the pH, there will be more of the acid (or conjugate acid) form in the solution. If the pKa is equal to the pH, there will be an equal amount of acid and base in the solution. If the pKa is less than the pH, there will be more of the base (or conjugate base) form in the solution.

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51 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Lab

Alternately if an equimolar amount of acid and basic forms of a drug are added to solution and the pH is measured immediately, the resultant pH should be close or equal to the pKa of the drug.

Calculation of pHp

To prepare a solution of weak electrolyte, the formulator would usually use the salt form of the drug, such as the sodium or hydrochloride salt. However, the addition of other ingredients may alter the final pH of the solution, causing the active ingredient to precipitate from the solution. To prevent unwanted precipitation, the pH of the solution should be buffered so that the drug remains dissolved. A calculation of pHp, the pH below which a weak acid will precipitate from the solution or above which a weak base will precipitate from the solution, must be made and the pH of the solution adjusted accordingly. How is this pH calculated?

We start with an expression of total solubility. The total solubility (ST) of a weak acid is the

summation of the un-ionized form, [HA], and the ionized form, [A-]:

(5) ST = [HA] + [A-]

We would like to derive a relationship between total solubility, pH, and pKa. We can re-arrange equation (2) to solve for [A-]:

(6) ][H

[HA]K][A a

Now substitute (6) into equation (5):

(7)

][H

[HA]K[HA]S a

(8)

][H

K1 [HA]S a

Using the identities in (2) and (3),

(9)

-pKa

T10

101 [HA]S

(10) pKapH

T 101 [HA]S

0 1 2 3 4 5 6 7 8 9 10 11

Volume NaOH Added (mL)

pH Titration Curve

5 6 7 8 9 10 11 12dV

/dp

H (

/pH

it)

dV/dpH vs pH

pKa

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52 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

0.1

100

1000

0 2 4 6 8

Tota

l Solu

bilit

y (

mg/m

Weak Acid:

Total Solubility vs. pH

100

0 2 4 6 8% o

f d

rug i

n i

ized

form

Weak Acid:

% Ionized vs. pH

We define S0 as the intrinsic solubility of the acid. This is, quite literally, the solubility limit of the drug if it were completely in the un-ionized (HA) form. Therefore, equation (10) becomes:

(11)

pKapH

0T 101SS

Finally, we can re-arrange this equation into a form similar to the Henderson-Hasselbalch equation:

(12)

0T pKpHS

SSlog

(for weak acids)

What does this mean for a weak acid? Let’s take a step backwards. Water is polar, and would form hydrogen bonds with a molecule that is ionized. So, it makes sense that the charged form of a drug (A-) will be more soluble than the un-ionized form (HA). If the pH of the solution keeps the drug un-ionized, the total solubility will be equal to the intrinsic solubility. As the pH of a solution of weak acid increases, more and more of the drug will be in the ionized form. From Equation (12), we can see that when pH=pKa, the total solubility of the drug should be double the intrinsic solubility. When the pH rises above the pKa, Equation (12) implies that the solubility keeps increasing without limit. In reality this does not occur, because eventually there are not enough counter-ions in solution to support more ions dissolving.

The whole concept can sound rather confusing, but becomes simple when you think of it in terms of percent of drug in ionized form. Here is an example of the solubility of a weak acid (pKa = 4, intrinsic solubility = 0.1 mg/mL) at different pH values, and a corresponding graph of the percent of drug ionized:

The left graph was calculated using Equation (12). Upon visual inspection, it becomes evident that when the pH dips below the pKa, the acid is mostly un-ionized (right panel), and the solubility reduces asymptotically to the intrinsic solubility (left panel). So if you have a saturated solution of this drug at pH 5 and the pH drops, you can expect precipitation.

A similar derivation can be performed for a weak base, yielding the equation:

(13)

pHpKS

SSlog a

(for weak bases)

A weak base behaves the opposite way. The base will be in the ionic form when the pH is less than the pKa. Therefore, the total solubility of a weak base increases as the pH decreases. Here

0.1

100

1000

0 2 4 6 8

Tota

l Solu

bilit

y (

mg/m

Weak Acid:

Total Solubility vs. pH

100

0 2 4 6 8% o

f d

rug i

n i

ized

form

Weak Acid:

% Ionized vs. pH

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53 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Lab

is an example of of the solubility of a weak base (pKa = 8, intrinsic solubility = 0.1 mg/mL) at different pH values, and a corresponding graph of the percent of drug ionized:

In summary, a weak acid will be more soluble at a high pH, and a weak base will be more soluble at a low pH. You can see that a small shift in pH can have a drastic effect, particularly when the pH is close to the pKa. Equations (12) and (13) may be used to calculate pHp, the pH below which a solution of a weak acid is likely to precipitate, and above which a saturated solution of a weak base is likely to precipitate. Simply substitute the drug concentration of your solution as “ST” into the equation, and calculate the pH at which this concentration is the total solubility limit.

A saturated solution of a weak acid will precipitate if the pH falls below the pHp

A saturated solution of a weak base will precipitate if the pH rises above the pHp

Chemical Stability

Discussed above are the physical aspects of a drug substance. Attention should also be paid to its chemical properties during the pre-formulation stage. For example, the stability of the drug in various storage conditions should be determined. (e.g. under sunlight, at room temperature and 70% relative humidity) A drug might undergo hydrolytic degradation, oxidation, and could exhibit incompatibility with certain excipients. The analysis of stability data will be discussed in Laboratory Exercise #2.

Proteins and peptides, produced by the commercialization of biotechnology and used as radiopharmaceuticals, present a greater analysis and formulation challenge. These proteins and peptides are intrinsically unstable and denature easily. While the same pre-formulation principles apply, more sophisticated techniques are required to characterize and evaluate these drug products.

Melting Point

An important part of characterizing the stability of a drug is determining its melting point. Knowledge of the thermostability of a drug is a critical factor in compounding, as the process may involve high temperatures and pressures (e.g. high shear granulation, tableting). The melting point of a

0.1

100

1000

10000

5 6 7 8 9 10 11 12 13 14To

tal S

olu

bil

ity

g/m

Weak Base:

Total Solubility vs. pH

100

5 6 7 8 9 10 11 12 13 14

% o

f d

rug

niz

ed f

orm

Weak Base:

% Ionized vs. pH

0.1

100

1000

10000

5 6 7 8 9 10 11 12 13 14To

tal S

olu

bil

ity

g/m

Weak Base:

Total Solubility vs. pH

100

5 6 7 8 9 10 11 12 13 14

% o

f d

rug

niz

ed f

orm

Weak Base:

% Ionized vs. pH

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54 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

substance is the temperature at which it changes from a solid to a liquid state. The capillary method is commonly used, as it is reproducible, relatively inexpensive, fast, and simple. Briefly, a sample is loaded into a capillary tube, which is inserted into a capillary tube melting point apparatus. The temperature is increased at a controlled rate while an observer (or video recording device) monitors the sample.

Relatively small amounts of impurities can change the melting temperature of a substance, or broaden its range. As a general guideline, 1% of a foreign substance will result in a 0.5 °C depression in the melting point. That is why most melting point units have three chambers: one for the unknown test substance (to be identified), one for the reference substance (100% pure), and one for an equal combination of both. If the melting ranges and melting point in all three chambers agree, then the conclusion of the test is that the test and reference substances are the same.

However, the effect of mixing two substances together does not always broaden the melting point range. In general, mixing two substances together will result in a depression of the melting temperature. For the mixing of two substances, the melting temperature hits a minimum at a specific combination of both substances. This is known as the “eutectic point”, and is not necessarily at a 50/50 mixture.

It is important to mention, because the mixture of two substances will not necessarily exhibit a broad melting point range. A eutectic mixture has the characteristic of a sharp melting temperature. The dash line in the above phase diagram indicates where melting begins, and the solid line indicates the clear point.

Important observations are made throughout the melting process, which should be recorded. The following distinct stages are usually present:

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55 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Lab

Stages of Melting Image Description

Starting Point

No liquid or condensation present. Sample is completely solid/crystalline.

First Signs of Change Early changes may be due to solvent loss, dehydration, change in crystallization state, decomposition (darkening of colour), condensation of solvent on cool parts of the capillary.

Sintering Point A few surface crystals melt.

Onset Point / Collapse Point

The “official” start of the melt: liquid clearly appears in equilibrium with crystals. This is the lower temperature recorded in the melting point range.

Meniscus Point

Enough crystals melt to form a clear meniscus in the capillary tube. There is a solid phase at the bottom and a liquid phase at the top. Sometimes bubbles will prevent a clear meniscus from forming. In Europe, it is also recorded as the melting point.

Clear Point / Liquefaction Point

The substance becomes completely liquid. There are no longer any solid crystals. This is the higher temperature recorded in the melting point range. In North America, it is also recorded as the melting point.

Chemical Degradation

It is important to note that some substances will degrade before they melt, and thus a melting temperature for these compounds cannot be recorded. Darkening or blackening during the melting process is a clear indication of thermal degradation. Degradation may also occur after the Clear Point.

The melting point and melting range are dependent upon the heating rate used in the melting point apparatus (also called the ramp rate). The ramp rate must be recorded along with the melting range in order to ensure reproducibility and in order to identify the test. In general, a slower ramp rate will provide better resolution and a narrower melting range.

Other parameters that need to be considered during pre-formulation include the type of sterilization used and the choice of packaging materials.

Polymorphism

Many drugs when dissolved and then are re-crystallized will form new crystals with a different molecular packing or arrangement than the original form. They are polymorphic forms of the

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56 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

drug or polymorphs. The new forms are called metastable and over time will often return to the original more stable state. These new crystal forms are often less stable than the original and exhibit a different melting point and sometimes greater solubility. This is important for drug dissolution and availability to the patient. The metastable polymorph’s different properties will also affect the milling and the tablet compression settings during dosage form production.

It is recommended that the descriptions of polymorphism in the text by Aulton and the paper by Apperley be studied before this laboratory exercise.

Sulfathiazole has been extensively studied and the many polymorphs that exist after crystallization have been studied. The most common is polymorph I with a melting point of 202 oC. The melting points of the other common polymorphs II to V are 197, 175, 175, and 175 oC respectively. Polymorphs I and V will be prepared in this laboratory.

Experiment Protocol

Chemicals Supplies Special Equipment Sulfathiazole (free acid, MW 255.31 g/mol) Ethanol 95% 1-Propanol Sorenson’s Phosphate Buffer pH 7.4 (from Lab 2) Potassium acetate (MW 98.14 g/mol) Calcium acetate (MW 158.17 g/mol) Sodium Hydroxide (MW 40.0 g/mol)

Glass Cover Slips Capillary Tubes Grid Slide Scintillation Vials

pH Meter Graduated Cylinder 25 °C Water Bath 80 °C Water Bath Light Microscope Melting Point Apparatus 50 mL Burette, Burette Clamp, and Retort Stand

The following solutions are prepared or provided by the TA:

Hydrochloric Acid (1.0 N)

Ammonium hydroxide (0.1 M)

pH Meter Standardizing Buffers (pH 4, 7)

Part A. Intrinsic Solubility Determination

Prepare a series of ten glass liquid scintillation vials each containing different quantities of sulfathiazole (the free acid) - 2 mg to 20 mg.

Add 20 mL of Sorenson’s phosphate buffer (pH 7.4) to each vial.

The vials are capped and allowed to equilibrate with agitation in a constant temperature bath at 25 °C. NOTE: usually they are agitated overnight and then allowed to equilibrate 3 hours. Start Part D as early as possible.

At equilibrium (approximately 1-2 hours, record the actual time), visually inspect each vial and record the presence and relative magnitude of un-dissolved crystals of drug in the vials.

Record the drug content, i.e., concentration, for the crystal-containing vial having the least amount of drug.

Record the drug content for the crystal-free vial having the greatest amount of dissolved drug.

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57 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Lab

Average the upper and lower limits to obtain the approximate solubility of the drug, ST,

under the conditions of the determination.

Determine the pH of the buffer solution of the first vial in the series containing crystals.

Calculate the intrinsic solubility for the drug at 25 °C using the pKa determined in Part D (below) and the ST value determined in this section.

Part B. Preparing Different Salts of Sulfathiazole

Weigh two 200 mg samples of sulfathiazole. Place each sample in a 50 mL Erlenmeyer flask. Label the flasks K and C .

In flask K, add 10 mL of 1-propanol. Gently heat in an 80 mL water bath (in a 150 or 250 mL beaker) at ~ 80°C until dissolution is complete.

While flask K is on the hot plate, add an amount of anhydrous potassium acetate equimolar to sulfathiazole to the flask.

Dissolve the anhydrous potassium acetate by continuing to slowly titrate small aliquots of 1-propanol, and stirring, until the potassium acetate crystals disappear.

Remove the flask from the water bath, and allow the content to cool to room temperature. Cover the flask with Parafilm. Let the flask stand for a few days or until crystallization occurs.

Repeat the above procedures for flask C except adding an amount of anhydrous calcium acetate equimolar to sulfathiazole.

The salts produced in this exercise will be examined during the second part of this exercise one week from now.

Part C. Preparing Different Polymorphs of Sulfathiazole

Weigh two 200 mg samples of sulfathiazole. Place each sample in a 50 mL Erlenmeyer flask. Label the flasks I and V.

Add 10 mL of 1-propanol to flask I, gently heat in a water bath at ~ 80°C until dissolution is complete.

NOTE: The propanol is volatile; you may need to add more than 10 mL in order to dissolve the drug.

Remove the flask from the water bath and allow the content to cool to room temperature. Cover the flask with parafilm and let it stand for a few days until crystallization occurs.

Add 10 mL of ammonium hydroxide 0.1 M into flask V and dissolve. Neutralize the solution using 1ml of 1 N HCl and allow it to stand until crystallization occurs. This may take a few days.

NOTE: You may need to add more than 10 mL of ammonium hydroxide in order to dissolve the drug.

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58 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

The crystals produced in this exercise will be examined during the second part of this exercise one week from now.

Part D. pKa Determination

NOTE: Use the sensitive analytical balances in the laboratory balance area.

Each group will prepare a set of two solutions at a single ethanol concentration, and perform their own titration. Team up with 2 other groups, and decide who will titrate 1A with 1B, 2A with 2B, and 3A with 3B. You will need the pKa values at all three ethanol concentrations to perform the final analysis.

Make 100 mL of each assigned solution (use 100 mL volumetric flasks, add the calculated amount of ethanol, sulfathiazole, and/or NaOH, then dilute to the mark with de-ionized water). The starting materials will be sulfathiazole powder, and sodium hydroxide pellets.

Solution: 1A 1B 2A 2B 3A 3B

% Ethanol by volume*

10% 10% 20% 20% 30% 30%

Sulfathiazole 0.001 M - 0.001 M - 0.001 M -

NaOH - 0.015 M - 0.015 M - 0.025 M

*Take into account that the stock concentration of ethanol is 95% v/v in water.

NOTE: Solution A will take a while to dissolve. Continue shaking until no crystals are present in solution. You may sonicate the solution if a sonicator is available. To prepare Solution B, you will need to grind 3 – 4 sodium hydroxide pellets in a mortar and pestle in order to weigh a precise amount. Transfer the required amount of the NaOH powder from the weighing boat into the volumetric flask by angling the corner of the boat into the spout of the volumetric flask, and washing the crystals into the flask with de-ionized water from your plastic wash bottle.

Wear protective gloves when working with 1 N HCl, and sodium hydroxide pellets.

Calibrate a pH meter with the standard buffer solutions.

Set up a burette on a retort stand. Place a stir plate at the base of the stand.

Pour all 100 mL of your assigned Solution A (1A, 2A, or 3A) into a 150 mL beaker. Place the beaker on the stir plate.

Load the burette with 50 mL of your assigned Solution B (1B, 2B, or 3B).

Insert the pH probe into the 150 mL beaker.

Add a magnetic stir bar to the beaker. Set the stir plate to a low stable speed, making sure that the stir bar does not knock against the probe tip or side of the glass beaker.

Adjust the pH of Solution A to below pH 4 with 1 – 2 drops of 0.1 N HCl.

Record the first pH with zero volume of NaOH added.

Add 0.5 mL of Solution B. Allow the pH to equilibrate (at least 20-30 seconds). Record the volume added and pH.

Continue adding 0.5 mL aliquots of Solution B and recording volume added and pH, until the pH is above 10. This should require at least 20 aliquots.

Download the Excel spreadsheet titration.xls from the laboratory website to create a graph of your titration. Use the “Titration” worksheet to find the pKa at your ethanol concentration. Then use the “Y-S” worksheet to use your pKa value, along with the pKa

http://phm.utoronto.ca/~ddubins/DL/titration.xls

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59 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Lab

values determined at other ethanol concentrations by other groups, to extrapolate the pKa to zero alcohol concentration. This is known as the Yasuda-Shedlovsky extrapolation.

Report the value of the apparent pKa at zero alcohol concentration and the r2 value to show the linearity of your data.

Part E. Melting Point Determination

Place a small amount (~0.1 g) of sulfathiazole on a clean, dry watch glass.

Crush the solid to a fine powder by gently rubbing it with the flat end of a spatula or pestle.

Open a glass capillary tube by scoring it around the middle, and gently bending.

Tap the capillary, open end downwards, repeatedly onto the dry powder.

Turn the capillary over (closed end down) and gently tap so that enough powder (1-2 mm) falls to the bottom.

Load the capillary, closed-end downwards, into the melting point apparatus.

Set the melting point apparatus to heat. Your TA or instructor will demonstrate proper use of the melting point apparatus.

A “coarse” or preliminary run may be conducted to obtain the approximate range.

On a new sample, a “fine” run is then conducted, with a slowed heating rate once the temperature is within 20 °C of the coarse melting point. For the fine run:

Do not insert the sample until the temperature is about 10 °C below the coarse melting point. This will minimize product degradation.

Do not exceed 1-2 °C/min.

Observe and record the melting temperature range of the solid powder. (HINT: The melting point of sulfathiazole is above 200 oC.)

Once a sample has been melted, discard it (decomposition, oxidation, or polymorphism conversion are likely)

Notes:

only use a small amount (overfilling will result in uneven heating, broader ranges)

Pack the sample well (loose samples will heat unevenly)

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60 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa

Part F. Macroscopic Evaluation

Obtain some anhydrous sulfathiazole powder. Record the colour, shape and general appearance of the crystals using the stereo microscopes.

Questions

1. Phenobarbital is a weak acid. It has a pKa of 7.41 and an intrinsic solubility of 1 g in 987 mL of water at 25 oC. You have to formulate a 5 mg/mL solution. Calculate what pH will be required to solubilize the drug.

2. Phenobarbital is a weak acid. Calculate the pHp of a 10 mg/mL phenobarbital sodium solution. What would happen if while in storage, the pH of the solution rises to 9? How about if the pH falls to 7?

3. What is the pHp of a 7 mg/mL dobutamine hydrochloride solution? Dobutamine increases cardiac output during short-term use. Its pKa and its intrinsic solubility are 9.4 and 1 mg/mL, respectively. What happens if the pH of the solution is adjusted to 10.2?

4. How could propylene glycol be used in this experiment?

5. Derive the formula for the fraction of drug ionized in solution if the drug is a weak acid, given the pKa and of the drug and the pH of the solution.

Hint 1: Fraction of drug ionized = ][A[HA]

][A

Hint 2: For a weak acid, pHpK

-a10

][A

[HA]

Hint 3: There shouldn’t be any concentrations in the final formula.

6. If a weak base (pKa=8.5) is in a solution buffered at pH 8.5, what proportion of the drug will be in the ionized form?

7. Does a weak base precipitate above or below the pHp, assuming that the basic form is un-ionized and the conjugate acid form is ionized?

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61 Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification

Lab

Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 3 students (work in the same groups as Lab 4)

What You’ll Be Doing

Part A: Determining the solubility of benzocaine in different concentrations of ethanol Part B: Melting point, visual inspection of the salts from Lab 4 Part C: Melting point, visual inspection of polymorphs from Lab 4

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/benzo.xls

What You’re Handing In Lab 5 Worksheet (due at the beginning of the next lab)

Introduction

Co-solvency

A drug administered in solution is immediately available for absorption and, in most cases, is more rapidly and more efficiently absorbed than the same amount of drug administered in a tablet (with the exception of sublingual tablets) or capsule. However, many pharmaceutical agents have limited solubility in water and may precipitate from solution due to the presence of other ingredients where a pH adjustment is not possible. In these instances, it is often necessary to utilize a blend of solvents for formulation purposes. Water is usually the main component of a mixed solvent system, with just enough co-solvent to keep the drug solvated. It is therefore necessary to know the solubility characteristics of an agent as a function of solvent composition over a wide range. This can be determined on a semi-quantitative basis by a water titration method. Although the method is not exact, it does provide a relatively rapid means for estimating solubility without recourse to time-consuming analytical procedures.

In addition to the active ingredient, a solution may also contain solvents, co-solvents, preservatives, buffers, sweetening agents, viscosity control agents, and flavouring agents.

Salt Selection and Polymorph Identification

Modern drug discovery involves the screening of a large number of compounds obtained by either structure-based design or combinatorial chemistry techniques. These compounds are generally dissolved in an aqueous solution of a water-miscible solvent such as DMSO and screened against various disease targets. Those lead candidates (aka new chemical entities or NCEs) showing desired biological activities are subsequently evaluated for their solid state characteristics and their physicochemical properties as pharmaceutical materials. This is an important process as new chemical entities considered for development as a drug candidate must have optimal physical and chemical properties to ensure stability during processing and storage in the final dosage form.

One of the first steps in optimizing the lead candidate is to select an appropriate salt form of the new chemical entity such that it would have desired properties for subsequent formulation development. Because most of the drug candidates are a weak base or a weak acid, the selection of suitable counter ions to form salts provides the opportunity for pharmaceutical scientists to modify certain physical properties of a compound without affecting its biological activity. As a result, salts are often employed to improve the aqueous solubility. During the salt

http://phm.utoronto.ca/~ddubins/DL/benzo.xls

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62 Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification

selection, a range of salts is prepared for each new chemical entity and their solid state properties characterized and compared. When an appropriate salt or the free molecule has been selected, the crystalline form of the solid needs to be properly defined. Polymorphs exist as a result of the ability of a compound to exist as two or more crystalline phases that have the same chemical composition but different arrangements of the molecule in the crystal lattice. These different polymorphic forms while similar in chemical structure possess different physical and chemical properties such as solubility, stability and bioavailability. Polymorph screening generally involves the exploration of various re-crystallization solvents and/or experimental conditions to obtain various crystalline forms for further identification and characterization.

The salt and polymorphic form selected will influence various properties of the solid such as melting temperature, hygroscopicity, physical and chemical stability, and mechanical properties. These properties can in turn affect the processing and manufacturing as well as the stability of the drug product. Therefore, a good understanding of the influence of drug, salt and crystalline form properties on the final drug product is essential to ensure successful selection of the best salt and polymorph for formulation development.

In Lab 4, sulfathiazole and its sodium salt were examined, and two salts and two polymorphs were prepared. In this exercise the polymorphs will be examined.

References

1. Edmonson, T.D. and Goyan, J.E., J. Am. Pharm. Assoc. Sci. Ed., 47, 810 (1958).

2. Lachman L, Lieberman H A and Kanig J L, ed., The Theory and Practice of Industrial Pharmacy, 2nd

ed., U.S.A.: Lea & Febiger, 1976, p. 32-77, 541-566.

3. Ware, E.C. and D.R. Lu, An Automated Approach to Salt Selection for New Unique Trazodone Salts, Pharmaceutical Research, 21, 177-184 (2004)

4. Apperley DC, et al, Sulfathiazole Polymorphism Studied by Magic-Angle Spinning NMR, J. Pharm Sci., 88, No. 12, 1275-1280 (1999)

Background

Co-solvency

Weak electrolytes and non-polar molecules frequently have poor water solubility. Their solubility usually can be increased by the addition of a water-miscible solvent in which the drug has good solubility. This process is known as co-solvency. Ethanol, sorbitol, glycerin, propylene glycol, and several members of the polyethylene glycol polymer series represent the limited number of co-solvents that are both useful and generally acceptable in the formulation of aqueous liquids. Co-solvents may also be used to improve the solubility of volatile constituents used to impart a desirable flavor and odour to the product.

Salt Selection

Generally, salt imparts unique properties onto the parent compound. The selection of the best salt form depends on several factors such as the physical form of the solid (does it form crystals?) and physical properties such as melting point and solubility. In this laboratory exercise, the physical appearance and the melting point as well as solubility will be examined for different salts of sulfathiazole.

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63 Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification

Lab

Polymorph Identification

Polymorphs of sulfathiazole will usually have a different physical appearance than the parent free acid. There are 5 common polymorphs each exhibiting slightly different properties. In this laboratory exercise the physical appearance and the melting point will be examined in order to establish the presence of distinct polymorphs of the drug.

Experiment Protocol

Chemicals Supplies Special Equipment Sulfathiazole Polymorphs (from Lab 4) Benzocaine Ethanol 95%

Capillary Tubes Filter Paper

Burette Burette Clamp Retort Stand 10 mL Graduated Pipette 50 mL Graduated Cylinders 100 mL Graduated Cylinders Microscope Melting point Apparatus

The following solutions are prepared or provided by the TA:

Not Applicable

Part A. Co-solvency

Prepare a stock solution of benzocaine in 95% alcohol:

Accurately weigh 8 g of benzocaine (don’t forget to record the actual weight).

Dissolve the benzocaine in 35 mL of 95% alcohol in a 100 mL graduated cylinder and add sufficient alcohol to produce a final 50.0 mL stock solution volume.

Agitate until dissolved (it will take a while). You may use the mechanical agitator.

Pipette an aliquot of the stock solution, as noted in trial 1 of the table below, into a 50 mL graduated cylinder (supplied by your TA).

Pipette the corresponding amount of alcohol, as noted in trial 1 of the table below, into the 50 mL graduated cylinder.

Record the total volume in the cylinder

Slowly titrate with water from a burette, while stirring vigorously, until the first definite permanent precipitate is evident.

Record the number of milliliters of water required to cause precipitation.

Record the total volume in the cylinder.

Repeat for the remaining trials as outlined in the table:

Trial # Stock Solution 95% Ethanol

1 10.0 mL 0 mL

2 8.0 mL 2.0 mL

3 6.0 mL 4.0 mL

4 5.0 mL 5.0 mL

5 4.0 mL 6.0 mL

6 3.0 mL 7.0 mL

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64 Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification

Record the following results:

– Volume of stock solution used (mL)

– Volume of alcohol, 95%, added (mL)

– Water added to precipitation (mL) (reading from the burette)

– Total volume of solution at precipitation

– % v/v of 95% alcohol in solution at precipitation

– % w/v of benzocaine at precipitation

By means of a graph, plot the calculated concentration of benzocaine (essential solubility) on the X-axis versus with ethanol concentration (%v/v) on the Y-axis. The spreadsheet benzo.xls should be used to plot your graph.

Part B. Salt Selection

Isolate the sulfathiazole crystals (from Lab 4) by decanting the supernatant, or by filtration. Air dry, or dry the crystals in a laboratory oven.

Describe both the gross and the microscopic appearance of both of the salts prepared in Laboratory 2. Draw the crystal shape as observed under the microscope.

Measure and record the melting point of each salt.

Part C. Polymorph Identification

Isolate the crystals by carefully decanting the supernatant (pour off the liquid without disturbing the sediment) or by filtration. Air dry or drying the crystals in an oven.

Describe both the gross and the microscopic appearance of both of the polymorphs prepared in Laboratory 2. Draw the crystal shape as observed under the microscope.

Measure and record the melting point of each polymorph.

NOTE: Based on the number of students in the course, melting point determination of the salts and polymorphs may be allocated in groups.

Questions

1. What is the effect of alcohol on the solubility of benzocaine? How does alcohol exert this effect?

2. What properties of the solution would be affected by the addition of alcohol in order to solubilize a drug?

3. What additional methods could be employed in order to produce additional polymorphs of sulfathiazole?

4. What solvents, other than alcohol, can be used to increase the solubility of a drug?

5. Design a 1% benzocaine solution suitable for use in emergency first degree burn situations. Benzocaine: MP 88-92 oC, 1 gram dissolves in: 2500 mL water, 5 mL acetone, 4 mL alcohol, 5 mL ether, 30 – 50 mL of almond or olive oil and in 2 mL chloroform.

http://phm.utoronto.ca/~ddubins/DL/benzo.xls

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65 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

Lab

Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

Preparing for the Lab Read the introduction and lab protocol completely Calculate the mass of potassium hydroxide required for Part C

Group Allocation You will be working in groups of 2 students

What You’ll Be Doing

Part A: Calibrating a Thermos Calorimeter Part B: Determining the Specific Heat Capacity of Copper Metal Part C: Determining the Heat of Reaction of Solid KOH and HCl Part D: Determining the Heat of Reaction of Liquid KOH and HCl Part E: Observing volumetric changes when mixing:

Solid NaOH + water

95% EtOH + water Spreadsheets You Will Need Not Applicable

What You’re Handing In Lab 6 Worksheet (due at the beginning of the next lab)

Introduction

In the preparation and evaluation of pharmaceuticals, the dissolution and mixing of components are very important: medicines are almost never administered as pure substances. For this reason, understanding the physical chemistry and thermodynamics underlying dissolution and mixing is central to most aspects of dosage form design. As with many aspects of chemistry, the change in free energy arising from dissolution or mixing may be used as a qualitative measure of these processes. It is also useful to access the conditions present when vessels are used in large scale production and when acids and bases are neutralized or mixed. In this laboratory you will use calorimeter to examine some thermodynamic principles. The calorimeter will be calibrated, a specific heat capacity will be determined of copper, and dissolution and acid/base neutralization studied. You will also examine the volume of mixing two liquids as an introduction to the concepts of partial molar quantities, specifically, the partial molar volume.

References

1. Aulton, ME, Pharmaceutics, the Science of Dosage Form Design, Churchill Livingstone, Edinburgh, 2

nd Ed, 2002

2. http://www.southernct.edu/departments/ftrc/chemistry/videos/coffeecup.htm

3. http://web.umr.edu/~gbert/cupCal/Acups.html

4. http://www2.stetson.edu/~wgrubbs/datadriven/fchen/bartender/partialmolarvolumechenpdf.pdf

Background

Specific Heat Capacity

Heat capacity is the ability of a material to change temperature depending on how much energy is added. Sometimes, energy can be added to a system and the temperature can rise very rapidly. Other times, you can add heat to a system, and the temperature won’t change at all (e.g. during an ice to liquid water transition).

The general equation for the amount of energy required to raise the temperature of substance “i” is:

(1) Qi = mi × Ci × T Where Qi is the energy gained or lost as a result of the change in temperature,

http://www.southernct.edu/departments/ftrc/chemistry/videos/coffeecup.htm

http://web.umr.edu/~gbert/cupCal/Acups.html

http://www2.stetson.edu/~wgrubbs/datadriven/fchen/bartender/partialmolarvolumechenpdf.pdf

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66 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

mi is the mass of substance x, Ci is the specific heat capacity of substance x,

T is the change in temperature of substance i (expressed as Tfinal – Tinitial).

When energy transfers from hot water to cold water at a constant pressure, we can write Equation (1) twice. The energy lost by the hot water (Qhot) is:

(2) Qhot = mhotCp,w (Tfinal - Thot)

Note the subscript on Cp,w to denote the specific heat capacity of water at constant pressure.

We associate a negative value of Q with a loss of energy (exothermic). This makes sense, because when the system cools down, it is losing energy.

The energy gained by the cold water (Qcold) is:

(3) Qcold = mcoldCp,w(Tfinal - Tcold)

We associate a positive value of Q with gain in energy (endothermic). When a system heats up, it is gaining energy.

If the system was perfectly adiabatic (no heat loss), then all of the energy lost by the hot water should equal the energy gained by the cold water. In other words:

(4) Qhot + Qcold = 0, or Qhot = -Qcold.

This is the main idea behind the first law of thermodynamics: the energy in a closed system remains constant.

Calibrating the Calorimeter (Finding Cp,cal)

In reality, the calorimeter is not perfect. It absorbs some heat. So we add an extra term into our system to account for this heat loss: Qcal:

(5) Qcal = Cp,cal(Tfinal – Tcold)

If system loses heat: (exothermic) Q = mcp(Tfinal – Tinitial) Since Tfinal < Tinitial, Q < 0.

If system gains heat: (endothermic) Q = mcp(Tfinal – Tinitial) Since Tfinal > Tinitial, Q > 0.

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67 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

Lab

The heat absorbed by the calorimeter (Qcal) won’t be the same for every experiment; it will depend on the temperature. That’s why we will solve for Cp,cal, the heat capacity of the calorimeter. This will remain constant.

Question: What happened to the mass term in Equation (5)? Shouldn’t it be Qcal=mcalCp,cal(Tfinal-Tcold)?

Answer: Yes, technically it could. You could weigh the calorimeter, and obtain a Cp,cal in units J/g*K. However, since the mass of the calorimeter won’t change, we can ‘lump’ the mass together with the specific heat capacity out of convenience, and report Cp,cal in J/K.

Consider the system now with an added term for the heat absorbed by the calorimeter:

Item Initial Temperature Final Temperature Q (J)

Hot Water Measured Thot (Temp of hot water, ~60°C)

Measured Tfinal Observed temperature at equilibrium

Qhot = mhotCp,w(Tfinal-Thot)

Cold Water Measured Tcold ~ambient Measured Tfinal Observed temperature at equilibrium

Qcold = mcoldCp,w(Tfinal-Tcold)

Calorimeter Measured Tcold ~ambient Measured Tfinal Observed temperature at equilibrium

Qcal = Cp,cal(Tfinal-Tcold)

System Qhot + Qcold + Qcal = 0

First you can use the energy balance to solve for Qcal:

(6) Qcal = - Qhot - Qcold

Now Cp,cal can be solved for:

(7) )T(T

coldfinal

cal

calp,

Once Cp,cal is known, you can use it in your future calorimeter calculations. Qcal is estimated in future experiments using Equation (5).

For the purpose of performing the calculation, the specific heat capacity of water (Cp,w) is 4.184 J/(g*K).

Using the Coffee Cup Calorimeter

Once Cp,cal is determined in Part A, you are ready to use the calorimeter to measure the specific heat capacities of other materials. In Part B, you are placing a heated piece of copper tubing into the cold fluid in the calorimeter. So your system is slightly different:

Item Initial Temperature Final Temperature Q (J)

Copper Tube Measured TCu (Temp of boiling water, ~100°C)

Measured Tfinal Observed temperature at equilibrium

QCu= mCuCp,Cu(Tfinal-TCu)

Cold Water Measured Tcold ~ambient

Measured Tfinal Observed temperature at equilibrium

Qw = mwCp,w(Tfinal-Tcold)

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Calorimeter Measured Tcold ~ambient

Measured Tfinal Observed temperature at equilibrium

Qcal = Cp,cal(Tfinal-Tcold)

System QCu + Qw + Qcal = 0

You can see how your calibration comes into play. Your ‘lumped’ heat capacity of the calorimeter is used to estimate how much energy the calorimeter absorbed when it was heated from ambient to the final equilibration temperature. The only unknown in this system is the heat capacity of the copper tube, which you can solve for:

Use the value of Cp,cal from Part A to estimate Qcal :

Qcal = Cp,cal(Tfinal-Tcold)

Use the energy balance from the table above to estimate the heat released by the copper tube (Qcu):

QCu + Qw + Qcal = 0

You can solve for Cp,Cu , because we know:

QCu= mCuCp,Cu(Tfinal-TCu)

Enthalpy

In Exercise (1), one fluid loses heat (Q is negative) while one fluid gains heat (Q is positive). When you use the calorimeter to measure the heat of a reaction, the heat of a reaction is equal to the heat absorbed by the water plus the heat absorbed by the calorimeter. However, since the reaction is giving off the heat, and the calorimeter water is absorbing it, the sign changes. Consequently, the expression for the enthalpy (heat) of reaction is:

(8) Hrxn = - (Qwater + Qcal)

When the reaction is exothermic, heat is given off, and Hrxn is negative.

When the reaction is enothermic, heat is absorbed, and Hrxn is positive.

Typically, heats of reaction are reported per mole of reactant, and called “molar heat of reaction”).

The standard heat of reaction (H°rxn) is the enthalpy change occurring when 1 mole of substance in its standard state reacts at standard temperature and pressure (25 °C, 1 atm).

When you report heat of reactions for Parts C and D, report your answers in terms of molar heat

of reaction, H.

Partial Molar Quantities

Thermodynamically, any extensive property Y of a mixture such as volume or enthalpy, which is proportional to the amount of the phase under consideration, can be expressed by:

(9)

............_

1 ii YnYnYnY

i this equation, Yi and ni are the partial molar quantity and the number of moles of component i, respectively. This partial molar quantity is the contribution per mole of the component i to Y in a mixture and is defined by:

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(10)

.....,,,

21 nnPTi

In Part E, the partial molar volume Vi is the change in volume of a mixture, at a constant temperature and pressure, resulting from the addition of 1 mole of component i to such a large reservoir of solution that there is no appreciable change in the concentration. The magnitude of Vi may vary with the concentration of i in the mixture. Such variation depends upon the nature of interactions between the components of the mixture at the given concentration, temperature, and pressure and must be evaluated experimentally. In the special case of an ideal solution, the partial molar volume Vi is equal to the molar volume V°i of the pure component.

For a binary mixture, the total volume V at a constant temperature and pressure is given by:

(11)

11 VnVnV

And just like Equation (10), the partial molar volumes of a two component system can be defined as:

(12)

,,n

nPT

(13)

,,n

nPT

When you mix the volumes of two different liquids, it is a common misconception to think the final volume will just be their individual volumes added together. However, the interaction of these liquids may result different molecular interactions, which could cause volumes to change. For instance, adding salt to a volume of water will cause the volume to contract, since the effect of electrostriction is increased in the water.

However, the partial molar volumes of two different fluids are additive, which allows Equation (11) to hold true, even if V ≠ V1 + V2.

You will not need to use Equations (9) to (13) for the results section of this laboratory. They are provided for your reference only.

Experiment Protocol

Chemicals Supplies Special Equipment Potassium Hydroxide Pellets (MW 56.11 g/mol) Sodium Hydroxide Pellets (MW 40.00 g/mol) Ethanol (95%) Hydrochloric Acid (1.0 N)

Styrofoam cup N95 Mask

Hot plate Thermometer Thermos Copper tube Tongs

The following solutions are prepared or provided by the TA:

Hydrochloric Acid (1.0 N)

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70 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

WARNING: The acids and bases in this lab are very concentrated. Extreme care should be taken when handling 1 N HCl, potassium hydroxide pellets, and sodium hydroxide pellets. In addition to the standard protective lab gear (goggles, lab coat, etc.) thick kitchen gloves are recommended for concentrated solutions. Never pour acids down the lab sinks.

Parts A to D of today’s exercise involve the use of a Thermos unit containing a thermometer, into which you will be causing the temperature of the liquid inside the Thermos to change. The initial temperatures and weights of substances are recorded, and then the two are mixed in a Thermos unit. The final equilibration temperature (Tfinal) is observed.

Part A. Calibration of the Calorimeter

NOTE: Part A (Calibration) is performed IN TRIPLICATE to obtain a reliable measure.

Add 200 mL of de-ionized water in a previously tarred Styrofoam cup. Weigh the water, and record the mass.

Pour the water into the Thermos unit. Do not put the Styrofoam cup into the Thermos.

Insert the thermometer unit into the Thermos unit and record the temperature (Tcold).

Open the Thermos unit.

Weigh the copper tube, and put it on its side inside a 600 mL beaker. (This is a preparation step for Experiment 2).

Add a fresh 200 mL of de-ionized water to the 600 mL beaker with the copper tube, and place on a hot plate.

Add 150 mL of de-ionized water to a 250 mL beaker, and place on top of the copper tube in the 600 mL beaker.

Put a thermometer in the inner (250 mL) beaker.

Turn on the hotplate and heat the water in the inner beaker to 55 – 60 °C by heating on a hot plate.

Remove the thermometer from the inner beaker. Push the thermometer through the lid of the thermos.

NOTE: The top of the lid should be lined up with the 20 °C mark of the thermometer.

Place the water from the inner beaker into a tarred Styrofoam cup. Weigh the water, and record the temperature (Thot).

Quickly, without spilling, transfer the water to the Thermos unit. Screw the lid with the thermometer into the top of the thermos, and record the temperature and time immediately. This is time = 0.

Record the temperature every 10 seconds thereafter until the temperature is equilibrated (equilibration temperature = Tfinal).

Plot the temperature in °C vs. cumulative time in seconds.

In your calculations, for Cp,cal report the three calculated values, the average, and the standard deviation. Use the average value for Cp,cal in Parts B, C, and D.

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Part B. Specific Heat Capacity of Copper Metal

Add 200 mL of de-ionized water in a previously tarred Styrofoam cup. Weigh the water, and record the mass.

Place the water in the Thermos unit.

Insert the thermometer unit into the Thermos unit and record the temperature (Tcold).

Open the Thermos unit.

Using tongs, quickly remove the copper tube from the boiling water bath, shake it once and place it carefully into the Thermos unit.

Immediately attach the top with the thermometer, and record the temperature.

Prepare a plot in a similar manner to Part A.

Determine the Specific Heat Capacity of copper metal (CpCu).

Part C. Heat of Reaction and Heat of Hydration

Using a mortar and pestle, grind 25.0 grams of potassium hydroxide into a fine powder. Calculate the amount required to achieve a 1.0 N KOH solution in 200 mL and weigh this accurately. (This should be done before Part C begins.)

NOTE: Use an N95 mask when working with KOH.

Accurately weigh 200 mL of 1 N HCl using a tarred 200 or 400 mL beaker and put it into the Thermos unit. Record the temperature and mass of the liquid.

Quickly place this powder into the Thermos unit and attach the top with the thermometer. Measure and record temperature every 10 seconds.

Will this be an endothermic or exothermic process?

Part D. Measurement of Molar Enthalpy of Reaction

Calculate the amount required to achieve a 2.0 N KOH solution in 100 mL of deionized water, and prepare the solution. Ensure the KOH is completely dissolved. Record the mass of the KOH powder used.

Accurately weigh 100 mL of 1 N HCl using a tarred 200 or 400 mL beaker and put it into the Thermos unit. Record the temperature and mass of the liquid.

Measure the temperature of 2.0 N KOH solution. Quickly pour the solution into the Thermos unit and attach the top with the thermometer. Measure and record temperature every 10 seconds.

What difference (if any) do you observe?

Part E. Illustration of Partial Molar Volume

De-ionized Water + Sodium Hydroxide:

Place 50.0 mL of de-ionized water into a 100 mL graduated cylinder.

Weigh 10 grams of NaOH pellets.

Grind the NaOH pellets in a mortar and pestle.

Place the NaOH into the cylinder and record the volume.

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72 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

Place Parafilm securely (and quickly) on the top of the cylinder. Ensure there is an airtight seal between the parafilm and the mouth of the cylinder. Hold the graduated cylinder in your hand with your hand resting on the 5-10 mL level. What do you observe?

Holding the palm of your hand on the top of the cylinder, slowly mix the solution by swirling it. Does the volume of the liquid change? Is the Parafilm convex or concave on the top of the cylinder? What is the final volume? What is the molarity of the final mixture?

Continue to agitate the liquid, until the sodium hydroxide is completely dissolved.

Record the final volume of liquid in the cylinder.

De-ionized Water + 95% Ethanol:

Place exactly 50.0 mL of de-ionized water into a 100 mL graduated cylinder.

In a separate 50 mL graduated cylinder, measure exactly 40.0 mL of 95% ethanol.

In one pour, transfer the 40 mL of 95% ethanol into the 100 mL graduated cylinder.

Record the final volume of the mixture. Physically, what is happening? This is known as the “bartender’s conundrum”.

Is the Parafilm convex or concave on the top of the cylinder?

Name other extensive and intensive properties of a system.

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Lab 7: Examination of Viscosity and Suspending Agents Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 2 students

What You’ll Be Doing

Part A: Using the Ostwald viscometer to measure the viscosities of a dilution series of methylcellulose to estimate the intrinsic viscosity. Examining the effect of adding an acid and a salt to methylcellulose solution. Part B: Using the Brookfield viscometer to characterize the fluid behvaiour of 2% methylcellulose solution Part C: Timing the clear point of glass beads in 0.4% , 0.04%, and 0% methylcellulose solution Demonstration: Using the Ostwald and Brookfield viscometers

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/viscosity.xls

What You’re Handing In Lab 7 Worksheet (due at the beginning of the next lab)

Introduction

Having the drug solubilized does not assure a high quality product. For instance, topical gels and some oral solutions need to have higher viscosity to promote the ease of use. The use of suspending agents is essential to the stability of suspension and cream formulations. Polymer solutions of natural or synthetic origins are usually used to impart viscosity in pharmaceutical preparations. Many suspending agents are polymeric in nature. Some natural polymers include alginates, gum Arabic, carrageen, guar gum and xanthan gum, all of which are polysaccharides. Cellulose derivatives are obtained from purified cotton or wood cellulose and then further processed. Examples of such derivatives are carboxymethylcellulose (an anionic polymer), methylcelluose, ethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Cellulose itself is not a good thickener. However, microcrystalline cellulose can be combined with carboxymethylcellulose to produce thicker aqueous solutions. For example, Avicel RC-591 is produced by co-processing cellulose microcrystals with sodium carboxymethylcellulose and then it is spray-dried. Chitosan is a derivative of chitin, the important organic component in the skeletal material of invertebrates. It may be best formulated at pH 2-3 region. Other synthetic polymers include polyacrylic acid (Carbomer), polyvinylpyrrolidone, polyvinylalcohol, and magnesium aluminum silicate.

Polymers when used in suspension, emulsion, or dispersions can minimize particle sedimentation. Only a small amount of polymer is needed to produce liquid products which exhibit pseudoplastic and thixotropic properties of which are advantageous for sedimentation resistance and processing ease.

In this exercise, the properties of a polymer solution will be explored.

References

1. Schott H, “Rheology”, in Gennaro AR ed., Remington: The Science and Practice of Pharmacy, 20th

ed., Mack Publishing Company: U.S.A. (2000), p. 335-355.

2. Parrott EL, Pharmaceutical Technology: Fundamental Pharmaceutics, Burgess Publishing Company: U.S.A. (1970), p.341-363.

3. Asthana, R., Kumar A., Dahotre N. Materials Science in Manufacturing. Academic Press: USA (2003), p.193.

http://phm.utoronto.ca/~ddubins/DL/viscosity.xls

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74 Lab 7: Examination of Viscosity and Suspending Agents

4. Lamb, H. (1994). Hydrodynamics (6th edition ed.). Cambridge University Press. ISBN 9780521458689. Originally published in 1879, the 6th extended edition appeared first in 1932.

Background

An Overview of Rheology: What is Viscosity?

Viscosity is a measure of a liquid’s resistance to flow. It is the resistance to the relative motion of adjacent layers of liquid. Viscosity is defined as the tangential force per unit area required to maintain a difference in velocity of 1 cm/s between two parallel layers of liquid 1 cm apart. Its

unit is therefore dynes/cm2 or g/(cms), which is called a poise (P). Viscosity is often reported in centipoise (100 cP = 1 P). Centipoise is often also abbreviated to “cps”. In the SI system, the unit

of viscosity is Newtons/m2 or Pascals, which equals 10 P. As the value of viscosity, ,

decreases, less and less stress, σ, is required to maintain the shear rate, γ.

(1) = σ

The unit of viscosity is pascal-second (Pas), or 10 P.

1 Pas = 10 P = 10 dynes/cm2 = 1000 cP.

If we plot the shear rate of a fluid (γ) as a result of a shear stress applied to it (σ) we can observe that the slope is the viscosity. The viscosity of some fluids do not change with respect to the shear rate applied. These fluids are called Newtonian (e.g. water is a Newtonian fluid).

Some fluids become more ‘runny’ (less viscous) when greater stresses are applied. These fluids are known as pseudoplastic, and the behavior is called “shear thinning”. In the graph above, you can see the slope of a pseudoplastic fluid decreases at higher shear stresses.

Dilatent fluids behave in the opposite manner. At higher shear stresses, dilatent fluids become more viscous (“shear thickening”). An example of a dilatent fluid is a thick suspension of corn starch in water.

Occasionally, fluids will be so viscous that at rest, they require a yield stress in order to start moving. If you were to touch these fluids lightly, they would feel solid, but if you were to push

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the fluid strongly, it would deform. These fluids are known as Bingham fluids. An example of a Bingham fluid is ketchup – it needs to be squeezed hard to start moving, in order to get it flowing out of the bottle. Once a Bingham fluid starts moving, it could exhibit Newtonian, pseudoplastic, or dilatent behaviour.

The viscosity of a liquid may be determined by measuring the time required for the liquid to pass between two marks as it flows by gravity through a capillary tube – Ostwald viscometer.

1. Pour the test fluid into arm A. Tilt the tube slightly and allow the sample to run smoothly down the side to avoid entrapping air. Add enough fluid until Bulb C is full (stop at D).

2. Clamp the viscometer securely on a retort stand.

3. Attach a pipette bulb to arm B. Suck up the fluid slowly up arm B, and stop at G (when the upper bulb is filled).

4. Cover the hole in arm A with your thumb, and remove the pipette bulb from arm B. Get a stopwatch ready to start timing.

5. Release your thumb from arm A. The liquid will flow down arm B due to gravity. Record the time it takes for the test fluid to travel from mark F to mark E.

The time of flow of the test liquid is compared with the time required for a liquid of known viscosity (usually water) to pass between the two marks. If η1 and η2 are the viscosity of the unknown and the standard liquids, and t1 and t2 are the respective flow times in seconds, the absolute viscosity of the unknown liquid, η1, can be determined. ρ is the density of the liquid. The value η1/η2 is also called relative viscosity, ηrel.

Relative viscosity:

(2) waterwater

testtest

water

test

1rel

tρ

ηη

Please note that Equation (2) in no way implies that test testttest. The units of density*time will not provide a unit of viscosity (try it and see for yourself).

The dynamic viscosity of water at 25 °C is 0.8903 cP, at 20 °C is 1.0020 cP. The densities of the solutions in this lab will be measured using an Anton Paar DMA-35 handheld density meter. Your TA or instructor will assist you with these measurements.

Re-arranging equation (2), we can solve for ηtest, the dynamic viscosity of the test fluid:

(3)

waterwater

testtest

watertesttρ

tρηη

One problem with the use of a capillary viscometer is that only the viscosity at a single shear rate can be measured. Since the viscosity of a pseudoplastic liquid depends on the shear rate, the apparent viscosity obtained can be misleading as the effect of thixotropy is neglected. Alternatively, the viscosity of a liquid can be measured by one of the rotational viscometers such

A B

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76 Lab 7: Examination of Viscosity and Suspending Agents

as the Brookfield viscometer. A solid rotating body immersed in a liquid is subjected to a retarding force due to the viscous drag, which is proportional to the viscosity of the liquid. The advantages of rotational viscometers are that the shear rate can be varied over a wide range of values, and that continuous measurements at a given shear rate or shear stress can be made for extended periods of time, affording measurements of time-dependent or shear-dependent viscosity. The principle of a rotational viscometer is outlined below:

The viscosity is calculated by means of the Margules equation

(4) = (1/Rb

2 – 1/Rc2)

= K*S

where Rb and Rc are radii as shown in the diagram; h is the height of the immersed solid; T is the torque;

is the angular velocity in radian/s ( X 60 / 2 = rpm)

A rotational viscometer is typically calibrated by providing the equipment constant, K. Upon immersion into the liquid, a torque measurement, S, is displayed. The product of K and S would yield the viscosity of the liquid.

There are other expressions of viscosity other than the relative viscosity. For example: specific viscosity, reduced viscosity, and intrinsic viscosity.

Specific viscosity:

(5) sp = rel –1

Most viscosity enhancing agents are polymeric in nature. With the use of dilute solution and that the effect of intermolecular entanglements is neglected, the Huggins equation describes the relationship between the reduced viscosity and polymer concentration, c.

Reduced viscosity:

(6) red = sp/c = [η] + k [η]2c

A plot of reduced viscosity versus c would give a straight line with the y-intercept as the intrinsic viscosity, [η]. In other words, the intrinsic viscosity is the reduced viscosity of a polymer solution

h Rb

liquid sample

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at infinite dilution. The intrinsic viscosity is a characteristic of a particular temperature-polymer-solvent system.

Intrinsic viscosity:

(7) c

cred

00limlim

Moreover, the intrinsic viscosity of a polymer solution is proportional to its viscosity-averaged molecular weight, Mv, according the Mark-Houwink equation:

(8) [ ] = K×Mv

where K and a are constants.

Consequently, the molecular weight of the polymer can be determined by viscometry.

Suspending Agents

Suspending agents impart viscosity to the suspension and hinder the sedimentation of dispersed solids. There are many suspending agents. It is essential to select one or a combination of suspending agents so that the formulation is compatible with other excipients.

1. Cellulose derivatives

A summary table of methocellulose products is provided in the appendix of this manual.

i. Microcrystalline cellulose with carboxymethylcellulose (Avicel RC591), at a concentration of 0.5 - 2% w/v produces pseudoplastic/thixotropic flow behaviour (i.e., shear-thinning behaviour) with a viscosity of <2000 cP. It is stable in a pH range of 3 – 10, but is incompatible with cationic surfactants, concentrated salt solutions and sucrose. This incompatibility is due to a lower degree of hydration of the polymer which leads to phase separation. Avicel RC591 produces a liquid dispersion with an average particle size of 0.15 microns. It can tolerate glycols and alcohols and is compatible with other hydrocolloids.

ii. Sodium carboxymethylcellulose (NaCMC) is an anionic macromolecule which produces a protective coating around the particles thereby preventing the close approach of particles (steric stabilization and charge stabilization). It also acts as a thickening agent. NaCMC is usually used at a concentration of 0.2% w/v.

iii. Hydroxypropylmethylcellulose 2910 (HPMC 2910) USP (Methocel E4M premium), has a viscosity of 4300 cP, is pseudoplastic, and has an average particle size of 79 microns. The pH of HPMCs in water is in the range of 6 - 8 and is stable over a pH range of 3 - 11. As a suspending agent HPMC 2910 is used at a concentration range of 0.3 - 2% w/v.

iv. Hydroxypropylmethylcellulose 2906 (HPMC 2906) USP (Methocel F4M premium), has a viscosity of 5100 cP and an average particle size of 65 microns. As a suspending agent HPMC 2906 is used at a concentration range of 0.3 - 2% w/v.

v. Hydroxypropylmethylcellulose 2208 (HPMC 2208) USP (Methocel K4M premium), has a viscosity of 4000 cP and an average particle size of 65 microns.

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As a suspending agent HPMC 2208 is used at a concentration range of 0.3 - 2% w/v.

vi. Hydroxypropylmethylcellulose 2208 (HPMC 2208) USP (Methocel K100M premium), has a viscosity of 16-19000 cP and an average particle size of 65 microns. As a suspending agent HPMC 2208 is used at a concentration range of 0.3 - 2% w/v.

vii. Methylcellulose USP (Methocel A4M premium), has a viscosity of 4800 cP, is pseudoplastic with an average particle size of 85 microns. It is stable over pH 2 - 12 and is used at a concentration range of 1 - 5% w/v.

2. Clays

i. Bentonite (colloidal aluminum silicate) produces a dispersion having a viscosity of less than 800 cP and with plastic/thixotropic flow behaviour. It is stable over a pH range of 3 - 10 and is used at a concentration range of 1 - 6% w/v. In solution it is incompatible with calcium ions and polyvalent cations.

ii. Attapulgite (colloidal magnesium aluminum silicate) is used at a concentration range of 0.5 - 5% w/w. It produces a dispersion having a viscosity of less than 2200 cP with plastic/thixotropic flow behaviour. In solution it is stable over a pH range of 3 - 10.

Preparation of Suspending Agents

The following is a description of how the suspending agents above are prepared. You are not required to perform this in the lab, it is provided for your information only.

Method of preparation of Methocel colloidal solutions

Thoroughly mix the Methocel powder with about one third of the required amount of water as hot water (80 - 90°C). This is to wet the particles and not to dissolve them. Allow the mixture to cool to room temperature and add the remainder of water as cold water. Place the solution in the refrigerator, continuously stirring until fully hydrated (usually overnight).

More information is available from: http://ww.colorcon.com/pharma/excipients/methocel

Method of preparation of colloidal silicate w/w magma

Place water (about 500 g) in a blender and add bentonite or attapulgite (50 g) while the machine is running. Blend the mixture for 5 to 10 minutes; add purified water to make 1000 g and mix. This procedure must be done by weighing and not by making up to volume.

Stoke’s Law

Stoke’s Law describes a relationship between the settling rate of particles in a liquid to particle size, their respective densities, and the viscosity of the liquid. Inherently, larger/heavier particles will fall out of suspension faster. Settling rate will also depend on the relative density of the particles and the fluid they are suspended in. For instance, if the particles are less dense than the fluid, they will rise instead of fall. Stoke’s law is expressed using the following mathematical relationship:

http://ww.colorcon.com/pharma/excipients/methocel

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9η

)gρ(ρ2rV Ls

NOTE: Watch your units!

V is the particles' settling velocity (m/s)

r is the radius of the particle

g is the gravitational constant (9.81 m/s2)

ρs is the density of the particles (kg/m3)

ρL is the density of the liquid (kg/m3)

is the dynamic viscosity (Pa*s, or kg/(m*s))

Although most drugs in suspensions are not perfect spheres and some suspensions are not dilute enough to follow Stokes’ law, the equation is still useful qualitatively. Three methods can be used to control sedimentation: 1. particle size reduction; 2. density matching; 3. viscosity building.

Experiment Protocol

Chemicals Supplies Special Equipment Sodium Chloride (NaCl MW 58.44 g/mol) Methylcellulose stock solution (1.0% Methylcellulose USP 1500 cps in de-ionized water) Hydrochloric Acid (0.1 N)

Glass beads

Size 300 Ostwald Viscometers (100 too narrow for this lab) Brookfield Viscometer Anton Paar Handheld Density Meter Test tubes, test tube rack Retort Stand

The following solutions are prepared or provided by the TA:

10 L of 1.0% Methylcellulose USP in de-ionized water).

Hydrochloric Acid (0.1 N)

Part A. Characteristics of a Polymeric Solution: Intrinsic Viscosity

Prepare 100 mL of 0.1%, 0.2%, 0.3%, and 0.4% methylcellulose solution and measure the viscosity of each solution, using the Ostwald viscometer.

The stock solution available to you will be 1% methylcellulose in water. Due to its high viscosity, you will not be able to use glass bulb pipettes to create your dilutions. Use a graduated cylinder to measure out the required stock for each solution, then empty the

NOTE: To prepare this solution, heat ~2L of water to almost boiling, while stirring with a large magnetic stir bar in a 4 L beaker. Slowly add the methocellulose USP 1500 cps powder with about one third of the required amount of water as hot water (80 - 90°C). This is to wet the particles and not to dissolve them. After you have added all of the methylcellulose to the hot water, allow the mixture to cool to room temperature. Transfer the solution to a 10 L plastic tote with spigot. A the remainder of water as cold water to the tote, bringing up the volume to 10L.

Place the solution in the large walk-in refrigerator on the 8th floor, set on a stir plate. The mixture needs to stir continuously while cooling, until it is fully hydrated – this takes overnight, so make the stock solution a few days before the lab to allow enough time to make sure it works. Preparing this solution correctly is crucial for the success of this lab.

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80 Lab 7: Examination of Viscosity and Suspending Agents

stock into the required volumetric flask. Use sequential wash steps to rinse the graduated cylinder out with the diluent (de-ionized water) and add this to the volumetric flask, in order to transfer all of the methylcellulose stock.

Using the “Reduced Viscosity” worksheet on viscosity.xls (available on the laboratory website), plot sp/C vs. C and determine the intrinsic viscosity, [ ], for methylcellulose

solution.

Add 0.4 g of sodium chloride to 20 mL of 0.4% methylcellulose solution, and measure the viscosity again, using the Ostwald viscometer. Discuss what happened to the viscosity of the polymer solution after adding the salt.

Add 3 mL of 0.1 N HCl to 20 mL of 0.4% methylcellulose solution, and measure the viscosity again, using the Ostwald viscometer. Discuss what happened to the viscosity of the polymer solution after adding the acid.

Plot ηsp /c vs. c and determine the intrinsic viscosity, [ ], for methylcellulose solution.

Part B. Characteristics of a Polymeric Solution: Fluid Type

Using the Brookfield viscometer, measure the viscosity of 1% methylcellulose solution at each speed of the viscometer (ranging from approx. 3-60 rpm, depending on the model used).

Using the “Brookfield” worksheet on viscosity.xls, plot a graph of viscosity versus spindle speed (rpm). What type of fluid behaviour is the methycellulose solution exhibiting in the range of spindle speeds you tested: Newtonian, Dilatent, or Pseudoplastic?

Part C. Measurement of the Sedimentation Rate of an Ion Exchange Resin (Glass Beads)

Accurately weigh three aliquots of 100 mg of the glass beads.

Brookfield Viscometer Notes:

Make sure your suspensions are properly mixed before measuring.

A reliable measurement on the Brookfied will require the %Torque to be between 10-100%. If the torque reading is below this value, the viscosity will not be accurate.

Make sure there are no bubbles under the spindle surface that would contribute to an erratic reading.

Allow each reading to stabilize before taking the measurement.

Use the largest spindle available for the Brookfield viscometer. If the solution is too viscous for the spindle (EEE message), then you will need to switch to a smaller piston.

Ostwald Viscometer Notes:

When using the Ostwald viscometer, measure the time for de-ionized water first, and then measure the other fluids in increasing order of concentration.

The Ostwald Viscometers have a very fine capillary bore. Do not force mixtures through them. The viscometers are expensive and difficult to replace.

The Ostwald Viscometers MUST be thoroughly cleaned with water after use. Failure to perform this cleaning will result in a loss of 10 marks for this laboratory.

http://phm.utoronto.ca/~ddubins/DL/viscosity.xls

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81 Lab 7: Examination of Viscosity and Suspending Agents

Lab

Measure 10 mL of 0.4% methylcellulose solution and place in a screw capped test tube.

Add one of the 100 mg samples of glass beads.

Place the cap on the top of the cylinder and mix thoroughly until hom*ogeneous.

Record the time taken for the resin to settle to the bottom of the cylinder.

Repeat the experiment with 0.04% methylcellulose, and then with de-ionized water as a control sample.

The “end-time” is reached when the methyl cellulose samples become as clear as the water control sample.

Could this result be predicted by using Stoke’s law?

Remember to rinse out the Ostwald viscometer before storing, or the tip will become clogged with solidified methylcellulose.

Questions

1. What is the difference between “solubilized” and “dispersed”?

2. Why is Avicel RC-591 added to cold water, not hot water?

3. What is pseudoplastic flow?

4. What is thixotropy? Why is it a desirable property for pharmaceutical liquids to have?

5. Explain why the Reduced Viscosity, red = sp/C, is used in the graph of sp/C vs C.

Why are the viscosities of pseudoplastic materials difficult to measure?

6. What is the effect of the particle size of the sphere on the rate of sedimentation? Would a sphere with a density of 1.5 g/cm3 and a particle diam

Hint: See Stoke’s Law, Equation (9) in the Background section, assume L = 1 g/cm3.

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82 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Preparing for the Lab

Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:

Measuring pH (http://phm.utoronto.ca/~ddubins/DL/pH.wmv)

Group Allocation You will be working in groups of 3 students

What You’ll Be Doing

Part A: Pre-heating an assigned buffer to 3 different temperatures Preparing a standard curve for salicylic acid, and examining the effect of temperature on ASA hydrolysis Part B: Repeating the Part A experiment ASA hydrolysis using a lower concentration at a single temperature/pH value Part C: Repeating the Part B experiment ASA hydrolysis using a saturated solution of ASA at the same temperature/pH value in Part B

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/calibration.xls

What You’re Handing In Individual formal lab report, due at the beginning of the next lab (see Guidelines for Writing Individual Laboratory Reports for details)

Introduction

In product development, it is critical to assess the rate and course of drug decomposition. This kinetic information enables the formulator to prepare more stable products.

An understanding of the kinetics of drug decomposition enables the pharmaceutical scientist:

to determine proper storage conditions for the drug (e.g. temperature, humidity, protection from light)

to make predictions regarding the stability of drugs (e.g. half-life, shelf life)

to select drug products from different manufacturers, and

to estimate stability when a drug is mixed with different solvents or solutions of other drugs.

The kinetics of aspirin hydrolysis have been investigated extensively. The experiment may be carried out at a single pH at various temperatures. Also the hydrolysis may be carried out at different pH values to examine the influence of pH on stability.

References

1. Mitchell, A.G. and Broadhead, J.F., Hydrolysis of solubilized aspirin, J. Pharm. Sci.,56, 1261-1266 (1967)

2. Garrett, E.R., J. Amer. Chem. Soc., 79, 3401 (1957).

3. Blanch, J. and Finch A., Amer. J. Pharm. Ed., 35, 191 (1971).

4. Zimmerman, J.J. and Kirschner, A.S., Amer. J. Pharm. Ed., 36, 609 (1972).

5. Alibrandi, G. et al., Variable pH kinetics: an easy determination, J. Pharm. Sci., 90, 270-274 (2001)

http://phm.utoronto.ca/~ddubins/DL/pH.wmv

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

PHC 340Y Lab Manual 2017-2018

83 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Lab

Background

Introduction to Chemical Kinetics

Pharmaceutical solutions undergo degradative chemical reactions at definite rates. The rates are dependent on conditions, such as concentration of reactants, temperature, moisture, pH, and the presence or absence of catalysts and light. You may want to review the principles of chemical kinetics in a typical first-year chemistry text. A summary is provided here.

Degradation is assumed to be an irreversible, monomolecular reaction:

Zero-order reaction: The reaction rate is independent of the concentration of the reacting substance.

In this type of reaction, the limiting factor is something other than concentration, such as solubility, or absorption of light in certain photochemical reactions.

(1) Reaction Rate =

Integrating equation (1):

(2)

(t)C

(0)C

A dtk(t)dCA

Where:

t is time,

CA(0) is the concentration of drug at t=0,

CA(t) is the concentration of drug at time t,

k is the zero-order reaction rate constant

(3) t

(t)C

(0)CA consttkconst(t)CA

(4) 0)k(t(0)C(t)C AA

(5) kt(0)C(t)C AA

From equation (5), we can see that the drug starts out at a concentration of CA(0), and degrades linearly with time, with a slope equal to k. Here is a zero-order degradation plot of a drug with k=1.5 mg/(mL×h), and CA(0) = 100 mg/mL (right panel).

For a zero-order reaction, a plot of the concentration of A versus time is a straight line, with a slope equal to k.

A B drug degradation

products

kdt

(t)dCA

y = -1.5x + 100

100

120

0 10 20 30 40 50 60

(mg/

mL)

Time (h)

Zero-Order Degradation

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84 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

First-order reaction: The reaction rate is dependent on the first power of concentration of a single reactant.

(6) Reaction Rate = (t)kCdt

(t)dCA

Integrating equation (6):

(7)

(t)C

(0)C A

A dtk(t)C

(t)dCA

(8) t

(t)C

(0)CA consttkconst)(t)ln(CA

(9) 0)k(t(0))ln(C(t))ln(C AA

(10)

kt(0)C

(t)Cln

Equation (10) can be re-arranged to solve for CA(t), the concentration of “A” as a function of time:

(11) kt(0)C

(t)Cln

ee A

(12)

A e(0)C

(t)C

(13) kt

AA (0)eC(t)C

Plotting concentration vs. time for a first order reaction gives an exponential decay curve, which is a straight line on a semi-log plot. Here is a first-order degradation plot of a drug with k=0.08 h-1, and CA(0) = 100 mg/mL:

Pseudo-First order reaction: The reaction rate is dependent on the concentration of two reactants. However, if one is retained at a constant concentration as compared to the other reactant (i.e. if it is in excess), the reaction rate will proceed as if it were first order.

y = 100e-0.08x

100

120

0 10 20 30 40 50 60

(mg

/mL)

Time (h)

First-Order Degradation

y = 100e-0.08x

100

0 10 20 30 40 50 60

(mg/

mL)

Time (h)

First-Order Degradation(semi-log plot)

y = 100e-0.08x

100

120

0 10 20 30 40 50 60

(mg

/mL)

Time (h)

First-Order Degradation

y = 100e-0.08x

100

0 10 20 30 40 50 60

(mg/

mL)

Time (h)

First-Order Degradation(semi-log plot)

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85 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Lab

Half-Life and Shelf-Life

Once the order of the reaction for degradation is known, the time necessary for a fraction of the material to degrade can readily be calculated. Usually, first-order degradation is assumed. The half-life, t1/2, of a drug is defined as the time required for 50% of the drug to degrade. For first-order degradation, half-life can be calculated as follows:

At the half-life (t1/2), exactly half the drug has degraded. Therefore, CA(t1/2) = 0.5*CA(0). Substituting this into equation (10):

(14)

1/2

A tk(0)C

(0)C0.5ln

(15)

1/2tk0.5ln

Solving for t1/2:

(16)

ln(2)

0.5ln-t1/2

In the pharmaceutical field, the time required for 10% of the drug to degrade is often called the shelf-life of the product. Substituting CA(t0.9)=0.9*CA(0) into equation (10) yields:

(17)

0.9

A tk(0)C

(0)C0.9ln

(18)

0.9tk0.9ln

(19)

0.9ln-t0.9

Knowledge of the rate constant, k, permits an estimation of the amount of drug that will degrade within a given amount of time in a given condition.

Temperature dependency of Kinetics: The Arrhenius Equation

In order for the rate constants of degradation to be of use in the formulation of pharmaceutical products, it is necessary to evaluate the temperature dependency of the reaction. This permits the prediction of the stability of the product at ordinary shelf temperature from data obtained under exaggerated conditions of testing. It is generally believed that the rate of reaction doubles for each 10 oC rise in temperature. A better way to estimate the influence of temperature on the rate constant is by setting up a planned schedule of accelerated tests for each formulation in order to ascertain the temperature dependency of the chemical changes in the product.

A generally satisfactory method for expressing the influence of temperature on reaction rate is the quantitative relation proposed by the Swedish chemist, Svante Auguste Arrhenius:

(20)

Aek

where: A: frequency factor Ea: activation energy

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86 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

R: gas constant, 8.314 J/(mol*K), or 1.987 cal/(mol*K) T: temperature (in Kelvin)

The frequency factor is a measure of the frequency of collisions which can be expected between the reacting molecules for a given reaction.

The logarithmic form of the Arrhenius equation is:

(21) RT

E-A)ln(ln(k) a

A plot of ln k vs. 1/T will yield the activation energy, which represents the energy the reacting molecules must acquire in order to undergo reaction. Although the accelerated stability experiment is carried out at elevated temperature, the prediction of stability at shelf temperature is possible by extrapolating the curve to the lower temperatures and reading off the k value for the lower temperature. Once the k value is obtained, it can be used to estimate t0.9, the shelf-life of the product at room temperature, by substituting equation (20) into equation (19):

(22)

K) R(294.15

E-

90%a

ln(0.9)-t

Kinetics of ASA Hydrolysis

The reaction of interest in this lab is the degradation of acetylsalicylic acid to salicylic acid in water:

(23)

Acetylsalicylic Acid(ASA)

+ OH2

Salicylic Acid (SA)

O CH3

Acetic Acid

It is known that ASA degradation is influenced by the presence of solvent (H2O) and specific (H3O

+, OH-) acid-base catalysts. Furthermore, the weak acid aspirin can exist in two forms in solution non-ionic (ASA) and anionic (ASA-) both of which are subject to hydrolysis.

The rate of hydrolysis which accounts for these factors is:

(24)

]][ASA[OHk]O][ASA[Hk]][ASAO[Hk

][ASA][OHkO][ASA][Hk][ASA]O[Hkdt

])ASA[d([ASA]

32231

Simplifying Equation (2):

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87 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Lab

(25)

][OHkO][Hk]O[Hk][ASA

][OHkO][Hk]O[Hk][dt

])ASA[d([ASA]

62534

32231

ASA

This complicated rate expression can be studied by investigating the decomposition of ASA in a buffer solution which maintains both a constant pH and also a constant ratio of [ASA] to [ASA-] Furthermore, since H2O is the major component present, its concentration does not change appreciably during the decomposition. Making these assumptions in the above expression and letting [ASA] + [ASA-] = [ASAT] (the total concentration of aspirin in solution), the kinetics can be approximated by a first order rate expression at constant pH:

(26)

]k[ASAdt

]d[ASAT

The value of k depends upon the pH, the individual k values listed above (k1 k2 etc.) and the temperature. The purpose of this experiment will be to determine the value of k at different values of pH and temperature.

Calculating the Amount of ASA as a Function of Time

The expression for the amount of acetylsalicylic acid in this experiment may be derived from a mass balance. The ferric nitrate solution reacts with salicylic acid, and not acetylsalicylic acid. Every mole of salicylic acid produced accounts for a mole of ASA which has degraded. We start out by weighing an initial amount of drug (mdrug), which contains both ASA and salicylic acid. Our first sampling time gives us an OD, which we convert to a concentration of salicylic acid ([SA]t=0) in mM. Therefore, the initial mass of salicylic added to the 50 mL volumetric flask (in mg) is:

(27) mSA = [SA]t=0 (mmol/L) * 0.050 L * 138.12 mg/mmol (MW of SA)

We added the initial mass of drug mdrug = 50 mg. By subtraction, the initial mass of ASA is:

(28) SAdrugASA mmm

Finally, the initial concentration of ASA ([ASAT]t=0) in mmol/L is:

(29)

L 0.050mg/mmol 180.16

m][ASA ASA

0tT

The amount of salicylic acid appearing in solution (in mmoles) is equal to the amount of ASA degraded. So an expression of the amount of [ASAT] present as a function of time (in mmol/L) is:

(30) [ASAT] = [ASAT]t=0 – ([SA] – [SA]t=0)

You will use Equation (30) to solve for the concentration of ASA in mmol/L as a function of time.

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88 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Experiment Protocol

Chemicals Supplies Special Equipment Acetylsalicylic acid (MW 180.16 g/mol) Sodium Salicylate (MW 160.11 g/mol) Hydrochloric acid (0.1 N) Ethanol 95% Sodium Hydroxide Pellets (MW 40.00 g/mol) Sodium Phosphate Dibasic (MW 268.07 g/mol) Ferric Nitrate (2% Fe(NO3)3 in de-ionized water) Boric Acid (MW 61.83 g/mol) Potassium Phosphate Monobasic (MW 136.09 g/mol) Citric Acid (MW 192.12 g/mol) Potassium Chloride (MW 74.55 g/mol) Prepared buffers (pH 2, 4, 6, 8)

0.45 µm syringe filters 3 cc plastic syringe Plastic test tubes Aluminum Foil

Helios UV/VIS spectrophotometer and cuvettes Three constant temperature baths (40 °C, 50 °C, 60 °C) Stopwatch

The following solutions are prepared or provided by the TA:

Ferric Nitrate (2% Fe(NO3)3 in de-ionized water)

Clark Lubs buffer (KCl - HCI) buffer, pH 2: KCl (MW 74.56 g/mol) HCl (12 N)

Mclivaine's Citric Acid - Phosphate buffer, pH 4 (C6H8O7. 1 H2O, Na2HPO4. 2H2O): Citric Acid (210.14 g/mol) Sodium Phosphate Dibasic (MW 268.07 g/mol)

Potassium Phosphate buffer, pH 6 Potasium Phosphate Monobasic (Potassium dihydrate orthophosphate) (MW 136.09 g/mol) NaOH pellets (MW 40.00 g/mol)

Clark - Lubs Borate buffer, pH 8, 10 (H3BO3, KCl, NaOH): Boric Acid (61.83 g/mol) KCl (MW 74.56 g/mol) NaOH pellets (MW 40.00 g/mol)

Buffer Preparation – To Be Performed by TAs

NOTE: 500 mL of each of the following buffers will be prepared for you ahead of time by your TAs: (*Buffers need to be prepared the day before the lab. Adjust to final pH with pH meter.)

Buffer Type Volume of Stock Solution A

Volume of Stock Solution B

q.s. to Final Volume

Clark Lubs Buffer KCl/ HCl 125 mL of 0.2 N KCl 26.5 mL of 0.2 N HCl 500 mL 2

McIlvaine’s Buffer Citric Acid/ Phosphate

307.5 mL of 0.1 M citric acid

192.5 mL of 0.2 M sodium phosphate dibasic

Do not q.s. 4

Potassium Phosphate Buffer

428.6 mL of 0.2 M potassium phosphate monobasic

71.4 mL of 0.2 M NaOH

Do not q.s. 6

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89 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Lab

Clark Lubs / Borate Buffer

250 mL of 0.1 M boric acid in 0.1 M KCl

13 mL of 0. 1 N NaOH

500 mL 8

Clark Lubs / Borate Buffer

250 mL of 0.1 M boric acid in 0.1 M KCl

220 mL of 0.1 N NaOH

500 mL 10

Part A. Acetylsalicylic Acid Hydrolysis – Effect of Temperature

Pre-Heating the Assigned Buffers

You will study the hydrolysis of ASA at three different temperatures and at one pH value. The instructor will assign different systems to be studied.

Effect of pH: pH 2, 4, 6, 8, 10

Effect of Temperature: 40, 50, 60 °C

Effect of Concentration: For one of your pH/Temperature reactions, you will be varying the concentration of ASA to observe the effect on reaction rate (see below).

Pre-heat the buffers you are assigned to use at the desired temperature, by placing them in the appropriate temperature bath, in a large volume Erlenmeyer flask. Cover the flask with aluminum foil. Do not allow the flask to float or tip over in the temperature bath – reduce the volume of the bath if necessary.

The hydrolysis can be determined by measuring the concentration of salicylic acid, since each mole of aspirin yields one mole of salicylic acid. The color reagent is a 2% ferric nitrate solution. The absorbance of the solution is measured at 525 nm using a UV/VIS spectrophotometer.

Preparing a Standard Curve for Salicylic Acid

Create a standard calibration curve for sodium salicylate at 525 nm in deionized water, as previously described in Lab 3, using the following concentrations, in triplicate:

0.5, 1.0, 1.5, 2.0, and 2.5 mM sodium salicylate.

A test tube is prepared for each standard solution. Each test tube contains:

1 mL of the standard solution

5 mL ferric nitrate solution

Create a blank solution, with 1 mL water and 5 mL of ferric nitrate solution.

Zero the spectrophotometer using the blank solution, and measure the OD of each standard solution.

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90 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Prepare the following table to collect and organize your data, using the following format:

Data for ASA Hydrolysis in pH____Buffer at ____°C Time (hours) Absorbance (OD) [SA]T

Salicylate Concentration (mM) [from the standard curve]

[ASA]T ASA Concentration (mM) [corrected for salicylate present at t=0]

ln[ASA]

Setting up the Hydrolysis Reaction

Weigh 50 mg of finely powdered ASA accurately, and transfer to a 50 mL volumetric flask.

Add 5 mL of 95% ethanol.

Dilute to the mark with the selected pre-heated buffer solution. Agitate gently.

Remove a 1.0 mL sample and transfer to a test tube immediately. Add 5 mL of ferric nitrate solution. This will be your first sample, at at time=0.

Place the flask in the selected constant temperature bath.

Spectroscopy Time Trials

Withdraw 1 mL samples from the volumetric into separate test tubes at 10, 20, 30, 40, 50, and 60 minutes from time=0. If possible, withdraw your sample while the reaction is still in the constant temperature bath. If you need to remove it from the bath, put it back as soon as possible.

For each 1 mL sample withdrawn, add 5 mL of ferric nitrate solution to the test tube. Mix well. Zero the spectrophotometer with the Blank solution, and determine the absorbance at 525 nm quickly (since the ASA will continue to hydrolyze).

Calculating Salicylate and ASA Concentrations

Calculate the concentration of salicylic acid using the best fit line of the standard curve.

The concentration of ASA remaining in the solution is then calculated using the method described in Equations (31) to (34) in the Background section.

Part B. Acetylsalicylic Acid Hydrolysis – Effect of Concentration

For one of your temperature / pH values, repeat the experiment using 25 mg ASA. Would you expect the rate of hydrolysis to increase or decrease?

Part C. Acetylsalicylic Acid Hydrolysis – Effect of a Suspension

For the same temperature you selected in Part B (Effect of Concentration), the following steps are performed:

Pre-heat 100 mL of 0.1 N hydrochloric acid to your selected temperature.

3.000 g of ASA is weighed and transferred to a 100 mL volumetric flask.

Dilute the 100 mL volumetric flask to the mark with the pre-heated 0.1 N hydrochloric

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91 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Lab

acid are added.

Shake the flask vigorously.

Withdraw 1 mL samples from the volumetric into separate test tubes at 10, 20, 30, 40, 50, and 60 minutes from time=0, as performed before.

NOTE: The 1 mL samples are collected using a 3 cc syringe fitted with a 0.45 µm filter.

In this experiment the concentration of drug against time should be plotted in order to determine the rate constant for the zero order process.

Interpretation of Data

Plot the logarithm of concentration of ASA remaining against time. A straight line indicates a pseudo first-order reaction (Fig. 1 - Title - Pseudo first-order hydrolysis of ASA at specified pH values and temperatures). The observed rate constants and the half-lives, and t90 can be evaluated from the line.

Using the two different rate constants (k) obtained at the same pH value the apparent energy of activation is calculated by means of the Arrhenius equation - plot log k against 1/T, T being the absolute temperature. Now calculate k25°C. Predict the amount of ASA remaining in your buffered solution at room temperature after 30 days.

The influence of pH on the hydrolysis of ASA may be determined by using the buffer solutions at different pH values. Use the results provided by your section to plot a pH profile at the temperature given by the instructor (Fig.2. Plot the pseudo first-order rate constant and half-life against the pH values. Title of graph - observed rate constant and half-life for ASA hydrolysis at____°C as a function of pH).

Depending upon the experimental conditions selected, various graphs can be prepared.

a) Log or concentration of ASA remaining against time, different lines representing different temperatures.

b) Log of concentration of ASA remaining against time, different lines representing different pH values.

c) Log of concentration of ASA remaining against time, different lines representing different original concentrations of ASA.

d) What is the value of the rate constants? Does this properly hold for zero, first and second order reactions?

e) Plot of concentration of ASA remaining or of concentration of salicylic acid formed against time for a zero order process. Interpret both the significance of the curved portion and linear portion of the line. Explain the phenomenon observed.

NOTE: For Part A, you will analyze data from the whole class in your final report. You will not be sharing data for Parts B and C.

Plot log [ASAT] vs. time.

Calculate k from the slope.

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92 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

Match log C. vs concentration of ASA used.

Determine the half-life (t1/2) of ASA at pH and temperature assigned.

Determine the shelf-life (t0.9) of ASA at pH and temperature assigned.

Determine Ea.

Determine k25°C using Ea equation.

Calculate amount of ASA remaining in your buffer solution at 25°C after 30 days. Repeat the calculation in another pH condition and compare.

Plot the pH profile.

State your conclusions.

What are the possible sources of error in the experiments?

PHC 340Y Lab Manual 2017-2018

93 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

Lab

Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 5 students

What You’ll Be Doing Part A: Preparing a standard curve for salicylate Part B: Setting up and running the diffusion experiment Demonstration: Hydrating and opening dialysis tubing

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/calibration.xls

What You’re Handing In A combined individual formal lab report for labs 9 and 10

Introduction

The permeability of salicylate through a dialysis membrane will be determined from the change of salicylate concentration as a function of time, in a membrane diffusion experiment.

References

1. C.K. Colton, K.A. Smith, E.W. Merrill, and P.C. Farrell, J. Blomed. Mater. Res. 5, 459. (1971)

2. Experimental Physical Chemistry, Daniels and Alberty, 1970, PP. 498-502.

3. G.L. Flynn, S.H. Yalkowsky, and T.J. Rosemarr, J. Pharm. Sci., 63, 479 (1974).

4. Diffusion: Mass Transfer In Fluid Systems, 2nd

Ed. E.L. Cussler, 1996, Chapter 17.

5. Physicochemical Principles of Pharmacy, 3nd

Ed., A.T. Florence and D. Attwood, 1998, PP. Chapters 3 and 8.

6. Riviere, J.E. Comparative Pharmaco*kinetics: Principles, Techniques, and Applications. Iowa State Press, 1999. P15.

Background

Membrane diffusion plays a key role in regulating cellular transport and gastrointestinal absorption processes in living systems. In addition, it has important applications in diverse areas such as industrial separations, hemodialysis, and controlled-release drug delivery systems. The key process involved is the diffusion of solutes and solvents across a thin membrane along their respective concentration gradient, usually at constant temperature and pressure. Rigorously, the driving force for diffusion should be the chemical potential gradient. However, in most practical applications, it can be approximated by the concentration gradient which is more accessible experimentally. Mechanistically, diffusion is a redistribution of molecules toward concentration equilibrium as a result of the random Brownian motion of the dissolved molecules.

“What the Flux?”

Flux may sound like a strange word, but it simply answers the question, “how fast is the drug diffusing through a surface area (like a membrane)?” Expressed mathematically, the definition of flux is the rate of change of moles per unit time divided by the surface area:

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

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94 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

(1)

sec

mol

seccm

mol

1 J(t)

Where, J = molar flux per unit area A = the surface area through which diffusion occurs (in this case, the total

surface area of the membrane) dn/dt = the rate of diffusion the drug in moles per unit time

Keep in mind that “flux” is the speed of drug movement. It is literally a measure of the speed of diffusion. A larger flux means faster diffusion. The expression which relates the flux of matter “J”, to the concentration gradient across a membrane “dC/dx” is Fick's first law:

(2)

cmcm

mol

sec

seccm

mol

dC(t) D J(t)

where D is the diffusion (or diffusivity) coefficient.

The simplest arrangement for illustrating membrane transport consists of two finite-volume compartments (V1 and V2) containing aqueous solutions of different solute concentrations (C1 and C2) separated by a membrane of thickness “h” (Fig. 1). It is assumed that each compartment is well stirred (no boundary layer), and there is no concentration or hydrostatic pressure gradient within each compartment. The initial solute concentration in Compartment 1 is larger than that in Compartment 2 (C1° > C2°). It is also assumed that since only early diffusion data points will be collected, the volume change due to osmotic water flow from Compartment 2 to Compartment 1 can be neglected.

Figure 1. Schematic Diagram of a Membrane Diffusion System.

x 0 h

Fluid 1 Compartment 1

Fluid 2 Compartment 2

Membrane

2C V1 V2

Flux

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95 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

Lab

Note that the concentrations in the membrane are different than the concentrations in the fluids. This is because our model takes into consideration that the drug might preferentially partition into the membrane, as it is a different polar environment than the surrounding fluids.

Mass Balance

If we add a known concentration of drug in Compartment 1, we may write a mass balance on the drug. At any point in time, the total number of moles in the system is equal to the initial number of moles added:

(3) ntotal = n1 + n2 + nmembrane

Since the membrane has such a tiny volume compared with the overall system volume, we can assume nmembrane = 0 without adversely affecting our model. We can express Equation (3) in terms of concentrations, since n = C×V:

(4) 22111

1 V(t)CV(t)CVC

Thus, at any point in time we can re-arrange Equation (4) to solve for C1:

(5)

221

V(t)CC

(t)VCVC(t)C

Integration of Fick’s Law

In order to solve for the flux as a function of concentration, we need to integrate Equation (2), as it is in the derivative form:

(6)

dC(t)DJ(t)

In our model, we assume Compartments 1 and 2 are well stirred, and the volumes V1 and V2 do not change throughout diffusion.

(7)

dC(t)DJ(t)dx

(8)

0C(t)DxJ(t)

(9)

(t)C-(t)CD0h J(t) m

(10)

(t)C(t)Ch

DJ(t) m

Rapid Partitioning into the Membrane

Recall in Lab 3 that we had to wait for the drug to partition and equilibrate between oil and water phases. The membrane in this experiment is so thin, that we may make a simplifying assumption of rapid equilibration. In other words, the concentrations of drug in the solutions touching both sides of the membrane rapidly partition and equilibrate into the membrane, according to their respective equilibrium partition coefficients. We thus ignore the “lag” effect

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96 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

that was present in Lab 3. If there were two different types of fluids in Compartment 1 and Compartment 2 (e.g. oil and water), we would define two partition coefficients:

(11)

CK ;

Making this assumption of rapid equilibration allows us to substitute the membrane equilibrium concentrations (K1C1 and K2C2) into Equation (10):

(12)

2211 CKCKh

This is more convenient, because it would be difficult measure the concentration of drug within the membrane. Note that J, C1, and C2 in Equation (12) still change with time. If the fluids in Compartments 1 and 2 are the same, as they are in this experiment, then we can set K1 = K2 = K. Then, Equation (12) becomes:

(13)

21 CCh

DKJ

From Equation (13), the math tells us the following about flux with respect to our particular system:

(C1-C2): A larger concentration difference (C1-C2) will make diffusion faster. Conversely, diffusion will stop (flux will be zero) when C1 = C2.

h: A thicker membrane (larger h) will make diffusion slower.

K: poor membrane partioning (K << 1) will make diffusion slower. Diffusion will be promoted if the drug partitions into the membrane (K>=1). Factors affecting K: drug HLB, drug ionization, membrane HLB.

D: A poor diffusivity constant of the drug in the membrane will make diffusion slower. Factors affecting D: membrane pore size/molecular weight cut-off (MWCO), drug size, shape, molecular weight.

Initial: Diffusing: Equilibrium: ● largest concentration difference ● (C1-C2) decreases with time ● C1 = C2 ● fastest flux (J) ● flux decreases with time ● flux = 0

Figure 2. Three stages of diffusion.

Is your head swimming in parameters? Since we will not have enough information to measure the membrane thickness h, diffusivity constant, or membrane partition coefficient, we will be

x 0 h

0C0

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collecting all of the coefficients into one, and calling it the bulk membrane permeability parameter, Pm:

(14) 21m CCPJ

Where h

DKPm

Going back to Equation (1), the flux (the number of moles per second×cm2) leaving Compartment 1 can be expressed as:

(15)

n/Vd

1J 1111

Similarly, the flux entering Compartment 2 can be expressed as:

(16)

n/Vd

1J 2222

Let’s focus on Equation (16) instead of (15), since we are not measuring the concentration of drug in Compartment (1). Since J1 = J2 = J, we can equate Equations (14) and (16), to derive an expression for Compartment 2:

(17)

21m22 CCP

We can use Equation (5) to express C1 in terms of C2 in Equation (17):

(18)

1m22 C

VCCP

Simplifying:

(19)

CAP

Similar to that of a first-order rate equation, Equation (19) can be integrated to yield the following working equation, relating the membrane permeability to the time-dependent solute concentration differences across the membrane:

(20)

dtAPdC

(21)

tAP V

Cln

(22)

0)-(tV

1AP

Cln

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98 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

(23)

1AP

ln21

(24)

1AP1

C1ln

Experimentally, by plotting

C1ln

vs. time, the solute permeability Pm can be

evaluated from the slope of the linear plot according to:

(25)

1APslope

NOTE: Don’t get dismayed by the calculus in the background section. The important concepts are in understanding our use of Fick’s Law: Equation (14). You will prepare a plot using the form in Equation (24), and calculate the slope of this plot to solve for Pm.

Expressions for C2(t) and C1(t):

Now that we’ve integrated and solved the entire system, we can derive expressions for the compartment concentrations as a function of time. We just need to re-arrange Equation (24) to solve for C2.

Taking the exponential of both sides of Equation (24):

(26)

1AP

2 21m

(27)

1AP

2 21m

11V

(28)

1AP

1221

e1VV

VC(t)C

Instead of creating the plot using Equation (23), you could use a fitting program to fit the model C2(t) to your data set. This exercise is not required for the laboratory, and is only mentioned for interest’s sake.

Equation (28) is interesting, because we know that at time=0, there is no drug in Compartment 2, and the term with the exponential is (1-e0) = 1-1 = 0. As time approaches infinity, the term with the exponential becomes (1-e-∞) = 1-0 = 1, and C2 approaches the value it would have been if you simply mixed both volumes together, with no membrane present:

(29)

12VV

VC)(tC

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Even though we never measured the concentration as a function of time in Compartment 1, our mass balance allows us to solve for it, using Equation (5). Provided Compartments 1 and 2 contain the same fluids, C1

= C2 at equilibrium (as t ∞). This is illustrated in Figure 2, in the “Equilibrium” panel.

Experiment Protocol

Chemicals Supplies Special Equipment Sodium Salicylate (MW 160.10 g/mol) Sodium Salicylate Stock Solution (1.0 M)

Plastic UV cuvettes Pipettes (1 and 5 mL) Dialysis tubing (7-8 cm) with closures

UV/VIS spectrophotometer 2 L beaker Volumetric Flasks (50, 100, 500, and 1 L) Glass rods

The following solutions are prepared or provided by the TA:

Sodium Salicylate Stock Solution (1.0 M in de-ionized water)

Part A. Standard Curve: Salicylate

Dilute the 1 M sodium salicylate stock solution by a factor of 100, by adding 1 mL of stock to a 100 mL volumetric flask, and diluting to the mark. This will make a 10 mM solution of sodium salicylate.

Prepare 50 mL of each of the following concentrations from the 10 mM sodium salicylate solution in triplicate:

0.1, 1.0, 1.5, 2.0, and 2.5 mM

Carefully pipette 1 mL of each solution into a test tube followed by the addition of 7 mL of de-ionized water. Use 8 mL of de-ionized water as the blank solution.

NOTE: Plastic UV cuvettes are not rectangular. They are tapered at the bottom because they are smaller volume. The fill line is just above the clear part of the cuvette window. The “V” shaped arrow on the Plastic UV cuvette indicates the side of the cuvette that the UV beam will travel through the entire 1 cm path length (not widthwise, which is only 0.5 cm):

Use the spectrophotometers in room 819. Obtain a UV scan of the blank solution and the highest concentration (2.5 mM), spanning wavelengths 210-300 nm. Compare the two wavelength scans, and select a convenient experimental wavelength. Measure the absorbance of each standard solution at your selected wavelength to construct a calibration curve. The standard curve will be a plot absorbance vs. molar concentration of salicylate at your selected wavelength. Indicate the wavelength on the Y-axis label (e.g. OD210).

Fill line (fill to at least here)

Spectrophotometer beam travels this way

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100 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

Part B. The Diffusion Experiment

Preparation of the Diffusion Cell

The next phase of the experiment involves setting up the membrane diffusion experiment.

Calculate the length of tubing you will need for 5 mL of filling solution. Since the closures are 1 cm long, the length required will be:

5.1Ends Protruding ofLength Closures ofLength (mL/cm)Capacity Volume

(mL) Volume Filling length Tubing

(Multiplying by 1.5 at the end is a safety factor that accounts for slack taken up by the closures.)

The volume capacity of the tubing will be listed on the box (most of the tubing in the pharmaceutics lab is approx 3 – 6 mL per cm tube). For example, to fill dialysis tubing (volume capacity of 3.3 mL/cm) with 5 mL of solution, the length required would be:

cm .385.12cm 12cm 1mL/cm 3.3

mL 5 length Tubing

Prepare the dialysis tubing by pre-soaking it in a beaker with de-ionized water.

Open up the tubing by firmly rubbing it between your thumb and index finger.

Lay the dialysis tubing flat. Lay one end across an open closure with about 1 cm of tubing protruding.

Snap the closure shut.

Hold the tubing with the open end up. Pipette 5 mL of 1 M sodium salicylate solution into the tubing.

Carefully flatten the open end, and squeeze out entrapped air without losing any liquid.

Fold a closure over the flat part of open end, and snap shut.

NOTE: During this last step, make sure not to place the closure too close to the main body of the filled dialysis tubing as possible leakage may develop from the resulting excessive bulging, or too close to the edge where it could slip off.

Weigh the filled dialysis tubing assembly and document the weight.

Accurately measure and transfer 1.5 L of de-ionized water using a 2 L graduated cylinder. Transfer the de-ionized water to a 2 L beaker or Erlenmeyer flask.

Gently place the filled dialysis tubing assembly into the 2 L beaker. Record the time when the assembly is introduced (t=0).

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NOTE: The dialysis tubing assembly tends to float in water. Make sure the entire assembly is always submerged so that the whole membrane surface area can be available for diffusion. This is achieved by stirring with a glass stirring rod or magnetic stir bar. Do not set the magnetic stir bar too high or the dialysis membrane will be pulled down to the bottom of the beaker and will hit the stir bar.

At time=0, withdraw a 1 mL sample from the 2 L beaker. Dilute it with 7 mL de-ionized water. Measure the OD of the sample at your selected experimental wavelength.

Continue sampling and measuring the OD every 10 minutes for at least 1 hour.

At the end of the experiment, remove the dialysis tubing assembly from the beaker, gently blot dry with paper towels, and quickly weigh the assembly again on the analytical balance.

Using a ruler, carefully measure the width of the flattened dialysis tube near the closure and the length of tubing between the two closures. This will be used in your calculation of surface area.

Calculations

With the present experimental setup, Compartment 1 corresponds to the concentrated solution phase inside the dialysis tubing and Compartment 2 corresponds to the aqueous phase in the beaker. The experiment described above measures the change of sodium salicylate concentration in the solution external to the dialysis membrane.

Based on the measured C2, calculate and tabulate

2 11V

C as a function of time (t in

sec.).

Plot ln

2 11V

C versus time, and draw a best-fit line through all data points.

Calculate the slope of this straight line, and use Equation (25) to calculate the permeability Pm of sodium salicylate through the given dialysis membrane at room temperature. The total membrane area A should be calculated by doubling the product of the width and length of the dialysis tubing since both sides of the flattened dialysis tube are involved in the membrane diffusion process.

Questions

1. In this lab, you are adding sodium salicylate to unbuffered water. Using the Henderson-Hasselbalch equation, calculate the percent of sodium salicylate expected to remain in the ionized form at pH 5.5, if the pKa of salicyclic acid is 2.97.

2. From the difference between the initial and final weights and the total amount of salicylate diffused out of the dialysis tube at the end of the experiment, estimate the volume change in the dialysis tubing compartment due to osmotic water flow. How valid is our assumption of no volume change during the course of the diffusion experiment?

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102 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

3. If the partition coefficient Km of sodium salicylate is taken as 1, and the thickness of the dialysis membrane h is 2.5 x 10-3 cm, calculate the diffusion coefficient of sodium salicylate in the membrane phase. Do you expect this to be larger or smaller than the diffusion coefficient of salicylate in water?

4. The present diffusion experiment is run at room temperature. What would you expect to happen to the diffusion coefficient if the experiment is conducted at 37 °C?

5. Create a C2 vs. time plot in a spreadsheet program. On the same graph, plot the equation of the line:

1AP

1221

e1VV

VC(t)C

Use your solved values for Pm and A in the model. Does the model fit the data?

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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

Preparing for the Lab Read the introduction and lab protocol completely Calculate the dilution scheme for the standard curve

Group Allocation You will be working in groups of 5 students

What You’ll Be Doing

Part A: Prepare a calibration curve for your salicylic acid release study Part B: Prepare 4 of the 6 ointment bases Demonstration: levigation, geometric dilution Part C: Formulating 3% salicylic acid preparations of 4 ointment bases; ointment release studies on 3 ointment bases Part D: Preparation of a 40% Urea Ointment and Demonstration of an Ointment Mill

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/calibration.xls

What You’re Handing In

Individual formal lab report, due at the beginning of the next lab (see Guidelines for Writing Individual Laboratory Reports for details) Four ointment bases, each dispensed in a suitable and properly labeled container:

1) 40% Urea in [Hydrocarbon (Base #1) or Absorption (Base #2)] 2) 1 of: W/O emulsion (Base #3) or O/W emulsion (Base #4) –

Non-medicated 3) Water Soluble (Base #5) – Non-medicated 4) 3% Salicylic Acid in Poloxamer Cream (Base #6)

Introduction

Semisolid dosage forms are those preparations intended for spreading on the skin for the purpose of: (1) providing lubrication (emollients); (2) bringing into contact with the skin drugs required for healing skin disorders; (3) acting as protective coverings to prevent contact of the skin surface with chemicals, solutions and organic solvents. The preparations include primarily ointments/creams (salves), cerates, jellies, pastes, plasters and poultices. Ointments are of such a consistency that they may be readily applied to the skin by inundation. They should be of such composition that they soften but not necessarily melt when applied to the body. Creams and jellies generally have a lower viscosity than ointments whereas cerates, pastes, plasters and poultices generally have a higher viscosity.

When semisolid preparations are applied to local areas of the skin, a special beneficial effect is the intended result. The effect produced may be due to the therapeutic action of the medicament (e.g., keratolytic agent, antipyretic agent, antiseptic) which must be released from the base, or due to the base itself. The base usually has a more general action on the skin, providing occlusion to water loss from the skin, emollient and lubricating action, or a drying action.

The purpose of this laboratory is to prepare examples of the pharmaceutical classes of ointments and investigate some of their physicochemical properties.

References

1. United States Pharmacopoeia XXII, United States Pharmacopoeia Convention, Rockville, MD, 1990. 2. The Pharmaceutical Codex, 11th ed., Pharmaceutical Press, London, England, 1983.

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

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104 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

3. Gennaro AR, ed., Remington’s Pharmaceutical Sciences, 20th

ed., Lippincott Publishing Co: U.S.A. 2000.

4. Martin AN, Physical Pharmacy, 4th

ed., Lea & Febiger: U.S.A. (1993), p. 233-235. 5. Parrott EL, Pharmaceutical Technology: Fundamental Pharmaceutics, Burgess Publishing Company:

U.S.A. (1970), p. 364-393. 6. Billups, N. F. and Patel, N. K., Am. J. Pharm. Educ., 34, 190-196, 1970. 7. Nakano, M. and Patel, N. K., J. Pharm. Sci., 59, 985, 1970. 8. Allen, L.V., Int. J. Pharmaceutical Compounding, 6, 58, 2002. 9. http://pharmlabs.unc.edu/labs/ointments/bases.htm

Background

There are several factors which influence the selection of the types of preparations to be used topically. Among these are:

the diagnosis

the effect desired

the condition of the skin area to be treated

its ability to release the medicament to the skin surface

the chemical and physical stability of active ingredients contained therein

In addition, cosmetic appearance and hypo-allergenic properties of the base can be important. The selection of bases for the extemporaneous preparation of a semisolid dosage form is the privilege of the physician, but in this area particularly, the knowledge and wisdom of the pharmacist is frequently called upon to assist the physician in making a suitable selection. It is the responsibility of the pharmacist to prepare a quality product that is pharmaceutically correct. In exercising this responsibility, the pharmacist may be required to make minor changes in composition in order to produce a superior product. This may involve the use of small amounts of inert materials as levigating and/or solubilizing agents. Levigation is the process of reducing the size of solid particles, made into a paste by the addition (with the aid of a spatula and ointment slab) of a small amount of a liquid or ointment base. This liquid or ointment base is known as a levigating agent.

The preparation of semisolid dosage forms often involves two procedures:

Fusion

Mechanical Incorporation

The medicament and the physical properties of the constituents of the base usually determine the extent to which each procedure is used.

Preparation by Fusion

Ointment bases consisting of hard, waxy ingredients, such as beeswax, spermaceti, paraffin, fatty alcohols, or high molecular weight polyethylene glycols, and soft or liquid ingredients such as petrolatum, mineral oil, glycols or hydrocarbons which are gently heated together over a water-bath until a melt is produced. Drugs and adjuvants which are soluble in the melt may be added at this point and mixed in. The melt is removed from the heat and stirred continuously as it cools until congealing has occurred. Heat-sensitive or volatile ingredients should be added just prior to the congealing point which is about 35 – 45 °C.

The method for preparing creams by the fusion process is slightly more complicated. In this case, both the aqueous phase and the oil phase are heated separately, to somewhere between

http://pharmlabs.unc.edu/labs/ointments/bases.htm

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60 – 80 °C. As a general rule, the oil phase should be heated to at least 5°C above the melting point of the highest melting waxy ingredient. The water phase is heated to 5°C above the temperature of the oil phase to prevent premature solidification prior to mixing and emulsification. Water-soluble adjuvant is dissolved in the heated aqueous phase with stirring while nonvolatile oil-soluble ingredients are dissolved in the heated oil phase. Generally, the internal phase is gradually added to the external phase and vigorously mixed. Mechanical dispersion techniques may be used to increase the state of dispersion. Many excellent creams have also been produced by the reverse order of combination, but it varies from one formula to another.

The method for the preparation of the poloxamer/lecithin isopropyl palmitate bases is unique. The poloxamers are white waxy free-flowing granules. They exhibit reverse thermal gelling. In other words they are free flowing when cold at refrigerator temperatures (4 – 8 °C). They are soluble in water and are prepared by placing the flakes in a closed bottle and adding cold water. The mixture is mixed gently and placed in a refrigerator overnight. More water is added up to the designated volume and the gel is ready for use. The soya lecithin is a yellow sticky granule. It is weighed and placed in a sealed bottle and isopropyl palmitate is added as the solvent. This dissolves overnight at room temperature. Usually either potassium sorbate or sorbic acid is added to each of the above as a preservative.

In order to prepare the cream/gel, the drug is dissolved or dispersed in one of either of the above and the second liquid component is added. At room temperature a smooth cream or gel is formed as an oil/water emulsion. Usually, the lipophilic portion of the final cream constitutes approximately 25% of the final weight of the preparation. Two methods are used to decrease the particle dimensions of the lipophilic phase: vigorous trituration in a mortar or extrusion through a small bore syringe opening.

Levigation: Preparing the Drug for Mechanical Incorporation

Mechanical incorporation may be performed with a mortar and pestle, or on a glass slab with a spatula. The drugs being incorporated into the base are frequently insoluble in the base, and it is necessary to reduce them to a fine paste by levigation. This is best accomplished using the slab and spatula technique by using a levigating agent. A legivating agent can be a wetting agent that is compatible with the base or simply a small portion of the base itself (or the melted base). Levigation will be demonstrated to you in the lab. If very small amounts of drug are to be incorporated, a small amount of mineral oil or glycerin can be used as a levigating agent. Using too much of a levigating agent can result in over-diluting or over-softening the finished product.

Geometric Dilution: Mechanically Incorporating the Drug into the Base

After the powder is levigated and worked to form a very smooth nucleus, the drug can then be incorporated (by geometric serial dilution) with the remainder of the base. Briefly, the first step of geometric dilution starts with mixing the drug with a roughly equal volume of base. Mixing is performed using a figure 8 pattern with the spatula, and periodically the mix is scraped to the centre of the glass slab to maintain a small working area. Once the drug is evenly distributed in the base with no evidence of lumps, more base is added, with a roughly equal volume to the current mix of drug+base. In this way, the amount of material doubles for each round of mixing (hence the term “geometric” dilution). Mixing is performed until uniform, and this continues until all of the base is incorporated into the drug+base mixture.

The mortar and pestle are probably not as efficient as the ointment slab and spatula for

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incorporating insoluble powders in an ointment base because of the small surface area levigated at any contact point of the mortar and pestle. However, when a liquid is to be incorporated into a base, the mortar is often preferred since the percentage of ointment exposed to the air is much less by this method, and the possibility of liquid loss by evaporation, due either to friction or thinness of film, is reduced.

Alternatively, the solid can be dissolved in a little solvent, usually water, and incorporated as a solution (i.e., its water number must be high enough). The base, of course, must have the capacity to take up the solution. Volatile aromatic materials, such as essential and perfume oils, camphor and menthol, will volatilize if dissolved in the hot oil phase. These ingredients are usually incorporated in creams as alcoholic solutions added with mixing at the point when the emulsion begins to solidify upon cooling. In the case of ointments, lanolin or some other w/o emulsifier may have to be substituted for a fraction of the base to allow aqueous solutions to be incorporated.

In the case of the poloxamers, the drug is incorporated into one of the two phases during gel/cream formation. It is usually levigated with a solvent such as alcohol or propylene glycol.

A summary chart of the properties of ointment bases is provided on the following page.

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SUMMARY CHART: PROPERTIES OF OINTMENT BASES

Property of Base

Oleaginous Ointment

Bases (Hydrocarbon)

Absorption Ointment Bases

Water/Oil Emulsion Ointment Bases

Oil/Water Emulsion

Ointment Bases

Water-miscible Ointment

Bases

Composition

oleaginous compounds

oleaginous base + w/o surfactant

oleaginous base + water (< 45% w/w) + w/o surfactant (HLB <8)

oleaginous base + water (> 45% w/w) + o/w surfactant (HLB >9)

Polyethylene Glycols (PEGs)

Water Content

anhydrous anhydrous hydrous hydrous anhydrous, hydrous

Affinity for Water

hydrophobic hydrophilic hydrophilic hydrophilic hydrophilic

Ability to Take up Water

very little some, forms a w/o emulsion

will take up more water, eventually the emulsion may break (phase separation) or form an o/w emulsion (phase inversion)

will take up a great deal of water becoming fluid

will only take up a small amount of water before becoming fluid

Spreadability difficult difficult moderate to easy easy moderate to

easy

Washability non-washable non-washable non- or poorly

washable washable washable

Stability

oils poor; hydrocarbons better

unstable, especially alkali soaps and natural colloids

unstable, especially alkali soaps and natural colloids; nonionics better

stable

Drug Incorporation

Potential

solids or oils (oil solubles only)

solids, oils, and aqueous solutions (small amounts)

solid and aqueous solutions (small amounts)

solid and aqueous solutions

Occlusiveness yes yes sometimes no no

Uses

protectants, emollients, vehicles for hydrolyzable drugs

protectants, emollients, vehicles for aqueous solutions, solids, and non-hydrolyzable drugs

emollients, cleansing creams, vehicles for solid, liquid, or non-hydrolyzable drugs

emollients, vehicles for solid, liquid, or non-hydrolyzable drugs

drug vehicles

Examples

White Petrolatum, White Ointment

Hydrophilic Petrolatum, Anhydrous Lanolin, Aquabase™, Aquaphor®, Polysorb®

Cold Cream type, Hydrous Lanolin, Rose Water Ointment, Hydrocream™, Eucerin®, Nivea®

Hydrophilic Ointment, Dermabase™, Velvachol®, Unibase®

PEG Ointment, Polybase™

Source: http://pharmlabs.unc.edu/labs/ointments/bases.htm

http://pharmlabs.unc.edu/labs/ointments/bases.htm

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Experiment Protocol

Chemicals Supplies Special Equipment Ointment Bases (see specific procedures for ingredients)

(Hydrocarbon) Simple Ointment USP, or white petrolatum

(Absorption) Hydrophilic Petrolatum USP, Aquaphor

(w/o emulsion) Cold Cream USP, or Nivea Cream

(o/w emulsion) Hydrophilic Ointment USP, or Unibase Dermabase

(o/w emulsion) Vanishing Cream

Water soluble PEG Ointment USP

Poloxamer Creams Salicylic Acid (MW 138.12) Urea (MW 60.06) Glycerin (MW 92.09) 95% Ethanol

Cellophane membrane (dialysis tubing), 45 mm flat width MWCO 12-14,000 Elastic bands One-ounce ointment jar Plastic Cuvettes Plastic Transfer Pipets

Dermamill 100 Ointment Mill Spectrophotometers Stopwatch Hot plate Evaporating Dish Hard Rubber Spatula (Green or Black) Burette, burette clamp, retort stand

The following solutions are prepared or provided by the TA:

Salicylic Acid (1.00 mg/mL)

Ferric Chloride TS USP (9% w/w FeCl3 in water)

Preparation of the Salicylic Acid Solution

(your TA will prepare this solution) NOTE: Due to the poor solubility of salicylic acid, the stock solution will be made the day before the lab, and set on a stir plate to dissolve overnight.

1. 2.000 g is weighed accurately and added to a 2 L volumetric flask. 2. The 2 L volumetric flask is diluted to the mark with de-ionized water. 3. A large stir bar is added to the flask, and it is set on a stir plate. 4. The solution is set on the stir plate and allowed to stir overnight.

Preparation of the Indicator Ferric Chloride TS USP (9% w/w FeCl3 in water)

(your TA will prepare this solution)

NOTE: If the ferric chloride doesn't dissolve completely, the solution should be filtered before using.

Part A. UV Absorbance Standard Curve of Salicylic Acid

Your TA will prepare a 1.00 mg/mL salicyclic acid stock solution. You will need to calculate the

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volume of stock required for each of the standard curve concentrations.

Preparing the Standards

1. Prepare 50 mL of each of the following salicylic acid concentrations in de-ionized water, in triplicate:

5, 25, 50, 75, and 100 μg/mL

To clarify, this means that you create each solution three times, rather than measure the absorbance of the same solution three times, to get an estimate of the error associated with creating the standard solutions. Measuring the standards in triplicate will allow you to report the average, standard deviation, and %RSD at each standard concentration. An efficient way to accomplish this is to have three different people run the same curve in parallel. The same spectrophotometer must be used.

2. Prepare each solution in a 50 mL volumetric flask, then once prepared, transfer the solution to a 50 mL Erlenmeyer flask or 50 mL beaker.

3. Add 2 drops of indicator solution to each concentration of your standard curve. NOTE: Allow 10-15 minutes for the colour to completely develop before measuring.

4. Show details of your preparation and calculation.

Preparing the Blank

5. Measure 25.0 mL de-ionized water into a beaker using a pipette and add 1 drop of the indicator ferric chloride solution.

6. As the colour of the blank may intensify with time, prepare a new blank as you start the drug release experiment in the second period.

Colourimetric Absorbance Measurement

Your TA will demonstrate how to use the spectrophotometer. A video is also available on the laboratory website.

Set the wavelength of the spectrophotometer to 525 nm (visible light).

Zero the absorbance of the spectrophotometer using the blank solution.

NOTE: Some time should be allowed for colour to develop in each of the samples: typically 10-15 minutes should suffice.

Measure the absorbance of each concentration of the standard curve and record the absorbance.

SPECTROSCOPY NOTES

Fill the cuvette to the etched line (approx ¾ full)

Make sure the cuvette is facing the correct way (the light path should go through the clear windows, not the ridged sides)

To avoid fingerprints, only handle the cuvettes by the ridged sides, not the clear windows.

Fill the cuvette slowly, and gently tap to release bubbles clinging to the sides of the cuvette

Gently wipe the clear windows with a Kimwipe prior to measuring

Make sure the sample door is closed before measuring absorbance

Make sure you use the same UV spectrophotometer for calibration and sample measurements.

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Plot a calibration curve of absorbance vs. known concentration of salicylic acid, using the laboratory computers. You may use the file calibration.xls in the “Downloads” section of the lab website.

Part B. Ointment Base Preparation

You will be melting components for your ointments on a steam bath. Set up the steam bath as follows:

1. Pour ~200 mL de-ionized water in a 400 mL beaker. 2. Set the beaker on a hot plate, and set it to medium/high heat. 3. Place a large evaporating dish on the mouth of the 400 mL

beaker.

When the water boils, the temperature of the evaporating dish will be approximately 60 °C. This will be sufficient for melting solid components in the bases.

4. Stir with a glass rod, not a thermometer 5. Materials should be weighed on weighing boats, waxed

weighing paper, or tared glass beakers. Never put chemicals directly on any scale or balance pan.

NOTE: Dispose of all ointments in the garbage not in the sink.

You will be compounding and testing 4 bases:

1 of: Hydrocarbon base (Base #1) or Absorption Base (Base #2)

1 of: W/O emulsion base (Base #3) or O/W Emulsion Base (Base #4)

Water Soluble (Base #5)

Poloxamer Base (Base #6)

http://phm.utoronto.ca/~ddubins/DL/calibration.xls

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1. Hydrocarbon Base

e.g. White Petrolatum USP

White petrolatum USP is a purified mixture of semi-solid hydrocarbons from petrolatum and is decolourized. It may contain a stabilizer.

Synonyms: white soft paraffin, white petroleum jelly, Vaseline®, white ointment USP

Yellow soft paraffin is often used in eye ointments, as it is not bleached and probably does not contain a stabilizer.

1. Prepare white ointment USP XXII

white wax 5 g white petrolatum 95 g to make: 100 g

2. Melt the white wax in the Ointment Melting Apparatus.

NOTE: boiling wax is extremely flammable. Do not bring the wax to a boil.

3. Add the white petrolatum, warming until liquefied.

4. Remove the evaporating dish from the steam bath.

5. Allow to cool, and stir until the mixture begins to congeal.

NOTES:

The fusion method is usually used in this class of ointments.

The material with the highest melting point is melted first and the other ingredients are incorporated in decreasing order of melting point (or range). Using this method the cooling process is quicker and all the ingredients are not subjected to the highest temperature.

If a lower temperature should be used, then the lowest melting ingredient is heated first and then the materials of highest melting point are added.

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2. Absorption Base

e.g. Hydrophilic Petrolatum USP

1. Prepare hydrophilic petrolatum as follows:

cholesterol 3 g stearyl alcohol 3 g white wax 8 g white petrolatum 86 g to make: 100 g

2. Melt the stearyl alcohol and white wax together in the Ointment Melting Apparatus.

3. NOTE: boiling wax is extremely flammable. Do not bring the wax to a boil.

4. Add the cholesterol, and stir until completely dissolved.

5. Add the white petrolatum, and mix.

6. Allow to cool, and stir until the mixture begins to congeal.

NOTES:

This base contains no water.

Absorption means that the base can absorb water i.e., it has nothing to do with drug absorption.

Because cholesterol is a surfactant with a low HLB, a certain amount of water can be added (see tests) to form a w/o emulsion.

The addition of stearyl alcohol, a surfactant with very low HLB, acts as a co-emulsifier and, along with white wax, gives firmness and heat stability to the product.

The anhydrous base is suitable for water unstable drugs.

A commercial absorption base is Aquaphor.

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3. Emulsion Bases W/O Type

e.g. Petrolatum Rose Water Ointment USP XVI (Cold Cream)

1. Prepare cold cream as follows:

cetyl esters wax (Spermaceti) 12.5 g white wax 12 g mineral oil 56 g sodium tetraborate 0.5 g purified water 19 g to make: 100 g

2. Reduce the cetyl esters wax and the white wax to small pieces.

3. Melt the cetyl esters wax and white wax together in the Ointment Melting Apparatus.

NOTE: boiling wax is extremely flammable. Do not bring the wax to a boil.

4. Once the waxes are melted, add the mineral oil.

5. Continue heating until the temperature of the mixture reaches 70C; maintain at 70°C for 5 minutes.

6. In a separate beaker, dissolve the sodium tetraborate in the purified water, warmed to

70C.

7. Gradually add this warm solution to the melted oil mixture.

8. Remove from heat. While the mixture is cooling, use a hand blender to emulsify the phases. Run the hand blender on the lower speed until the mixture appears milky.

9. Stir rapidly and continuously until it has congealed (thickened up). Otherwise the phases will separate.

NOTES:

The oil phase is prepared by fusion.

The aqueous solution is at about the same hot temperature as the oil phase, so when the aqueous phase is added, a suitable hom*ogeneous w/o emulsion will form without congealing.

In this method of preparation, the internal phase is added to the external phase and a suitable product is formed. Usually the aqueous phase is added to the oil phase because it is more convenient to pour the aqueous phase and thus a minimal loss of ingredients.

Nivea Cream and Pond’s Cold Cream are commercial examples of w/o emulsions.

The emulsifier is formed in situ and is the sodium salts of the acids in White Wax, any acids in the cetyl esters wax, and acids formed during heating.

Because of the phase volume ratio, an o/w emulsion is formed in spite of the high HLB of the emulsifier.

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4a. Emulsion Bases O/W Type

e.g. Hydrophilic Ointment USP

1. Prepare hydrophilic ointment as follows:

Alternate Formulas for a Softer Base

Methylparaben (methyl p-hydroxy benzoate)

0.025 g 0.025 g 0.025 g

propylparaben (n-propyl p-hydroxy benzoate)

0.015 g 0.015 g 0.015 g

sodium lauryl sulfate 1 g 1 g 1 g propylene glycol 12 g 24 g 20 g stearyl alcohol 25 g 19 g 25 g white petrolatum 25 g 19 g 17 g purified water 37 g 37 g 37 g to make: 100 g 100 g 100 g

2. Melt the stearyl alcohol and white petrolatum in the Ointment Melting Apparatus.

3. Dissolve the other ingredients in water, warmed to 75 C.

4. Discontinue heating. Add the water to the melted oil phase with agitation.

5. Allow to cool, and stir until congealed.

NOTES:

The parabens are used as preservatives. Ointments containing water should/must have a preservative.

Because the oil phase contains some solids, fusion is used.

This is an o/w emulsion because of the phase volume ratio and the high HLB of the surfactant.

Stearyl alcohol functions as the adjuvant emulsifier and provides smoothness (with the petrolatum) on the skin. (reference 3, p. 1575)

Propylene glycol, a humectant, tends to increase the viscosity of the aqueous phase and binds the water, thus lessening the evaporation.

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4b. Emulsion Bases O/W Type

e.g. Vanishing Cream

1. Prepare the vanishing cream as follow:

stearic acid 18.0 g potassium hydroxide 0.80 g glycerin 5.0 g methylparaben 0.020 g propylparaben 0.010 g purified Water 76.0 g to make: 100 g

2. Melt the stearic acid in the Ointment Melting Apparatus.

3. In a separate beaker, dissolve the remaining ingredients in the water at 75 °C.

4. Discontinue heating. Add the aqueous solution to the oil phase with agitation.

5. Allow to cool, and stir until congealed.

NOTES:

The stearic acid is reacted with alkali, e.g. potassium hydroxide, to form a soap. The unreacted stearic aid forms the oily dispersed phase, which is left as a film on the skin after the water evaporates.

The glycerin serves as a humectant.

5. Hydrophillic/Water Soluble Bases

e.g. Polyethylene Glycol Ointment

1. Prepare polyethylene glycol ointment as follows:

Carbowax® PEG 3350 (formerly polyethylene glycol 4000) 40 g PEG 400 (also known as PEG-8) 60 g to make: 100 g

2. Prepare 100 g of the above ointment.

3. Heat the PEG3350 in the Ointment Melting Apparatus (at 65C) in the ceramic dish until it completely melts.

4. Add the PEG 400, and mix until uniform.

5. Allow to cool, and stir until congealed.

NOTES:

A firmer preparation can be prepared by replacing up to 10 g of the PEG 400 with PEG 3350.

If more than 5% water is to be added, i.e., from 6 to 25%, then 5 g of PEG 3350 is replaced with an equal amount of stearyl alcohol.

The PEGs are characterized by their number referring to their approximate molecular weight. Smaller numbers indicate the number of ethylene glycol repeat units.

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6. Poloxamer Gel/Cream

The two precursors to poloxamer base cream are:

pleuronic gel 20% (F127)

lips (lecithin and isopropyl palmitate), or lipmax®

These precursors will be provided to you in the lab, but also can be made from scratch in your pharmacy. Why, might you ask, would you bother preparing them from scratch, when you can simply buy them pre- made? The answer lies in cost savings – translating a higher profit to your pharmacy. The method for preparing PLO 20% and LIPS is provided here for your future reference:

Preparation of the Poloxamer Gel/Cream

Poloxamer gels have wide-ranging applications from teeth-bleaching to artificial skin. Pluronic F-127 is a nonionic surfactant, MW 12,500, which forms gels of tailorable strength and gelling temperature, depending on the concentration and excipients used.

Poloxamer creams are very versatile. If the drug is hydrophobic, it is levigated with a suitable hydrophobic levigating agent and incorporated into the oily (LIPS) phase. If it is hydrophilic, it is levigated with a suitable hydrophilic levigating agent and incorporated into the watery (PLO) phase. The two phases are then combined. Use the following protocol to prepare the salicylic acid poloxamer cream:

1. In a glass mortar, combine 1.2 g salicylic acid in an equal quantity (1.2 g) of 95% ethanol, and mix with a pestle until smooth.

2. Add 8.8 mL of the Lipmax® solution, and continue mixing until smooth.

Add 28.8 g of PLO 20% (about 29 mL), and triturate until a smooth gel/cream is made.

Gel formation happens as the mixture heats to room temperature. You may have to place your hands around the mortar to bring the ingredients up to room temperature. The pestle should be

Poloxamer (PLO 20%)

Weigh 20 g of poloxamer 407 (pluronic F127) and place in an amber 220 mL graduated prescription bottle. NOTE: pluronic F127 powder is a respiratory irritant. Wear an N95 mask when dealing with poloxamer 407 powder.

Weigh 0.3 g of potassium sorbate and place in the same bottle.

Cap the amber bottle and gently mix the pluronic F127 and potassium sorbate together so that it is adequately blended.

Open the cap, and while gently agitating, slowly add cold de-ionized water (5-10 °C) to approximately 100 mL (use the graduated markings on the amber bottle).

Gently agitate the mixture, label, and place in a refrigerator.

The PLO will disperse and dissolve in the fridge in 1-2 days.

Lecithin/Isopropyl Palmitate (LIPS, or Lipmax®)

Weigh 22.7 g of soya lecithin and place in a 100 mL prescription bottle.

Add 0.45 g of sorbic acid to the bottle

Add 22.7 g (26.6 mL) of isopropyl palmitate to the bottle, label, and store.

The lecithin beads will dissolve at room temperature in 1-2 days.

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able to stand upright when the gel is finished.

Part C. Salicylic Acid Base Compounding and Drug Release

NOTE: Start the release experiment as soon as you finished making your base. Depending on your base type, it may take up to two hours to collect sufficient data points.

In the next part of the experiment, the prepared ointment base will be used as a vehicle into which a drug, salicylic acid, will be incorporated. The release characteristics of this dosage form will be evaluated using a simple dialysis cell method.

For bases 1-5, prepare 3.0% (w/w) Salicylic Acid Ointments:*

salicylic acid 1.253 g levigating agent (with or without) 0.500 g ointment base 40.00 g

*(base 6 already has the salicylic acid incorporated)

Neatly pack the remaining amount (60 g) of unused ointment base into a clean ointment jar. Label properly and retain. You will be handing your unused ointments in for evaluation with your final report. Do not discard the unused ointment.

The salicylic acid is levigated with 5 drops, approximately 0.5 g, of levigating agent to form a smooth mass using a plastic spatula. The levigating agent, usually liquid, should be compatible with the ointment base. Use glycerin for o/w emulsion base, mineral oil for a hydrocarbon base, PEG 400 for the PEG base. If the base is soft, it alone may be used as the levigating agent.

The mass is then incorporated into the ointment base by the method of geometric dilution. The drug must be uniformly distributed.

Your TA will demonstrate levigation, geometric dilution, and preparing the diffusion cell during the laboratory.

Preparation of the Diffusion Cell

The diffusion cell consists of a a suitable ointment jar, PC jar or snapsafe vial filled with the ointment under study. A membrane is used to allow for the diffusion of the drug (salicylic acid) but not the base itself.

The ointment jar cell is packed to avoid air-pockets and is rounded slightly with a spatula.

The dialysis cellophane membrane is moistened with de-ionized water, opened by vigorous rubbing, cut, and the excess water is removed by blotting between 2 sheets of tissue paper.

The moist membrane is then spread smoothly over the ointment, removing all wrinkles and air-pockets at the ointment-membrane interface. Care should be taken to avoid damaging the diffusion surface of the membrane (handle membrane by the edges).

Secure the membrane in place with an elastic band.

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Release and Analysis of Salicylic Acid

You will be conducting release studies on four of your bases. Retain the unused portion of your bases to hand in at the end of the lab.

Release Study 1 3.0% Salicylic Acid in Hydrocarbon (Base #1) or Absorption (Base #2)

Release Study 2 3.0% Salicylic Acid in W/O emulsion (Base #3) or O/W emulsion (Base #4)

Release Study 3 3.0% Salicylic Acid in Water Soluble (Base #5)

Release Study 4 3.0% Salicylic Acid in Poloxamer Gel/Cream (Base #6)

Using a retort stand and clamp, the completed diffusion cell is immersed in a large ceramic dish or 400 mL beaker containing exactly 100 mL of de-ionized water to which has been added 4 drops of ferric chloride TS.

Fill a cuvette with the above solution to blank the spectrophotometer with. Blank the spectrophotometer, and retain the cuvette with the blank solution in order to zero the spectrophotometer prior to each sample measurement. You will need to use a new blank for each release study.

The membrane is maintained approximately 0.5 cm below the surface of the solution. This permits the removal of 5 mL of the solution for analysis without exposing the membranes to the atmosphere and prevents bubbles at the solution-membrane interface.

Timing is started when the cell is in contact with the release solution. A purple colour is formed by the reaction of the salicylic ion with the FeCl3.

Gently agitate the release solution to make the colour uniform prior to taking your sample for assay.

Depending on the ointment base under study:

For White Ointment: (Base 1)

At 10 min intervals until 1 hr, remove approx. 5 mL of the release solution and place it in a curvette tube. Assay spectrophotometrically at 525 nm for salicylic acid content.

For Hydrophilic Petrolatum (Base 2), Cold Cream (Base 3), and Poloxamer (Base 6):

At 10 min intervals until 1 hr, remove approx. 5 mL of the release solution and place it in a curvette. Assay spectrophotometrically at 525 nm for salicylic acid content.

For Hydrophilic Ointment (Base 4a), Vanishing Cream (Base 4b), and PEG Ointment (Base 5):

At 5 min intervals until 45 min, remove approx. 5 mL of the release solution and place it in a curvette. Assay spectrophotometrically at 525 nm for salicylic acid content.

During the dissolution runs, measure the absorbance of the release solution as soon as it is collected. Re-blank the spectrophotometer prior to each sample measurement. Return the 5 mL sample to the original solution after each UV measurement, so that the volume of water in the dish remains constant.

Collect at least 5 data points for each release study.

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For the final report, plot the concentration of salicylic acid released vs. time for the six bases on the same graph.

Ointment Base Evaluation

Test a small amount of the prepared ointment base (on the tip of your finger) and tabulate the results in chart form for the following:

texture

ease of application

ease of removal

type of film, if any, remaining on the skin

is the base washable?

how is the ointment removed from the skin?

Store the bases in suitable containers for use in the second lab period.

Place 5 g of the ointment on a glass slab. Determine approximately how much water can be incorporated before a 2-phase preparation is observed or when the ointment becomes fluid.

Part D. Using an Ointment Mill

Ointment mills are used in compounding pharmacies to help reduce the particle size of insoluble APIs or excipients, and ultimately produce a smoother, more pharmaceutically elegant cream or ointment. In Part D, you will be preparing a 40%w/w urea ointment. With the assistance of your instructor, you will be running the cream through the ointment mill in order to reduce the grittiness of the preparation.

In a small glass mortar, triturate 16.0 g urea.

Using geometric dilution, incorporate 24 g of your remaining Base (1) or Base (2).

Mix with a pestle until uniform. Try to make the ointment as smooth as possible.

Note the grittiness of the formulation.

Bring your Part D base to the ointment mill. With the help of your instructor, mill your ointment, and note the final consistency.

SAFETY TIP: Make sure loose hair is tied back, and do not touch the ointment mill while it is operating. Do not operate the ointment mill without assistance.

Pack, label and hand in your final 40% urea ointment along with your final report.

Results & Questions

1. Plot the salicylic acid standard curve. (i.e., the UV absorbance vs. the concentration of salicylic acid in µg/mL)

2. Prepare a plot of the salicylic acid released in µg/mL vs. the time in minutes. The best smooth curve is drawn through the experimental points. Compare the rate of release of the drug from the different ointment bases. Explain the difference in the release rates in

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terms of the physical and chemical properties of the ointment bases.

3. What is Beer’s law? When is it valid?

4. Why is it important to use the same spectrophotometer for calibration and sample measurements?

5. Show the calculations and method for preparing the solution of various concentrations of salicylic acid for the standard curve.

6. Give some comments on each of the following factors which may affect release of salicylic acid from various ointment bases.

a) Characteristics of the drug b) Characteristics of the base c) Inclusion of various liquids in the base d) Nature of diffusion membrane used e) Type and amount of surface active agents used, i.e., HLB system

7. Water is used in the exercise as the release medium. Is it a good model to simulate the human skin? Why? What would you use to better simulate human skin?

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Lab 11: Tonicity and Pharmaceutics

Preparing for the Lab Read the introduction and lab protocol completely Prepare the dilution and tonicity calculations prior to the lab Lab 11 Quassignment to be handed in at the beginning of the lab

Group Allocation You will be working in groups of 5 students

What You’ll Be Doing

Part A: Determining the tonicity of a series of salt solutions using a freezing point depression or a vapour pressure osmometer Part B: Determining the tonicity of one concentration in a series of atropine solutions. Class data will be combined for the final report. Part C: Preparing an isotonic solution of atropine sulfate Part D: Preparing isotonic phosphate buffer at 2 pH values Part E: Observe the effect of extreme tonicities on erythrocytes under a microscope Demonstration: Proper use of the Advanced Instruments Osmometer

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/tonicity.xls

What You’re Handing In Lab 11 Worksheet (due at the beginning of the next lab)

Introduction

When preparing pharmaceutical solutions either for injection or for intra-venous administration or to sensitive tissues such as the eyes or mucous membranes, it is important that the osmotic pressure exerted by the ingredients in the solution do not cause a movement of water within the tissues. This laboratory examines the measurement of osmotic pressure and the determination of the quantities of ingredients in the drug that will render the solution isotonic.

Four colligative properties that depend on the number of particles in a solution are:

Osmotic pressure elevation

Boiling point elevation

Vapour pressure depression

Freezing point depression

If one of the above is known, the others may be calculated from this value. In the case of the pharmaceutical solutions, the osmotic pressure must be controlled in order to prevent damage to the above sensitive tissues. By knowing the freezing point depression of a particle in a solution that doesn’t penetrate the membrane it is possible to determine the effect of the effect of the solution on the membrane (in this case a cell) by determining whether it is a hypertonic solution (lower freezing point) and higher osmotic pressure or hypotonic (higher freezing point) and lower osmotic pressure. In order to prepare a solution that has a neutral osmotic pressure relative to the tissue involved, it is possible to calculate the amount of another therapeutically inert substance that may be added to render the solution isotonic.

References

1. Gennaro, AR (editor) Remington: The Science and Practice of Pharmacy, Chapters 16, 17,and 18, pages 208 – 262, 20

th Edition, Lippincott, Williams and Wilkins, Philadelphia, 2000.

2. Martin, A , Physical Pharmacy, 5th

edition, Chapters 5 and 6, pages 101-142, Lee and Febinger, Philadelphia, 1993.

3. https://online.epocrates.com/u/10a308/atropine

http://phm.utoronto.ca/~ddubins/DL/tonicity.xls

https://online.epocrates.com/u/10a308/atropine

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122 Lab 11: Tonicity and Pharmaceutics

Background

Tonicity Definitions

Osmosis This is the net movement of water across a selectively permeable membrane driven by a

difference in solute concentrations on two sides of the membrane. The membrane

generally will allow water to pass but will exclude electrolytes and small molecules.

Osmolality This is an expression of osmol concentration and a solution has an osmolol concentration of ONE when it contains 1 osmol of solute per kilogram of water. It is important to note that this is an expression of weight to weight therefore a 1 osmol solution of dextrose will contain 6.02 x 10

23 molecules/kg of water and a 1 osmol solution of sodium chloride will

also contain 6.02 x 10 23

total ions/kg of water with ½ being sodium and the other ½ being chlorine.

Osmolarity This is an expression of osmolar concentration and a solution has an osmolar concentration of ONE when it contains 1 osmol of solute per litre of solution. This is contrasted to osmolality in that it is a relation between weight to volume. Therefore a 1 molar and a 1 osmolar solution would be identical for non-electrolytes.

Tonicity This is the effective osmolality equal to the sum of the concentrations of the solutes which have the ability to exert an osmotic force across a membrane.

Isosmotic Any given two solutions are isosmotic if they have the same total osmolarity (or osmolality).

Isotonic An isotonic solution is isosmotic with physiological tonicity (i.e. 0.9% NaCl). A net flow of water across cell membranes is not observed in cells placed in an isotonic solution.

Hypertonic A hypertonic solution has a higher osmotic pressure when compared with physiological tonicity (i.e. 0.9% NaCl). Cells placed in a hypertonic fluid will lose cellular water, and shrink.

Hypotonic A hypotonic solution has a lower osmotic pressure when compared with physiological tonicity (i.e. 0.9% NaCl). Cells placed in a hypotonic fluid will gain cellular water, and will swell and possibly rupture.

Freezing Point Depression Method

The freezing point of normal healthy blood is -0.52 °C and the medium in which its components are dissolved or suspended is water.

Three steps are required to adjust the tonicity:

1. Identify a reference solution and its associated tonicity parameter. This is the solution whose tonicity you are trying to match.

2. Determine the contribution of the drug or additives to the total tonicity.

3. Determine the amount of NaCl required by subtracting the contribution of the actual solution from the reference solution.

This method makes use of the D values in Appendix A (Remington pages 256-261). The units of D are °C/ x% of drug. The relevant values for this lab are provided here:

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Appendix A: Sodium Chloride Equivalents, Freezing-Point Depressions, and Hemolytic Effects of Certain Medicinals in Aqueous Solution

0.5% 1% 2% 3% 5% ISO-OSMOTIC

CONCENTRATION

E D E D E D E D E D % E D H pH

Atropine Sulfate 0.13 0.075 0.11 0.19 0.11 0.32 8.85 0.10 0.52 0 5.0

Dexamethasone sodium phosphate

0.18 0.050 0.17 0.095 0.16 0.180 0.15 0.260 0.14 0.410 6.75 0.13 0.52 0 8.9

Dextrose 0.16 0.091 0.16 0.28 0.16 0.46 5.51 0.16 0.52 0 5.9

Sodium chloride 1.00 0.576 1.00 1.73 1.00 2.88 0.9 1.00 0.52 0 6.7

Sodium phosphate, monobasic

0.29 0.168 0.27 0.47 3.33 0.27 0.52 0 9.2

Sodium phosphate, dibasic (2 H2O)

0.42 0.24 2.23 0.40 0.52 0 9.2

Sodium phosphate, dibasic (12 H2O)

0.22 0.21 4.45 0.20 0 9.2

Potassium phosphate monobasic

0.44 0.25 2.18 0.41 0.52 0 4.4

Potassium phosphate dibasic

0.46 0.27 2.08 0.43 0.52 0 8.4

An example from Remington will illustrate how to perform this calculation:

Dexamethasone sodium phosphate 0.1% Purified Water q.s. 30 ml Mft isotonic solution

This prescription tells us to add water qs (quantum sufficiat: as much as is sufficient) and then in addition Mft (misce fiat: to mix and make) the solution isotonic.

Step 1. Identify a reference solution. The concentration of the reference solution used will be in the first column of the “ISO OSMOTIC CONCENTRATION” section. Its associated tonicity parameter is two columns to the right (“D”).

In this example, we select 0.9% NaCl as our reference solution. According to Appendix A above, sodium chloride is isotonic at 0.9%. If you are preparing a media for cells, you would typically choose 0.9% NaCl as your reference solution. At 0.9%, NaCl has a value for D of 0.52. This means:

D = Tf,ref = 0.52 °C

Step 2. Contribution of the drug.

Now we need to look up the D value for the dexmethasone sodium phosphate at the concentration in solution. Our concentration is 0.1%. Unfortunately, the lowest concentration in the Appendix is 0.5%, and reports a D value of 0.05. So we make the assumption that D varies linearly with concentration. We can multiply the closest D value (in this case, 0.5%) by the ratio of concentrations (Creqd/Cappendix):

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124 Lab 11: Tonicity and Pharmaceutics

C 0.01D

0.5%

0.1%C 0.05D

0.5%

0.1%DD

0.1%

0.5%0.1%

Step 3. Reference solution – Actual solution:

The drug will reduce the freezing point by 0.01 °C. In order to be isotonic with blood, we require the same freezing point with bood (-0.52 °C). So we need to add a sufficient amount of NaCl to make up the difference. That difference in temperature will be:

C 0.51 C 0.01C 0.52ΔT

ΔTΔTΔT

reqdf,

drugf,reff,reqdf,

Now we calculate the concentration of 0.9% NaCl needed to lower the freezing temperature by the difference, 0.51 °C:

NaCl 0.883%C 0.52

C 0.51NaCl 0.9%[NaCl]

ΔT

ΔT] [Reference [NaCl]

reqd

ref

reqdf,

reqd

Converting this to mass of NaCl required:

NaCl g 0.265mL 30mL 100

NaCl g 0.883mNaCl

To make the solution isotonic to the reference solution, you would need to add 0.265 g NaCl.

You need not use NaCl to adjust tonicity. Now that we calculated the Tf,reqd we can use other osmotic agents to make the solution isotonic. For instance, the D value for dextrose at 1% is 0.091. So the concentration of dextrose required to make the solution isotonic would be:

Dextrose % 5.60C 091.0

C 0.51Dextrose 1%[Dextrose]reqd

Dextrose g 1.68mL 30 5.60%mDextrose

Adding 1.68 g of dextrose would make the solution isotonic to the reference solution just as well as adding 0.265 g NaCl.

Sodium Chloride Equivalence Method

This method uses the weight of NaCl that will produce the same osmotic effect as 1 gram of the drug. These values are given as E in Appendix A above.

Using the same prescription as above, the following calculations follow:

1. Reference solution: 0.9% NaCl

Calculate what the mass of NaCl would be in your reference solution at the desired tonicity (in this case, 0.9% NaCl)

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NaCl g 0.270mL 30mL 100

NaCl g 0.9

Look up the value for E of the drug at the desired concentration. E is the mass of NaCl that will produce the same tonicity as 1 g of drug. Use the value for E that is closest to the concentration of your drug. In this case, the closest concentration to 0.1% Dexmethasone is 0.5%:

drug g 1

NaCl g 0.18

drug,0.5%

NaCl

2. Contribution of drug:

Calculate the contribution of the drug in solution by multiplying E by the mass of drug in 30 mL of solution:

NaCl g 0.0054mL 30mL 100

drug g 0.1

drug g 1

NaCl g 0.18

3. Reference solution – actual solution

Just as we did with the last method, we calculate how much NaCl is lacking to make the solution isotonic to the reference solution:

g 0.265 g 0.0054g 0.270m

mmm

difference

drugreferencedifference

So we would add 0.265 g of NaCl to make the solution isotonic. We arrived at the same results using the Freezing Point Depression method.

Isotonic Buffers

When preparing isotonic buffers, first you should calculate the quantities of buffer ingredients in the solution. Then using either of the above methods, determine the amount of NaCl required to make the solution isotonic.

Each of you will be given TWO isotonic buffers to calculate and prepare.

Erythrocytes

The observation of erythrocytes through a microscope while bathed with solutions of differing tonicities will give the best indication of whether isotonicity has been achieved. A simple experiment using blood will illustrate this during the class.

Atropine Sulfate

Atropine sulfate is a cardiac drug usually used in the treatment of bradycardia, cardiac arrest, and exposure to nerve gas. There are many movies (e.g. Pulp Fiction, Mission Impossible, The Rock) which illustrate a direct injection of atropine to the heart to revive a pivotal character. For intravenous use, atropine should be in an isotonic medium so that it is compatible with blood. Due to its potential toxicity, atropine gets its name from the Greek god Atropos, who in Greek mythology decided how a person would die. It’s a good idea to wear gloves when handling atropine powder and solutions, and only deal with the powder in a fume hood.

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Experiment Protocol

Chemicals Supplies Special Equipment Atropine Sulfate Sodium Chloride (NaCl MW 58.44 g/mol) Sodium Phosphate Monobasic (verify MW on the bottle used) Sodium Phosphate Dibasic (verify MW on the bottle used) 5.0% Atropine Sulfate Stock Solution 0.2 M Sodium Phosphate Monobasic 0.2 M Sodium Phosphate Dibasic

Erythrocytes Glass Slide Glass Cover Slip Test tubes Scintillation Vials

Freezing Point Depression Osmometer (Advanced Instruments 3250) pH meter Microscope

The following solutions are prepared or provided by the TA:

50 mL of 5.0% Atropine Sulfate Stock Solution

3 L of 0.2 M Sodium Phosphate Monobasic

1 L of 0.2 M Sodium Phosphate Dibasic

NOTE: This lab is calculation-intensive. In order to save time, you can prepare all of the dilution and tonicity calculations before the laboratory, so you will be ready to go.

Part A. Determination of the Tonicity of Sodium Chloride Solutions

Prepare 50 mL each of the following six sodium chloride standard solutions in de-ionized water:

0.1, 0.5, 1.0, 2.0, 3.0, and 4.0 %w/v NaCl in de-ionized water.

Measure the tonicity of the solutions on either the Freezing Point Osmometer or the Vapour Pressure Osmometer. You will be assigned an instrument by the TA or instructor. There will not be sufficient time to measure the samples in triplicate. Only obtain one reading per standard.

Plot the values obtained for each instrument using the “NaCl” worksheet in tonicity.xls. Are the values equal? How do the two instruments differ in their measurements?

Advanced Instruments Model 3250 Osmometer (Freezing Point Osmometer)

The 3250 osmometer works via measuring the freezing point of the solution tested. Do not touch the blue fluid in this machine with bare hands. Use the latex gloves provided.

Note to TA: Calibration instructions are provided in the User’s Guide.

Once the machine is on and calibrated, the following procedure is used to measure osmolality:

White, opaque sample tubes are used with the 3250 Osmometer. The sample size required is 0.2 – 0.25 mL.

http://phm.utoronto.ca/~ddubins/DL/tonicity.xls

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Remove the sample tube from the freezing point chamber.

Dampen a Kimwipe with de-ionized water. Gently wipe the probe tip until it is clean and there is no visible excess fluid.

NOTE: Be careful not to bend the probe tip!

A 0.2 mL pipette is provided for use with the osmometer. Select a clean sample tube. Using the micropipette, pipette your sample into the tube making sure it fills the lowest part, and place it into the freezing chamber.

Press start. The probe tip will lower, and the machine will say “Cooling Sample”.

At the freezing point, the machine will emit a shrill and startling buzzer sound, like when you answer wrong on a game show. This is normal.

Measurement takes about a minute per sample. The machine will print and display the reading in mOsm. The probe tip will now retract.

Record your osmolality measurement. You can also take the slip of paper.

Remove the plastic sample tube and gently clean the probe tip again with a Kimwipe, dampened with de-ionized water.

Rinse and wipe the plastic sample tube. Do not throw the sample tube away.

The 3250 is now ready for the next sample.

When you are finished, place a clean sample tube in the freezing point chamber. The chamber should not be left without a sampling tube in place.

Part B. Determination of the Tonicity of Atropine Sulfate Solutions

NOTE: Your TAs will prepare the atropine sulfate solutions.

NOTE TO TAs: Prepare atropine sulfate solutions in the fume hood before the lab. For Part C, prepare a 1% solution Part C for each lab group. Wear proper protective gear (gloves, goggles, lab coat). Add 2.5 g of atropine sulfate to a 50 mL volumetric flask, and dilute to the mark. Then prepare the following solutions in 20 mL scintillation vials:

3250 Plastic Sample Tube

Probe Tip

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128 Lab 11: Tonicity and Pharmaceutics

[Atropine] (w/v%)

VStock (mL)

VTotal (mL)

Part B:

0.50% 1 10

1.0% 2 10

2.0% 4 10

3.0% 6 10

4.0% 8 10

5.0% 10 10

Part C:

1.0% 2 10

Using the osmometer in Part A, measure the tonicity of the following atropine sulfate solutions:

0.5, 1.0, 2.0, 3.0, 4.0, and 5.0% w/v atropine in de-ionized water.

Plot your data using the “Atropine” worksheet in tonicity.xls.

Part C. Calculation and Preparation of an Isotonic Solution of Atropine Sulfate

Prepare 10 mL of 1.0% solution of atropine sulfate.

Using the Freezing Point Depression Method, calculate the amount of NaCl that must be added to the 1% solution in order to make it isotonic.

Add this quantity to the 1% solution, and measure the tonicity using the same osmometer you used in Part B. Using your graphs from Part A, what is the concentration of NaCl that results in the same tonicity value as this solution?

Part D. Preparation of an Isotonic Phosphate Buffer

NOTE: Two pH values will be assigned to each student in class (between pH 5.8 and 7.8, and include pH 7.4).

Prepare 100 mL of two Sorensen phosphate buffers using the protocol in Lab 2. Use de-ionized water only. The stock solutions (0.2 M sodium phosphate monobasic and 0.2 M sodium phosphate dibasic) will be prepared for the laboratory by your TA.

Using the Sodium Chloride Equivalence Method, determine the amount of NaCl required to make the buffers isotonic. If the value is negative, calculate the volume of de-ionized water required to dilute the buffer so that it is isotonic.

Add the respective amounts of NaCl to each of the two buffers and measure the tonicity using the same osmometer you used in Parts B and C. How does this value compare with the measurement obtained in the atropine sulfate experiment?

Part E. Demonstration of the Action of a Hypotonic, Isotonic, and Hypertonic Sodium Chloride Solution on Erythrocytes

A drop of blood will be placed on a haemocytometer slide placed under the lenses of a compound microscope. The cells will be bathed in turn by an isotonic, hypertonic and a hypotonic solution. Record the observations seen under each condition.

http://phm.utoronto.ca/~ddubins/DL/tonicity.xls

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Questions

A problem question will be handed out via email and will be due at the beginning of this laboratory period. The assignment will be graded as a quiz and must be an individual effort.

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130 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 3 students

What You’ll Be Doing

Part A: Preparing the precursor solutions, assembling the CMC apparatus, and determining the CMC of three sets of SDS solutions Part B: Detection of the phase inversion point of a W/O emulsion Demonstration: Assembling and testing the oscillator apparatus

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/oscillator.xls

What You’re Handing In Lab 12 Worksheet (due at the beginning of the next lab)

Introduction

This laboratory will examine a method of producing small clusters of surfactant molecules called micelles, which have applications in drug suspensions, and in soaps. Surfactants can stabilize emulsions by forming oil-filled micelles. If the micelles are small enough, the colloidal suspensions may be injected intravenously and carry a drug to sites in the body where the reticuloendothelial system is active (liver, bone marrow, spleen). This is convenient, since most drugs are lipophilic and dissolve readily in the oil phase of the micelles. Larger colloids may be injected subcutaneously and act as depots. Finally, suspensions of micelles may be the vehicle in oral suspensions for the delivery of lipophilic drugs.

Their ability to emulsify makes surfactants effective cleaning materials. The “dirt” often containing oil is attracted to the hydrophobic end of a monomeric surfactant. Several of these combine to surround the droplet of oil and suspend it in the surrounding water environment. Try placing a minimum amount of soap in the water used to wash dishes or to bathe. As “dirt” is freed from the dish or the body a fine colloid (micelle) is formed.

Alone in aqueous liquid, surfactants first populate the air/liquid interface, and lower the surface tension. As the concentration of surfactant increases, superstructures in solution are formed with the compatible moiety in solution and the incompatible moiety pointed inwards in an empty micelle. These micelles form at the Critical Micelle Concentration (CMC). When this occurs, several physical changes occur; density change, conductivity, surface tension, osmotic pressure, and interfacial tension. In this laboratory, we will examine conductivity and osmotic pressure in order to determine or estimate the critical micelle concentration. We will be examining the formation of micelles in aqueous media, in the absence of an oil phase.

References

1. Nash RA, “Pharmaceutical Suspensions” in Pharmaceutical Dosage Forms – Disperse Systems, Volume 1, Lieberman, H.A., Rieger, M.M. and Banker, G.S., editors, Marcel Dekker Inc., New York, NY (1988) p. 151-198.

2. Ofner CM, Schnaare RL and Schwartz JB, “Oral Aqueous Suspensions” in Pharmaceutical Forms – Disperse Systems, Volume 2, Lieberman, H.A., Rieger, M.M. and Banker, G.S., editors, Marcel Dekker Inc., New York, NY (1989) p. 231-264.

3. Lachman L, Lieberman HA and Kanig JL, eds., The Theory and Practice of Industrial Pharmacy, 2nd

ed., U.S.A.: Lea & Febiger, 1976, p. 141-183.

4. Aulton M. Aulton’s Pharmaceutics: The Design and Manufacture of Medicines. 3rd

ed., U.S.A.: Churchill Livingstone Elsevier. p. 85-90.

http://phm.utoronto.ca/~ddubins/DL/oscillator.xls

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5. Gennrbinro AR, et al., Remington: The Science and Practice of Pharmacy, 21st ed., USA: Mack Publishing Company, 2006, p. 311-314, 335-6.

Background

Surfactants

A surface-active molecule possesses approximately an equal ratio between the polar and non-polar portions of the molecule:

Surfactants in an Oil-Water System

When such a molecule is placed in an oil-water system, the polar groups are attracted to or oriented toward the water phase, and the nonpolar groups are oriented toward the oil phase. This physicochemical characteristic consequently lowers the interfacial tension between the oil and water phase:

If a molecule, such as glycerin, possesses a dominance of polar groups, it will not be surface-active as it will dissolve in the aqueous phase and will not be oriented at the oil-water interface. If a molecule, such as glyceryl tristearate, possesses a dominance of non-polar groups, it will also not be surface-active as it will dissolve in the oil phase. A molecule, such as glyceryl monostearate, which possesses approximately an equal balance between the polar and non-polar groups will be oriented at the interface and will be surface active. There are two general types of surfactants: nonionic and ionic surfactants. Glyceryl monostearate is a nonionic surfactant, whereas sodium lauryl sulfate is an ionic surfactant.

Surface-active agents (surfactants) form micelles in aqueous solution above a critical concentration called the critical micelle concentration (CMC). Since the surfactant molecules would much rather live at an interface than be in either solution alone, when the interface is saturated, the surfactant molecules create more interface by increasing the surface area real estate by creating micelles. In aqueous solution, the micelle has a hydrophobic core and a dielectric gradient towards the surface of the micelle making the micelle surface hydrophilic.

oil

water CH2OH

CHOH

CH2OH

glycerin

CH2

SO3- Na+

CH3

(CH2)10

sodium lauryl sulfate

CH2OC=O

CHOH

CH2OH

CH3

(CH2)16

glyceryl monostearate

glyceryl tristearate

CH2OOC17H35

CHOOC17H35

interface

Hydrophilic (water-loving) head

Lipophillic (oil-loving) tail

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Thus, the micelle can act as a soluble phase for non-polar solutes (core), semi-polar solutes (palisade layers) and polar solutes (surface). As a result, the efficiency of a particular surfactant as a solubilizing agent varies from substance to substance. The process of increasing the water solubility of a solute (drug) using a surfactant is called micellar solubilization.

Figure 1. Example of an O/W (oil in water) suspension being formed as the concentration of surfactant

increases above the CMC

Estimating the Amount of Surfactant Required to Surround Oil Globules in an Emulsion

Since surfactants are adsorbed at the oil-water interfaces, the minimum amount of surfactant required to form a complete monolayer around the oil droplets can be estimated. Sodium lauryl sulfate has a cross sectional area of 22 x 10-16 cm2 and has a molecular weight of 288 g/mole. Say you would like to emulsify 100 mL of oil in water, with an average oil droplet diameter of 1

µm (or 1 x 10-4 cm). We can calculate the average volume of an oil dropet:

Vi = 43 πr3 =

43 π(0.5 x 10-4 cm)3 = 5.24 x 10-13 cm3

The number of droplets in 100 mL is:

Total Volume

Volume of a Globule =/dropletcm10 x 5.24

cm 100313-

= 1.91 x 1014 droplets

The surface area of a drop is:

Si = 4πr2 = 4 × 3.14 x (0.5 × 10-4 cm)2 = 3.14 × 10-8 cm2

Total surface area of all the emulsified droplets of oil is the total number of drops of dispersed oil x surface area per drop:

= 1.91 × 1014 × 3.14 × 10-8 = 6 × 106 cm2

The number of surfactant molecules required to cover the surface is equal to:

Total Surface Area

Surface Area per Molecule = 16

1022

106

= 2.72 × 1021 molecules

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The number of moles of surfactant required is equal to the total number of molecules adsorbed at the interface divided by Avogardro’s number:

Total Number of Molecules

Number of Molecules per Mole = 32

1002.6

102.72

= 4.5 × 10-3 moles

The weight of surfactant required:

4.5 × 10-3 × 288 = 1.3 g

The actual amount used would be more than 1.3 g, because some surfactant would adhere to equipment and thus not be available to solubilize the oil.

Hydrophile-Lipophile Balance (HLB) System

The orientation and positioning of a surfactant molecule at the oil-water interface would depend on the interactions of the hydrophilic and lipophilic segments with the environment. The molecules’ hydrophilic portion can be expected to dissolve in, or associate with the aqueous phase of the system. On the other hand, the lipophilic portion would dissolve in the oil phase. The balance between the hydrophilic and lipophilic properties of a surfactant has been codified by the hydrophile-lipophile balance (HLB) system. Griffin, almost 40 years ago, established an empirical scale of HLB values for a variety of nonionic surfactants. The original concept defined HLB as the percentage (by weight) of the hydrophile, divided by 5 to yield more manageable values:

(1) HLB = wt.% hydrophile

The HLB system provides a rational means for identifying combinations of emulsifiers and facilitates the formulation of stable emulsion. Surfactants with a high HLB dissolve or disperse in water, while those with a low HLB dissolve or disperse in oil.

Surfactants with an HLB from 1-10 are considered lipophilic.

Surfactants with an HLB from 10-20 are considered hydrophilic.

A list of the average HLB values of some common surfactants is provided in the appendix of this manual. The HLB of a surfactant will help determine what application it will be most useful for:

Source: Gennrbinro AR, et al., Remington: The Science and Practice of Pharmacy, 21st ed., USA: Mack Publishing Company, 2006, p. 311-314, 335-6.

Surfactants in a Water-Only System

In the first part of this laboratory, we are examining the effect of increasing surfactant concentration on electrical conductance of the solution. This is because the solvent more

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strongly interacts with the hydrophilic moiety of the surfactant molecule. As there is no oil phase in this system, at low concentrations the surfactant molecules will tend to orient at the air-liquid interface. Like the Oil-Water diagram, as the surfactant concentration is increased, the interface will become saturated with surfactant, and eventually superstructures of surfactant molecules will form in solution:

Superstructures of surfactant are concentration-dependent. Above the CMC, other structures also form, including cylinders and sheets.

An important feature of the CMC is that at surfactant concentrations below it, the osmotic properties of the liquid change drastically with surfactant concentration. However, once the air-liquid interface is saturated, changes in osmolarity, solution conductance, and surface tension are much less pronounced. Consequently, the CMC may be found by measuring these properties as a function of surfactant concentration.

The figure to the right shows the several physical changes that occur near the CMC.

Cubic Phase (Spherical)

Hexagonal Phase (Cylindrical)

Lamellar Phase (Sheets)

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The square wave produced by the oscillator, shown on an oscilloscope.

Equipment

A.M. Halpern has described an experiment which uses an oscillating 0.5 volts produced from an oscillator which converts +9 volts DC into an alternating 0.5 V system, using an NE555 chip. We have modified Halpern’s circuit to produce an output AC voltage directly proportional to the current that travels between two metal electrodes. The voltage is amplified using a TL071 chip. Measurement of amplified voltage, rather than current, provides more sensitive and reproducible results.

In the oscillator circuit, when the electrodes are connected to the area on the schematic marked “probe 1 and 2”, and a digital multimeter (DMM) is connected to the area on the schematic marked “DVM 1 and 2”, a measureable voltage is detected when a weak salt solution is in the dish.

Briefly, the current (and consequently, the measured voltage) increases as the monomers are added to water in a 150 mL beaker. When the CMC is reached, micelles form, which add a much smaller contribution to the electrolyte population. The experiments are repeated in a 0.02 M NaCl environment. A higher ionic strength in the water is less compatible with the hydrophobic moieties of surfactant molecules. Can you hypothesize whether or not this will promote micelle formation?

Phase Inversion

Provided a system has enough surfactant to prevent phase separation, an interesting phenomenon occurs when an emulsion is diluted with the dispersed phase. Eventually, micelles of the disperse phase coalesce, resulting in a phase inversion. The dispersed phase becomes the continuous phase, and vice versa. This can be problematic in compounding, as a formulation scientist may believe they have created a W/O emulsion when in fact the opposite is present:

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The conductivity properties of the emulsion will drastically change over a phase inversion provided ionic surfactants are used. In the second part of this experiment, you will create a W/O emulsion with a handheld blender, while monitoring the conductance of the emulsion in order to find the point of phase inversion. A water-soluble colouring agent (FDC red) is added for microscopic qualitative evaluation.

Pharmaceutical Applications

The vast majority of drugs are hydrophobic. However, often aqueous solutions, suspensions, or emulsions are required (e.g. intravenous and topical formulations). Surfactants, when organized into micelles, solibilize drugs by entrapping them in their hydrophobic core. Drugs which would never exist in aqueous solution can be wetted and effectively dissolved using an appropriate type and concentration of surfactant. Micelle formation and phase inversion is depending upon the surfactant’s concentration. Too low a concentration will leave surfactant only at the interface of the formulation.

Particularly with emulsions, compounding must involve knowledge of the concentration of surfactant required in order to achieve the desired emulsion (e.g. O/W, W/O, O/W/O, W/O/W) as well as the target micellar size. Some negative consequences of diluting a carefully balanced emulsion could involve phase separation, phase inversion, or drug precipitation. For an intravenous formulation, drug precipitates can cause local irritation or perhaps stroke and death. This can occur simply because the formulation is diluted below the CMC. In the case of intravenous, the drug solubility not only in the formulation itself but at the site of delivery must be considered. For instance, a drug dissolved in an organic solvent can precipitate upon injection. Moreover, compounding intravenous admixtures is often practiced by hospital pharmacists. Instability and physiochemical stability can happen in these admixtures owing to the changes of surfactant concentration, pH, solvent properties, and ingredient compositions, etc.

Surfactants can also impart stability to the drug itself. If a drug is sensitive to hydrolysis or oxidation in aqueous medium, the addition of a non-ionic surfactant can protect the drug from degradation. In addition to improving the stability of formulation and the drug, determination of the phase inversion point and CMC is used to help characterize the HLB of surfactants. In particular, the HLB of non-ionic surfactants has been shown to influence the phase inversion temperature. Other constituents in the formulation can affect these values, such as the concentration of ionic species present (e.g. NaCl). Surfactant type, heat, time, evaporation, and surfactant/excipient or surfactant/drug interactions can affect the CMC and result in phase

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Lab

inversion and/or precipitation. This makes the selection of surfactant type and concentration a crucial component for a successful liquid or semi-solid formulation. Phase inversion can be minimized by using the proper emulsifying agent and keeping the volume ratio of the dispersed phase well below the phase inversion point.

Experiment Protocol

Chemicals Supplies Special Equipment Sodium Lauryl Sulfate (SDS MW 288.38 g/mol) Sodium Chloride (NaCl MW 58.44 g/mol) Heavy Mineral Oil

N/A DC Oscillator 4 connecting wires (with alligator clips) Digital Multimeter (DMM) Burette, Burette Clamp, Retort Stand, Vinyl Retort Clamp Metal electrodes Magnet 9V DC adaptor 150 mL and 500 mL Beaker 50 mL, 100 mL Volumetric Flasks 10 mL, 100 mL Graduated Cylinders Stir Plate and Medium-Sized (1.5”) Magnetic Stir Bar

Materials and Special Equipment

Part A. Preparing the Solutions

Water acts as an insulator. As a charged species is added, the current may more readily flow between the electrodes. The amount of current that flows can be proportional to the concentration of ionic species present.

Below the CMC, the addition of amphiphiles causes an increase in charge carriers, and we observe an increase in current. Above the CMC, there is an increase in micelle concentration, and the monomer concentration stays the same. Hence the relative levelling of the curve with the break occurring at about the point that [C] = CMC.

Prepare the following solutions in de-ionized water for Part A in 100 mL volumetric flasks. Wear an N95 mask when weighing and handling SDS in powder form, as it is a respiratory irritant.

Experiment Solution A (in 150 mL beaker)

Volume Required

Solution B (in burette)

Volume Required

Experiment 1 Solution 1A: (De-ionized Water)

100 mL Solution 1B: 0.04 M SDS

100 mL

Experiment 2 Solution 2A: 0.01 M NaCl

100 mL Solution 2B: 0.04 M SDS with 0.01 M NaCl

100 mL

Experiment 3 Solution 3A: 0.02 M NaCl

100 mL Solution 3B: 0.04 M SDS with 0.02 M NaCl

100 mL

NOTE: When you are working with SDS solutions, avoid shaking them. Agitate gently, in order to minimize bubble formation. Pour slowly and on an angle. When diluting to the mark, do not use

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the top of the bubbles as a guide. Wait for a clear liquid meniscus to form, and add fluid until the etched line at the neck of the volumetric mark.

Assembling and Testing the CMC Apparatus

The following is a step-by-step guide on assembling the CMC apparatus. If you have any questions, ask your instructor or TA.

Set up a burette on a retort stand with a burette clamp.

Place a magnetic stir plate at the base of the retort stand and plug it in.

Place a 150 mL beaker on top of the magnetic stir plate.

Locate the metal electrodes and affix them to the beaker using the magnets provided.

Locate the DC Oscillator, and the 9V DC Adaptor. Handle the DC Oscillator with care – the circuit board components are fragile.

Locate a RadioShack® or MASTECH® digital multimeter. Turn on the multimeter, and set it to measure voltage (V). Press the SELECT button, to select direct current (the “ ” symbol will appear on the LCD screen).

Metal Electrodes

Magnet

DC Oscillator

9V DC adaptor

Magnet

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Adaptor Test: Touch the free ends of the wires connected to the multimeter to the terminals of the adaptor, and verify that the adaptor is working. The voltage readings will typically be in the range of 9 V (see above right panel).

The following procedure will connect the equipment according to the following diagram:

The oscillator produces a pulsed, bipolar voltage having a frequency of ~1 kHz and a peak to peak amplitude of about 0.3 V.

The 9V DC Oscillator: Component Diagram

RadioShack® Multimeter

MASTECH® Multimeter

To multimeter

To metal electrode

Power switch

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NOTE: To connect the alligator clips, squeeze the widest part of the connector head, and attach the clip to the exposed part of the oscillator wire:

YES NO

Using two alligator-clip wires, connect the yellow (or white) leads on the right side of the oscillator (opposite the battery) to the graphite electrodes:

NOTE: To connect the alligator clip to the electrode, open the jaws as wide as possible, and clip them to the exposed copper wire on the end of the metal electrode.

Using another two alligator-clip wires, connect the leads on the right side of the oscillator (purple leads, grouped together) to the multimeter (it doesn’t matter which lead is connected to black or red):

Pour only 50 mL of Solution 1A (de-ionized water) into the 150 mL beaker.

Note: The extra is there if you need to repeat the experiment.

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Place a medium-sized magnetic stir bar into the 150 mL beaker and set the speed on low.

Pour about 40 mL of Solution 1B (0.04 M SDS) into a 50 mL burette.

NOTE: Unclamp the burette to load it, so that Solution 1B does not spill accidentally into 1A.

The final connected apparatus should look like the following picture:

The metal electrodes are protected by a layer of heat shrink tubing, allowing the internal conductivity of the solution to be measured, not at the surface. Ensure the exposed metal is completely submerged under the surface of the water, and not touching the bottom or sides of the beaker, or magnetic stir bar.

On the multimeter, press the SELECT button, to select alternating current (the “~” symbol will appear on the LCD screen).

Before you begin:

1. Verify switch is set (or rotated) to “V” (Voltage):

Submerged electrodes: under liquid level, not touching sides of beaker or magnetic stir bar.

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2. Verify “~” symbol in the LCD display:

If the multimeter auto-powers off, ensure these settings are selected again when you switch the multimeter back on.

3. Verify the LED light on the Oscillator is on. If not, press the blue power button.

4. Call your TA or instructor to verify that your apparatus is ready to go!

Experiment 1

Titrate the SDS into the 150 mL beaker in 0.5 mL aliquots (40 – 50 aliquots will be sufficient).

Record the voltage (in mV) after adding each 0.5 mL aliquot. Allow the reading to stabilize (5-10 seconds) before taking each reading.

NOTE: Depending upon the voltage reported, the multimeter may switch from “mV” to “V” in the display. Make sure you record your results in mV only.

Experiment 2

Repeat the above experiment using Solution 2A (0.01 M NaCl) in the 150 mL beaker, and Solution 2B (0.04 M SDS with 0.01 M NaCl) in the burette.

Experiment 3

Repeat the above experiment using Solution 3A (0.02 M NaCl) in the 150 mL beaker, and Solution 3B (0.04 M SDS with 0.02 M NaCl) in the burette.

Part B. Phase Inversion

Add the following to a 500 mL beaker:

240 mL Heavy Mineral Oil 10 mL of 1.0% FDC red in water

Using a handheld mixer, mix on low speed for 1 minute.

While continuing to blend, slowly add:

5 g Sodium Laurel Sulfate

Blend on high speed until mixture appears hom*ogenous (approx. 2 minutes)

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NOTE: Completely submerge the head of the mixer into the liquid. Do not let the head rise above the surface of the liquid, or foaming may occur.

From the centre of the solution, withdraw a sample of the emulsion using a disposable transfer pipette. Place a drop on a clean microscope slide. Place a cover slip over the drop. Observe the emulsion under a microscope and record your observations.

Place two drops of your starting emulsion on a clean slide or watch glass. Describe what happens when a drop of heavy mineral oil is added right next to (and touching) the emulsion.

Measure out 50 mL of the emulsion using a graduated cylinder and transfer to a 150 mL beaker.

Place the beaker on a stir plate and a medium-sized magnetic stir bar in the emulsion. Set the highest possible speed of stable stirring that doesn’t result in aspiration or foaming.

NOTE: You should be able to see a vortex in solution. A fast stirring rate is necessary in order for the titrant to be properly mixed into the emulsion. Do not let the stir bar knock against the side of the beaker while stirring. At fast speeds, the stir bar can break through glass.

Add 1 g of SDS to a 50 mL volumetric flask, and dilute to the mark with de-ionized water. Load this into a 50 mL burette.

Set up the electrode apparatus as described in Part A.

Check the “Before you begin” section again – check for “V”, “~”, and verify the LED light is on.

Titrate your emulsion with the SDS solution by 0.5 mL aliquots, and record voltage as a function of volume titrated. Stop titrating after collecting at least 10 data points after phase inversion has occurred.

From the centre of the solution, withdraw a second sample of the final emulsion using a disposable transfer pipette. Place a drop on a microscope slide. Place a cover slip over the drop. Observe the emulsion under a microscope and record your observations. How does this compare to your initial sample?

Place two drops of your starting emulsion on a clean slide or watch glass. Describe what happens when a drop of heavy mineral oil is added right next to (and touching) the emulsion.

Clean-Up

The head on the hand blenders is removable from the base. Please remove the head prior to rinsing it in the lab sink.

Return the probe and magnet to your instructor. The magnets are small and easy to lose track of – please don’t lose them or accidentally throw them away.

Data Analysis – Part A

On the “CMC” worksheet of oscillator.xls, enter your data into a spreadsheet as a table consisting of:

Volume of titrant added (mL), and

Measured voltage V (in mV).

http://phm.utoronto.ca/~ddubins/DL/oscillator.xls

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Adjust the spreadsheet baselines to fit your specific data. The spreadsheet will calculate the volume of titrant added at which the CMC was detected. From this volume, calculate the CMC. Label the CMC of each experiment with an arrow. Print a graph for each experiment, and attach it to your lab report.

Convert the aliquot numbers into final SDS concentration to create the column “[SDS] (moles/L)”. Subtract off the starting voltage value from each value, to create the column of baseline-corrected voltages, “Corrected V (mV)”.

Plot Corrected Voltage V/[SDS] vs. ][SDS for the Experiment 1 data, where [SDS] is

the concentration of SDS in moles/L. The CMC of this graph should be the point at which the slope changes. Label the CMC of each experiment with an arrow. Print the graph, and attach it to your lab report. There is no spreadsheet for this graph.

Does the CMC calculated using the first method (baseline fitting) agree with the V/[SDS]

vs. ][SDS method for Experiment 1?

Data Analysis - Part B

On the “Phase Inversion” worksheet of oscillator.xls, enter your data into the spreadsheet as a table consisting of:

Volume of titrant added (mL) - ascending, starting with your first reading, and

Measured voltage V (in mV).

Print a graph for each experiment, and attach it to your lab worksheet.

With an arrow, label the phase inversion point on the graph. The phase inversion point occurs at the first aliquot of volume added at which the conductivity of the solution changes from being zero to detectable, or vice-versa depending on the type of inversion observed.

Calculate and report the volume ratio of water:oil at the phase inversion point.

Calculate and report the volume fraction (Vwater/Voil+water) at the phase inversion point.

Questions

1. Compare the CMC for the aqueous samples and those salt medium. Explain the differences in the values if any.

2. The graphite electrodes are protected by a coating of paraffin wax to monitor conductivity below the surface of the emulsion. What would you expect to see happen in Part A if the electrodes were not coated?

3. If a non-ionic surfactant were used in Part A of this experiment, would the experimental design have to change? If so, how?

4. What happens to the surfactant molecules during a phase inversion?

5. If a non-ionic surfactant were used in Part A (CMC determination) of this experiment, would the experimental design have to change? If so, how?

6. If a non-ionic surfactant were used in Part B (Phase Inversion) of this experiment, would the experimental design have to change? If so, how?

7. Would you expect the type of surfactant to change the phase inversion point? If Part B were repeated with 0.02 M NaCl, would you expect the phase inversion point to change? If so, how?

http://phm.utoronto.ca/~ddubins/DL/oscillator.xls

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8. In Part B, the titrant included the same concentration of SDS as the emulsion. What

might have happened if de-ionized water was used as a titrant?

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146 Lab 13: Optimization of Powder Flow and Particle Size Determination

Lab 13: Optimization of Powder Flow and Particle Size Determination

Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 3 students

What You’ll Be Doing

Part A: Six powder blends are compounded and blended for a specified time Part B: Evaluate the tapped density of the 6 blends Part C: Measuring the Angle of Repose of the 6 blends Part D: Evaluate the powder flowability of the 6 blends Part E: Sieving the 1- and 5-minute blends for one of your formulations Demonstration: Using the Powder Flow Apparatus

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/tapdensity2.xls http://phm.utoronto.ca/~ddubins/DL/probit.xls http://phm.utoronto.ca/~ddubins/DL/probit.pdf

What You’re Handing In Lab 13 Worksheet (due at the beginning of the next lab)

Introduction

Powder Flow

Scale-up of a formulation from development to production requires a fundamental understanding of the interrelationships between ingredient behaviour and processing equipment parameters. Selecting the correct combination of ingredients to satisfy these interrelationships involves careful examination at the pre-formulation stage. Powder blends can be used for capsule formulations, insufflations, douche powders and dusting powders. Additives, such as diluents, binders, lubricants, glidants, disintegrants, and colourants, are usually included to facilitate handling, enhance physical appearance, improve stability, and aid in absorption. This exercise demonstrates the measure of a few experimental parameters to characterize powder flow properties.

Powder Size

In the preparation and characterization of many pharmaceuticals, one is vitally concerned with the size distribution of particles. In the preparation of a solution the time required for a given weight of material to dissolve depends on the degree of subdivision of the solute. The texture, taste, and rheology of an oral suspension depend on the size-frequency distribution of the dispersed phase. The automatic machine-filling of bulk powders into bottles and vials is affected by the shape and the size of the particles of the powder. The uniformity of weight of compressed tablets and hard gelatin capsules depends on the proper flow of the granulation from the hopper into the die cavity. Particle size affects the rapidity of extraction from crude drugs. Particle size is one of the major factors which influence the physiological availability of drugs from oral and parenteral pharmaceuticals.

Most starting materials used in the manufacture of solid dosage forms are fine powders with wide size distributions. Pharmaceutical wet granulation can agglomerate powders in order to enhance some of the material handling characteristics. Agglomeration (defined as the assemblage of particles in a powder) primarily serves to prepare powders for tableting by

http://phm.utoronto.ca/~ddubins/DL/tapdensity2.xls

http://phm.utoronto.ca/~ddubins/DL/probit.xls

http://phm.utoronto.ca/~ddubins/DL/probit.pdf

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147 Lab 13: Optimization of Powder Flow and Particle Size Determination

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rendering them free-flowing, non-segregating and easily compressible. It may also serve to modify solubility and dissolution rate and to reduce the formation of dust.

In this laboratory, the effect of particle size on tableting properties is investigated by comparing some of the physicochemical properties of un-granulated material. The results should indicate the importance of selecting the appropriate particle size of drug for the development of tablet dosage forms.

References

1. Carr RL, Chem. Engineering, 72(1), 163-168, 1965. 2. Brown RL and Richards JC, Principles of Powder Mechanics, Pergamon Press Ltd: Britain (1970), p.

13-37. 3. Jones TM and Pilpel N, J. Pharm. Pharmac., 18, 182S-189S, 1966. 4. United States Pharmacopoeia XXII, United States Pharmacopoeia Convention, Rockville, MD,

1990. <Dissolution>. 5. O’Connor, RE, and Schwartz, JB, Remington: The Science and Practice of Pharmacy, 20

th ed., Mack

Publishing Company: U.S.A., (2000), p.681-699. 6. Wells JI and Walker CV, Int. J. Pharm., 15, 97-111, 1983. 7. Alderborn G and Nyström C, Acta Pharm. Suec., 19, 381-390, 1982. 8. Riepma KA, Zuurman K, Bolhuis GK, de Boer AH and Lerk CF, Int. J. Pharm., 85, 121-128, 1992. 9. Rhodes M, Introduction to Particle Technology, John Wiley & Sons ltd, U.K., 1998, p. 55-80. 10. Parrott, E, Pharmaceutical Technology: fundamental pharmaceutics. Burgess Publishing

Company, 1970, U.S.A. p. 1-136.

11. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM218825.pdf

Background

Types of Additive in a Powder Formulation Examples

Diluents – increase the bulk to a practical size for

handling

dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, inositol, sodium chloride, dry starch, powdered sugar, hydroxypropylmethylcellulose

Binders or Granulators – impart cohesive qualities to the

powder – can be in solution or in a dry form

starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, acacia, sodium alginate, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone (occasionally, polyethylene glycol, waxes, water, alcohol)

Lubricants - prevent the adhesion of material to the

surface of equipment, reduce interparticle friction, may improve the flow of granulation

talc, magnesium sterate, calcium stearate, stearic acid, hydrogenated vegetable oils and polyethylene glycol

Glidants - improve the flow characteristics of a

powder mixture - usually added in the dry state just prior

to compression

colloidal silicon dioxide, talc

Disintegrants - facilitate the disintegration of a

tablet

starches, clays, celluloses, aligns, gums, and cross-linked polymers

Colourants - make the product identifiable

FD & C red, FD & C yellow, FD & C green, FD & C blue, iron oxide, titanium dioxide

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM218825.pdf

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148 Lab 13: Optimization of Powder Flow and Particle Size Determination

In order to make a capsule, it is necessary to (a) make the powder flow from a hopper into a feed frame, (b) make the powder flow from the feed frame into the die holding the capsule shell, (c) lock the capsule shell with the capsule cap without dislodging the filled powder, and (d) eject the filled capsule from the die. Flow rates of powders are a function of particle size, particle shape, and surface roughness. In addition to the inherent powder characteristics, there are numerous processing variables that must also be considered such as the time and speed of mixing, type of mixing dynamics, and temperature and humidity effects. Scale-up of a formulation from development to production remains an inexact discipline. Proportionality does not necessarily apply. In early development, supplies of bulk active are limited and capsule formulations must be developed on a small scale. Difficulty can be encountered if the formulations are not designed with consideration of the stresses of high speed manufacturing equipment.

The degree of mixing affects the lubricity and wettability of magnesium stearate-containing capsule blends, and stressing a powder blend in a mixer can mimic the changes in blend properties that may occur on scale-up. Measuring tapped bulk density, wettability and disintegration of stressed blends identifies robust formulations which are unaffected by long “lubrication” times and scale. In this fashion, the industrial formulating pharmacist can quickly assess the impact of various parameters on the suitability of a formulation.

Bulk Density

Bulk density is the mass of powder per unit of bulk volume which consists of the void volume and the true volume occupied by the particles. Although there is no direct linear relationship between the potential flowability of a powder and its bulk density, other properties of the substance can affect the bulk density and flowability. By comparing both the initial and final bulk volumes of powder subjected to tapped compression, Carr defined the compressibility index (CI):

Compressibility Index (or, Consolidation Index) =

1 – VtapVbulk

x 100

Where Vtap is the volume of the tapped powder

Vbulk is the volume of the bulk powder when placed in a

container (includes the true volume, volume of the internal pores, volume of spaces between the particles)

Compressibility is strictly a misnomer since compression is not involved and “consolidation” might be a more suitable description. It is a simple index and the interpretation is shown in the following table:

Consolidation Index (%) Flow

5-15 Excellent

12-16 Good

*18-21 Fair to passable

*21-35 Poor

33-38 Very Poor

>40 Extremely Poor

* adding a glidant should improve flow

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Blends having CI’s less than 15% usually exhibit good flow tendencies while those with a CI value greater than 26% most likely have poor flow characteristics. The index of Carr is a one-point determination and does not reflect the ease or speed of consolidation. Some materials may have a high index suggesting poor flow but may consolidate rapidly which is essential in tableting. An empirical relationship can be drawn between the percentage change in bulk density: (V0–Vn)/(V0–V50) x 100 and the log of the number of taps, (log n), where V0 is the initial bulk volume and V50 is the bulk volume after 50 taps. Non-linearity occurs up to two taps and after 30 taps, when the bed consolidates more slowly. The slope is a measure of the speed of consolidation and is useful for assessing powders or blends with similar indices, the beneficial effect of glidants, and the design of capsule formulations. Although counter-intuitive, when comparing two blends, a steeper slope indicates a slower speed of consolidation.

100VV

VVlog(n)

500

Angle of Repose

A static heap of powder, when only gravity acts upon it, will tend to form a conical mound. One limitation exists; the angle to the horizontal cannot exceed a certain value, and this is known as

the angle of repose (). The angle depends on the mutual friction between the particles. With an increase in the friction, there is an increase in the angle of repose. As the irregularity of the particles become greater, the friction and the resistance to flow is increased. Accordingly there

is an implied relationship between and flow and particle shape. By measuring the diameter of the base, D, and the height of the heap (h) the angle of repose can be calculated using the trigonometric relationship:

5.0tan)(θ Repose of Angle 1-

The exact value for depends on the method of measurement but in general the values in the table below may be used as a guide:

Angle of Repose Flow

<25 Excellent

25-30 Good

*30-40 Passable

>40 Very Poor

* adding a glidant should improve flow

Powder Flowability through a Hole in a Plate

Many powders are marketed with “viscosity” of the powder specified. This does not refer to the viscosity of the material when dissolved into a liquid, but rather the flowability of the powder itself. The viscosity of a powder may be estimated by observing its ability to fall freely through a

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150 Lab 13: Optimization of Powder Flow and Particle Size Determination

hole of known dimensions in a plate. The powder is tested with different hole sizes, ranging from small (e.g. 5 mm) to large (eg 22 mm). The diameter of the smallest hole through which the powder passes three times out of three is taken as the flowability index. The diameter may be used as an empirical measure, or converted into viscosity, which is a comprehensive industry standard.

Mathematically, a “core” cylinder of powder will flow through a hole if the weight of the powder above the hole is greater than the friction of the side surface of the powder:

(1) πr2hdg ≥ 2πrhK Where: h = height of core cylinder of powder r = radius of hole (πr2h = volume of core cylinder) g = acceleration due to gravity (981 cm/s2) d = non-tapped bulk density of powder (2πrh = surface area of core cylinder of powder) K = coefficient of friction/cm2 (powder viscosity)

The above equation can be simplified to:

(2) 𝑟 ≥𝐾(

𝑔𝑐𝑚∙𝑠2⁄ )

490.5 (𝑐𝑚𝑠2⁄ )×𝑑(

𝑔𝑐𝑚3⁄ )

Solving for K (powder viscosity):

(3) 𝐾(𝑃𝑜𝑖𝑠𝑒) ≤ 490.5 (𝑐𝑚

𝑠2 ) × 𝑟(𝑐𝑚) × 𝑑 (𝑔

𝑐𝑚3)

The answer in Poise (P) can be multiplied by 100 to obtain the measurement in centipoise (cP), a more typical viscosity unit. Thus the viscosity of the powder can be estimated by finding the minimum hole diameter the powder will freely flow through.

Particle Sizing

The major techniques for particle size determination are microscopy, sieving, and sedimentation. Sieving is the simplest and most widely used method for determining particle size since the analysis can be completed in a short time.

Core cylinder of powder

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The sieve number refers to the number of meshes to a linear inch of the sieve through which the powder will pass. The number of openings per linear inch, however, is not always an indication of the size of the openings in the sieve due to the variation in the diameter of the wire used in various sieve cloths. For this reason the Bureau of Standards has established specifications for standard sieves, as given in the following table:

U.S. Standard Sieves

Nominal Designation No.

(mesh #)

Sieve Opening (µm)

Nominal Designation No. (mesh #)

Sieve Opening (µm)

2 9500 45 355

4 4750 50 300

6 3350 60 250

8 2360 70 212

10 2000 80 180

12 1700 100 150

14 1400 120 125

16 1180 140 106

18 1000 170 90

20 850 200 75

25 710 230 63

30 600 270 53

35 500 325 45

40 425 400 38

The integrity of a tablet is dependent on the strength and resistance of the compacted powder in withstanding external disruptive forces until the tablet is administered. The purpose of compaction is to bring particle surfaces into close proximity and to enhance intermolecular forces, thereby enabling inter-particulate bonding. Compactibility of powder is dependent on both the intra- and inter-particulate bond strength and on the area of inter-particle bonding resulting from powder compaction and decompression. Compactibility may be affected by both physicochemical characteristics of material under consolidation as well as tableting conditions. Important functional characteristics include the ability of particles to bond following deformation, particle roughness and shape, particle size and size distribution, moisture and amount of elastic recovery occurring during decompression. In general, excipients that deform quickly and permanently facilitate inter-particle bonding, and produce tablets with improved mechanical strength. Several techniques used to enhance compactibility of excipients have been cited in the literature. Changes in process equipment or conditions can influence the compaction behaviour of the materials.

Particle size evaluation involves placing a sample of the test material on the upper stack of standard sieves and shaking the sieves for a given time. A Tyler or Cenco-Meinzer sieve shaker holds seven standard sieves and will classify a powder in five or ten minutes. The weight of powder retained on each sieve is determined. The size assigned to the powder retained is arbitrary, but by convention the size of the particles retained on a sieve is taken as the arithmetic or geometric mean size of the two sieves. Thus, for a powder passing a 30-mesh sieve and retained on a 45-mesh sieve, we look up their sieve openings on the table above:

Sieve # 30 has a sieve opening of 600 µm

Sieve # 45 has a sieve opening of 355 µm

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152 Lab 13: Optimization of Powder Flow and Particle Size Determination

The particles weighed on the #45 sieve will have an arithmetic mean diameter of:

μm 477.52

355600

The particles weighed on the #45 sieve will have a geometric mean diameter of:

μm 461.501 2

log(355)log(600)

Simple statistical analysis can be performed on sieve data. The student should be familiar with the following statistical calculations; arithmetic mean, geometric mean, median, mode, percentile, standard deviation.

A number of graphs may also be drawn with this data in order to facilitate evaluation. The simplest of these is the frequency distribution curve which plots the percentage of particles retained on a sieve versus the mean particle size for the particles. In order to make a visual estimation as to whether or not the particles approximate a normal distribution curve it is necessary that the range of sizes of the sieves be uniform. To do this, an adjusted frequency distribution curve is prepared by dividing the fraction retained by the size range of the two sieves between which the particles fell. This value is then plotted versus the mean particle size. A visual comparison of size distribution can now be made between different materials or the same material subjected to different agglomeration processes. From this data, a series of cumulative plots may now be drawn. Cumulative distributions are used to determine percentiles, i.e., the proportion of a test material which is above or below a specified size value.

Experiment Protocol

Chemicals Supplies Special Equipment Corn Starch Lactose Regular Avicel PH101 Dibasic Calcium Phosphate (CaHPO4)

Parafilm Cardboard Scintillation Vials

Powder Funnel Standard Sieves #20 Sieve

Materials and Special Equipment

The following solutions are prepared or provided by the TA:

Not applicable.

Part A. Compounding Powder Blends

Three separate capsule formulations are to be prepared and evaluated according to the following table:

Ingredients Blends (%w/w)

A1 A5 B1 B5 C1 C5

Dibasic Calcium Phosphate - - - - 10 10

Corn Starch 90 90 15 15 25 25

Lactose Regular 10 10 10 10 - -

Avicel PH101 - - 75 75 65 65

BLENDING TIME (min) 1.0 5.0 1.0 5.0 1.0 5.0

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Prepare 120 g of each blend above (6 in total). Please note that the proportions in the blends above are listed in (%w/w). Make sure to properly label each blend.

Place the blend into a laboratory blender.

Add 3 g of magnesium stearate.

Turn the blender on at the lowest speed and blend for the allotted time.

Do not rinse the laboratory blender between samples – this will alter the humidity of the powder. Simply remove as much as the previous blend as possible between runs.

Perform the following analysis on each blend sample (2 X 3 blends = 6 samples in total) and record your results.

Part B. Determining Tapped Density

NOTE: We are using 15 taps instead of 50 taps as detailed in the Background section.

Loosely place an aliquot of the blend into a dried, tared 25 mL graduated cylinder, filling the cylinder to the 25 mL graduation mark.

Weigh the cylinder again to determine the weight of powder. Cover the mouth of the cylinder with Parafilm. Calculate and record d, the untapped powder density.

Obtain a retort stand and rubberized ring clamp. Place a piece of corrugated cardboard on the retort stand base.

Set the rubberized ring clamped securely to the stand at 10 cm above the horizontal.

NOTE: Use the ring clamps with rubber tubing to prevent breaking the graduated cylinder.

Hold the 25 mL graduated cylinder within the ring clamp so that the base is at the level of the clamp. Allow the cylinder to drop onto the cardboard. (The ring clamp should act as a guide to prevent the cylinder from tipping over.)

Tap the 25 mL cylinder in this manner ONCE. Record the volume of the blend in the 25 mL cylinder.

Continue tapping using the fixed-height drop technique for a total of 1, 2, 5, 10, and 15 times. Record the volume of the blend at different times.

Calculate the tapped density of the blend powder: (powder weight/V2).

Use tapdensity2.xls on the laboratory website to plot log n vs. (V0-Vn)/(V0-V15), and to calculate the speed of consolidation for each formulation. Print off the graphs and staple them to your worksheets. Report the slope of the curve.

http://phm.utoronto.ca/~ddubins/DL/tapdensity2.xls

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154 Lab 13: Optimization of Powder Flow and Particle Size Determination

NOTE: Use the rubberized ring clamp (shown above) to determine tapped density.

Part C. Determining the Angle of Repose

Using the angle of repose platforms in the lab, measure the angle of repose of all 6 powder blends.

Part D. Determining Powder Flowability

Setting up the Powder Flow Apparatus:

The powder flow apparatus will be set up at each station.

Sign out a series of disks and a metal ruler from your instructor or TA. You will be responsible for returning the disks and ruler at the end of the lab. As these disks are custom made, take special care not to lose them. A 3 mark penalty will be associated with not handing all of the disks back.

Place the powder collection bowl at the base of a retort stand.

Attach the powder flow apparatus on a retort stand using a vinyl retort clamp.

Powder flow apparatus

Powder flow disks

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The apparatus should be high enough so that the trap door swings open freely. With the trap door shut, the body of the powder flow apparatus cylinder should be ~10 cm from the bottom of the ceramic bowl.

The trap door should be facing downwards.

The trap door release hinge should be pointing forward (towards you).

Attach the metal funnel above the powder flow apparatus using a ring clamp, so that the funnel is 4-5 cm above the top of the top of the powder flow apparatus. The final assembly should look like this:

Powder Flow Apparatus

The disks are labeled with their hole diameter in millimeters stamped on the disk.

Insert the 16 mm disk into the powder flow apparatus. Make sure the disk is flush with the bottom of the powder flow apparatus cylinder.

Close the trap door by sliding the middle hole of the trap door release hinge into the pin attached to the bottom of the trap door:

Powder flow assembly

Powder collection bowl

Funnel on rubber ring clamp

Trap door release hinge

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Test Procedure

1. Fill a clean, dry 400 mL beaker approximately half way with the powder test mix.

2. Pour the powder test mix into the metal funnel, until the powder flow apparatus is filled ~1 cm from the top. If the powder becomes trapped in the funnel, tap the funnel gently with a spatula until all of the powder falls loosely into the centre of the cylinder of the powder flow apparatus. Pouring the powder in the funnel disrupts powder aggregates due to long term storage or sitting.

NOTE: Be careful not to touch the sides of the powder flow apparatus or tap it once it is loaded.

3. After the cylinder is filled, allow 30 seconds for possible formation of individual flocculi or mass flocculation of the whole powder mass.

4. Flip the trap door release hinge up to open the trap door.

5. A “positive” result is deemed if the powder flows through the hole, and the hole is visible from the top of the cylinder. A “negative” result is deemed if the hole is not visible from the top of the cylinder:

Positive Result (hole visible from above) Negative Result (hole not visible)

Release hinge Trap door (closed)

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6. For positive results, repeat the test with smaller and smaller disks until a negative result is obtained. For negative results, repeat the test with larger and larger disks until a positive result is obtained.

7. Determining Flowability:

Three positive results in a row are required to determine flowability. Repeat the test two more times on the smallest disk that produces a positive result. If a negative result is obtained, advance to the disk having the next largest diameter, and proceed testing.

Calculate and record the viscosity of the powder.

Comment on the flowability of the sample.

Compare the values obtained with this instrument with your previous determinations. Are they complimentary?

Part E. Sieve Analysis

Pick a formulation (A, B, or C) to conduct a sieve analysis. You will be particle sizing the 1 and 5 minute blends, and comparing the results.

For each of your 1- and 5-minute blends:

Select 5 sieves (+ bottom), based on your anticipated particle size distribution. The goal is to try and capture the particle size distribution such that the top and bottom tray will contain a small amount of powder compared to the entire initial batch.

Pre-weigh the selected sieves on a lab scale that can accommodate larger weights (ask your TA or Instructor to locate the kilogram scale).

Assemble the sieves with a bottom tray, and place an accurately weighed fraction of your selected formulation on the top sieves. You may use your entire formulation (150 g) provided you conduct this test last.

Shake the sieve stack for 5 minutes.

Carefully remove the sieve stack, separate the sieves, and accurately weigh each sieve, to calculate the amount of powder retained from your empty sieve weights.

Complete the following table in the Laboratory worksheet:

Sieve # (Passed / Retained)

Mean Size, d* (µm)

Weight Retained (g)

Fraction Retained, n

Cumulative Fraction Retained (cfr) (frequency distribution)

Arithmetic Weighted Size, n x d

Geometric Weighted Size, n x log d

⋮ ⋮ ⋮ ⋮ ⋮ ⋮ ⋮

n = (nd) = (nlog d) = *use arthrimetic mean size

Use the graphs probit.xls from the laboratory website to prepare a graph to determine the Mean Mass Diameter using probit analysis. Also read the background information in probit.pdf.

Determine the arithmetic mean diameter:

(1) dav = nd / n

http://phm.utoronto.ca/~ddubins/DL/probit.xls

http://phm.utoronto.ca/~ddubins/DL/probit.pdf

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where n is the fraction retained and d is the mean pore opening.

What is the arithmetic average diameter of your sample?

Also, determine the standard deviation:

(2)

ddnσ

Determine the geometric mean diameter, dgeo:

(3)

geo

dnd

log

loglog

On the same graph, plot the weight fraction retained vs. the mean particle diameter for the 1- and 5-minute blends. Indicate the mode, median, arithmetic mean particle size and geometric mean particle size for each blend. There is no graph template for this plot. Compare the particle size distributions. Did blending time impact the particle size distribution for the formulation you tested?

Questions

1. Why do we prepare plots of ( V0 – Vn ) / ( V0 – V15 ) vs log n, what does the slope indicate?

2. Why do we tap the 25 mL cylinder 15 times?

3. How do we calculate the angle of repose? Why do we measure the angle of repose in this experiment?

4. Define bulk density.

5. What is the purpose of adding magnesium stearate to a powder mixture? How would the magnesium stearate help?

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Lab

Lab 14: Pharmaceutical Granulations Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 5 students. This lab will take two lab periods to complete. Stay in the same groups for both lab periods.

What You’ll Be Doing

Part A: Preparing a standard curve for acetaminophen Part B: Pre-milling acetaminophen. Preparing granulation mixes, and performing low- and high-shear granulation on Formulation A or B. You will pair up with another group to share data for the other formulation. Part C: Dry-milling granulates, determining powder characteristics, testing powder potency Demonstration: High and Low Shear Granulation

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/probit.xls http://phm.utoronto.ca/~ddubins/DL/probit.pdf

What You’re Handing In

Retain and store your powder blends for second part of Lab 14, and for Lab 15. Do not throw away your powder blends. Individual formal lab report, due at the beginning of Lab 15 (see Guidelines for Writing Individual Laboratory Reports for details)

Introduction

Pharmaceutical granulations are used primarily for the preparation of materials for tableting and or encapsulation. The main objectives of granulation are to improve the flow properties and, in the case of tableting, the compression characteristics of the mix, and to prevent aggregation of the constituents during the tableting process. In this laboratory exercise, wet granulation will be carried out on both low shear and high shear granulators. The difference in granule properties due to process differences will be evaluated from the powder flow, particle size, and bulk density data.

References

1. Pharmaceutics: The Science of Dosage Form Design, 2nd

Ed., M.E. Aulton (Ed.), Churchill Livingstone, 2001, Chapters 25 & 26.

2. S.M. Iveson, J.D. Litster, K. Hapgood, and B.J. Ennis, Powder Technology, 117, 3-39 (2001)

3. H.J. Kristensen and T. Schaefer, Drug Dev. Ind. Pharm., 13, 803-872 (1987)

4. Handbook of Pharmaceutical Granulation Technology, D.M. Parikh (Ed.), Marcel Dekker, 1997

Background

Granulation is a process of particle size enlargement such that small particles are agglomerated (or assembled) into larger, semi-permanent aggregates in which the original particles can still be distinguished. In essence, it is a controlled aggregation process by which fine particles are agglomerated into larger ones with defined sizes thereby preventing uncontrolled aggregation that would have taken place with un-granulated fine powder during processing. The end result is improved flow properties as uncontrolled aggregation tends to disrupt powder flow.

Granulation methods can be divided into two main types: wet methods which utilize a liquid in the process, and dry methods in which no liquid is utilized.

http://phm.utoronto.ca/~ddubins/DL/probit.xls

http://phm.utoronto.ca/~ddubins/DL/probit.pdf

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160 Lab 14: Pharmaceutical Granulations

Dry Granulation

Since the dry granulation process does not involve the use of liquid, it is primarily used as a means of granulation for moisture sensitive or heat sensitive drugs. In this process, dry powder particles are brought together mechanically by compression into slugs or by roller compaction followed by milling into the desired particle size ranges.

Wet Granulation

The wet granulation process is widely used in the pharmaceutical industry. It involves the use of a granulating fluid to facilitate the agglomeration process. The granulating fluid can be water, an organic solvent (such as ethanol), or a mixture of the two. A binder can also be included in the granulating fluid to improve the integrity of the resulting granules. Before the start of the wet granulation process, a powder blend of the drug and selected excipients is first prepared through mixing in a blender to achieve a uniform distribution of the dry powder. Subsequently, the granulating fluid is introduced by pumping, pouring or atomizing while the mixed dry powder bed is agitated in a tumbling drum, fluidized bed, high shear mixer or similar devices to form the desired granules. The agitation allows the liquid to distribute evenly and to wet and bind the particles together by a combination of capillary and viscous forces. Although, water is commonly employed in wet granulation as a liquid binder, non-aqueous volatile solvents such as ethanol are often employed in wet granulation when the drug is moisture sensitive or unstable in the presence of water.

In pharmaceutical processing, wet granulation converts fine powder with wide size distribution to larger granules with a narrower size distribution. The extent of such particle size control is dependent on the granulation equipment and properties of the granulating solvent, as well as properties of the feed material, especially its particle size distribution. The following stages may be observed during wet granulation, the desired endpoint typically being the capillary stage:

Wet granulation proceeds by various mechanisms of agglomerate formation and growth. More widely accepted view is that the process can be represented by a combination of the following three rate processes:

(1) Wetting and nucleation: A liquid binder is introduced into the powder bed and is distributed through the bed via mixing agitation to produce a distribution of nuclei granules.

(2) Consolidation and growth: The collisions between granules or between granules and the equipment during mixing agitation result in granule compaction and growth.

(3) Attrition and breakage: Wet granules break due to impact, wear or compaction in the granulating equipment.

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The formed wet granules can be dried in conventional pharmaceutical drying equipment such as fluid bed or tray driers. More permanent bonds are formed by the drying process through either solid bridge formation due to binder hardening and/or recrystallization of soluble component as well as granule densification due to other forces such as hydrogen bonding and mechanical interlocking.

Granulation can be carried out in fluid bed, low shear and high shear granulators. It is usually difficult for a given formulation to be successfully processed in each piece of equipment. However, it is recognized that the processing time required, the bulk density, and particle size obtained from these granulators will be different. In general, the bulk density of granules produced form low shear granulator (such as a planetary mixer) will be intermediate between those from a fluid bed and a high shear granulator, with the latter having the highest bulk density. Similar conclusion can be drawn on the granule morphology as lower shear granulators produce more porous granules than do high shear granulators. Additionally, the granule yield (reduced large and small fractions) is generally the highest from the fluid bed process, followed by the high shear, and low shear granulators.

Experiment Protocol

Chemicals Supplies Special Equipment Acetaminophen Lactose Microcrystalline cellulose (Avicel PH101) Polyvinlypyrrolidone (Povidone)

Aluminum trays (reusable – do not discard)

Planetary mixer (Erweka AR 402 with mixer attachment) High shear granulator (4M8 Granulator; Pro-C-epT) Fluid bed dryer (4M8 Fluidbed; Pro-C-epT) or drying oven Powder Mill (Quadro Comil or Erweka AR 402 with rotary mill attachment)

The following solutions are prepared or provided by the TA:

Not applicable.

NOTE: This lab will take two lab periods to complete.

Lab Period 1: You will be preparing your standard curve, granulation mixes, and you will be granulating them using low- and high-shear granulators.

Lab Period 2: You will be dry milling your granulated powder blends, testing powder characteristics of each blend (flowability, tapped density, etc.), sieving each blend to determine particle size distribution, and testing the potency of the sieved fractions.

DO NOT throw away your powder blends once testing is complete. You will be using your blends to tablet during Lab 15.

Part A. Preparing a Standard Curve for Acetaminophen

Prepare 100 mL of your own 1.00 mg/mL stock solution of acetaminophen, in de-ionized water.

You may need to sonicate to get the acetaminophen to dissolve.

Using your stock solution, prepare 50 mL of each of the following acetaminophen

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162 Lab 14: Pharmaceutical Granulations

concentrations in de-ionized water, in triplicate:

5, 25, 50, 75, and 100 μg/mL.

Show details of your dilution calculations.

The ultraviolet spectrum of acetaminophen in neutral water shows a major band at 242 nm and a minor band around 280 nm. Addition of acid (5 drops of 50% HCl) does not affect either absorption band. In neutral methanol the wavelength of maximum absorption appears at 243 nm and its position is not affected by acid. In ethanol the UV wavelength of maximum absorption is 250 nm.

Perform a wavelength scan of your blank and most concentrated standard solution, and select a wavelength that provides a useful difference in absorption (the curve should span 0 to 1.5 or 2 OD). Then at this wavelength, zero the spectrophotometer using the blank solution, then measure the absorbance of your standard samples. Construct a calibration curve using the laboratory computers. Fit a line of best fit using Excel, and force the line to cross the origin. Report the r2 and equation of the line using Excel. Print out the calibration curve for inclusion in your report.

Use plastic UV cuvettes only, to be provided by your TA.

Remember (and take note of) the correct orientation for the plastic UV cuvettes.

Part B. Preparing the Powder Blends and Granulating

Formulation A Formulation B

Ingredients weight (g) % w/w wt (g) % w/w

Acetaminophen 90.6 30.2% 90.6 30.2%

Lactose 118.2 39.4% 75.6 25.2%

Avicel PH101 75.6 25.2% 118.2 39.4%

Polyvinylpyrrolidone 15.6 5.2% 15.6 5.2%

Total: 300.0 g 100.0% 300.0 g 100.0%

Pair up with another group of 5 students, so that each group is doing a high- and low- shear granulation run on one single formulation.

Weigh out two sets of ingredients for each granule composition listed above for the low and high shear granulation experiments.

Premix acetaminophen, lactose, Avicel PH101, and polyvinylpyrrolidone on either a low shear laboratory mixer at ~100 rpm (e.g. Erweka planetary mixer) or a high shear granulator at ~500 rpm (e.g. 4M8 Granulator; Pro-C-epT) for 3 minutes.

Start the granulation process by increasing the blade speed to that of the normal granulation process (~200 to 300 rpm for the low shear and ~1000 rpm for the high shear). Introduce water as the granulating fluid via pumping (~10 mL/min) for the high shear process or intermittent pouring or spraying for the low shear process (up to 175 mL max.). Refer to the operating manuals for correct speed settings and the determination of granulation end point from torque measurement for the high shear process.

Stop the granulator and sweep the walls of the granulator. The wet mass should pack well and break apart easily at this point.

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Transfer granulate to an aluminum tray and break up any larger clods of granulate with a hard rubber spatula.

Dry in either the fluid bed dryer, laboratory oven, or stability chamber. Be sure to properly label your granulate with the formulation name, granulate method, lab members, and the date.

Part C. Milling and Sizing

Using the Quadro Comil or Erweka AR 402 with rotary mill attachment, mill the granulation mixes to a mesh size equivalent to a #12 mesh screen.

Test the resulting dried granules from the two processes for bulk density, angle of repose, flowability, and sieve analysis (to determine particle size distribution). Conduct the sieve analysis last.

After sieving, determine the drug potency of the powder for each sieve fraction for each blend:

Accurately weigh 0.1 g of the powder into a 500 mL volumetric flask.

Fill the flask half way with de-ionized water, and agitate for 5 minutes.

Dilute to the mark with de-ionized water and agitate. NOTE: As there are insoluble excipients in the granulate, not all of the powder will dissolve.

Measure the absorbance of the resultant solution by filtering with a 0.45 µm filter. Use the same wavelength you selected for your calibration curve in the first part of the lab.

If the absorbance of your assayed granulate is above the highest absorbance in your standard curve, dilute the sample accordingly to obtain a convenient reading, within in the range of your standards.

Calculate and report the expected %w/w of drug, the actual %w/w of drug, and the % potency in each blend. Show your work in the final report.

Each test is non-destructive. At the end of sieve analysis, recombine the sieve fractions and retain the product.

Appropriately label and store your granulations in the laboratory stability chamber. You may heat-seal the bag.

NOTE: Do not throw out your granulations, you will be using them for tableting in Lab 15. Wash and clean the aluminum trays, and return to your instructor or TA.

Results

Calculate the corresponding Consolidation Indices.

Tabulate the granule properties for each of the high and low shear granulation processes.

Questions

1. Looking at your results, what are some of the major differences in granule properties between the high and low shear granulation processes and why?

2. Is there a difference in the amount of granulating fluid required for these two different granulating processes? What are some the reasons for this observation?

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3. Can you think of a better way to determine the end point of drying for the granules?

4. Do you detect any difference in content uniformity (i.e. acetaminophen concentration) in different sieve fractions? In what way this might be related to the low and high shear processes?

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Lab

Lab 15: Tableting, Capsuling, and Dissolution Testing

Preparing for the Lab

Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:

Capsule Making (Cap-M-Quik Method) (http://phm.utoronto.ca/~ddubins/DL/Capsule_Making.wmv)

Capsule Making (Hand-Filling Technique) (http://pharmlabs.unc.edu/video1.php?pack_capsule.flv)

Group Allocation You will be working in groups of 5 students, and combining data with another group for the other formulation (A or B).

What You’ll Be Doing

Lab Period 1 Part A: Prepare tablet powder mixes from Lab 14 granulates, and press tablets -Determine the hardness, weight uniformity, thickness, friability, and disintegration time of the different tablet formulations Part B: Begin stability assays (1 at room temperature, incubate 10 units of each formulation at 40 and 60 °C Part C: Coat 100 placebo tablets with your own tablet coating formulation Demonstration: Tablet hardness, friability, tablet coating

Lab Period 2 Part C: Perform a tablet dissolution test on your formulation (A or B) Part D: Prepare a capsule formulation from one of your powder blends (high or low shear) using a capsule machine. Part E: Determine content uniformity of tablets and capsules (Continue stability assays for Part B) Demonstration: Dissolution, Capsule Hand-Filling, Capsule Machine

Lab Period 3 Part F: Perform a dissolution test on your compounded capsules (Continue stability assays for Part B) Part G: Perfoming TLC analysis of tablet formulations (room temp, 40°C, 60°C)

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/Capsule_Filling.xls http://phm.utoronto.ca/~ddubins/DL/shelflife.xls

What You’re Handing In Individual formal lab report, due at the beginning of Lab 16 (see Guidelines for Writing Individual Laboratory Reports for details)

Introduction

The compressed tablet is by far the most popular among all pharmaceutical dosage forms. Tablets are prepared by compacting a powder mixture of drug and excipients in a die under high compressive force. In addition to the drug, the powder mixture also contains diluents, binders, disintegrants. lubricants, glidants etc. For large scale production, the powder mixture needs to exhibit desired properties regarding hom*ogeneity, flowability, and compactibility. The resulting tablets also have to meet certain product quality standards such as uniformity of content, disintegration, and dissolution. In the first part of this laboratory exercise, granules obtained from the previous wet granulation experiment will be further converted into tablets. Through the evaluation of tablet properties, the effects of formulation and granulation process will be identified.

http://phm.utoronto.ca/~ddubins/DL/Capsule_Making.wmv

http://pharmlabs.unc.edu/video1.php?pack_capsule.flv

http://phm.utoronto.ca/~ddubins/DL/Capsule_Filling.xls

http://phm.utoronto.ca/~ddubins/DL/shelflife.xls

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166 Lab 15: Tableting, Capsuling, and Dissolution Testing

In the second and third parts of this laboratory, you will be making capsules using one of your formulation blends. Weight uniformity, content uniformity, and dissolution will be repeated, allowing you to compare results across dosage forms.

Stability of tablets will also be investigated. We will be storing tablets at three different temperatures to assess the shelf life at room temperature.

References

1. Pharmaceutics: The Science of Dosage Form Design, 2nd Ed., M.E. Aulton (Ed.), Churchill Livingstone, 2001, Chapter 27.

2. Modern Pharmaceutics, 2nd Ed., G.S. Banker and C.T. Rhodes (Eds.), Marcel Dekker, 1990, Chapter 10.

3. Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., L.V. Allen, H.C. Ansel, and N.G. Popovich, Lippincott Williams & Wilkins, 2004, Chapters 8 & 9.

4. The Theory and Practice of Industrial Pharmacy, 3rd Ed., L. Lachman, H.A. Lieberman, and J.L. Kanig, Lea & Febiger, 1986, Chapter 11.

5. http://www.dionex.com/en-us/webdocs/67573-LPN-2048-01-Uniformity-note.pdf

6. http://www.39hg.com/jp14e/14data/Tablet_Friability_Test.pdf

7. https://infohost.nmt.edu/~jaltig/TLC.pdf

Background

Tablets are manufactured by compressing a powder mixture into a coherent compact. To achieve reproducible dosage unit, it is important that each component of the powder mixture be uniformly dispersed within the blend and any tendency to segregate be minimized. Furthermore, the processing conditions require that the powder mixture have certain minimum flow properties and be cohesive enough when compressed. In general, to reduce segregation tendencies, the particle size distribution, shape and density of all the component ingredients in the powder mixture should be similar. The flow properties can be enhanced by having regular shaped smooth particles with a narrow size distribution. In general, the powder mixture may consist of either primary particles or aggregated primary particles (i.e. granules). When it is compressed, bonds can be established between the particles or granules, thus conferring mechanical strength to the compact.

The properties of the tablet such as mechanical strength, disintegration time and drug release profile can be affected by both the component materials and the manufacturing process. Excipients such as diluents, binders, disintegrants, lubricants and glidants are generally incorporated in a formulation in order to facilitate the manufacturing process as well as to ensure desired properties for the resulting tablets. For example, tablets should be strong enough to withstand handling during manufacturing and usage, but also disintegrate and release the drug in a predictable and reproducible manner. It is therefore very important to select appropriate excipients and manufacturing process when developing a new tablet formulation.

In addition to mechanical strength, disintegration time and drug release profile, the tablet formulation also has to exhibit sufficient stability to achieve a reasonable product shelf-life. Therefore the selection of suitable excipients becomes a critical step prior to the initiation of formulation development. This is usually achieved by conducting an excipient compatibility study wherein quantitative mixtures containing the drug substance and excipient(s) are

http://www.dionex.com/en-us/webdocs/67573-LPN-2048-01-Uniformity-note.pdf

http://www.39hg.com/jp14e/14data/Tablet_Friability_Test.pdf

https://infohost.nmt.edu/~jaltig/TLC.pdf

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Lab

subjected to accelerated stability conditions (elevated temperature and humidity). The excipients which exhibit minimum drug degradation are usually selected for the subsequent formulation studies.

Excipients

Tablet formulations normally consist of the following four categories of excipients. These normally serve very specific purposes with defined functions, however some of the excipients are multifunctional capable of serving several roles.

Tablet Excipient Function Examples

Diluent Used as a bulking agent (sometimes called filler).

microcrystalline cellulose, lactose, dicalcium phosphate, mannitol, starch

Binder Added to facilitate the granulation process and produce a stronger granule.

polyvinyl pyrrolidone (PVP), microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), starch, gelatin

Disintegrant

For immediate-release tablets which are intended to disintegrate upon entering the stomach, a disintegrant is added to overcome the cohesive strength introduced by the compression and by any binder present. Most disintegrants work either by enhancing the action of capillary forces to produce a rapid uptake of aqueous phase or by swelling in contact with water.

To facilitate the disintegration of the tablet including its component granules, it is common to add a portion of the disintegrant prior to granulation (intragranular disintegrant) and the remaining portion after granulation (extragranular disintegrant).

starch, croscarmellose sodium, crospovidone, sodium starch glycolate, microcrystalline cellulose, starch, Act-D-Sol

Lubricant Added to reduce friction at the tablet die wall and adhesion to punch faces.

Usually mixed with the granulation as the last step prior to tableting.

magnesium stearate, calcium stearate, stearic acid

Glidant Used to promote powder flow by reducing interparticle friction and cohesion.

Used in combination with lubricants as they have no ability to reduce die wall friction.

colloidal silicon dioxide, talc

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168 Lab 15: Tableting, Capsuling, and Dissolution Testing

Tableting Methods

There are three methods of making compressed tablets: direct compression, wet granulation, and dry granulation:

Direct Compression

In this method, directly compressible filler (serves as filler and binder) is blended with the drug, a disintegrating agent, and a lubricant. The powder mixture is then compressed directly into tablets. Such free flowing directly compressible fillers are necessary for direct compression. These include anhydrous lactose, unmilled dicalcium phosphate dehydrate, microcrystalline cellulose, and spray-dried lactose. Although this method is simple, it is limited by the fact that large differences in particle size and bulk density between the drug and the excipients can lead to segregation of powder components during handling or due to vibration on the machine. This can severely affect the resulting drug content uniformity of the tablets. Also, direct compression is usually only suitable for small dose tablet products because the amount of drug which can be added without affecting the compression properties of the filler is rather limited.

Granulation Processes

The operating principles of wet and dry granulation processes have been covered in the previous laboratory. These are often used for large dose poorly compressible drugs which are impractical for direct compression. A majority of pharmaceutical tablet products are produced by one of these granulation processes.

Tablet Properties

Hardness

Hardness may be defined as the resistance of a solid to attrition or breakage. It has been used in characterizing tablet as it provides a simple measure of the effectiveness of the compression process. Both the Strong Cobb and the Stokes hardness testers are used in the pharmaceutical industry to measure the force required to break the tablet. Hardness values obtained from these two instruments for a given set of tablets are not equivalent but they can be correlated. There appears to be a linear relationship between the tablet hardness and the logarithm of the compression force. One would anticipate that as a tablet becomes more dense, its hardness would increase. Other relationships among hardness, compression force and pack density are empirical and can be obtained experimentally.

The desirable tablet hardness depends on the formulation intended, and tablet weight. Small ones have low hardness (~5 kP for 100 mg tab.) and large ones have high hardness (15-20 kP for ~1000 mg tab). Tablet hardness should be high enough to keep tablet integrity in the further processing, such as coating and packaging, and in product transportation. The following table lists approximate values for different formulations, and serves only as an approximate guideline:

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Lab

Formulation N (Newton)

kg (kilogram) or kp (kilopond)

Sc (Strong-Cobb)

lb or lbf (pounds)

Fast-Disintegrating (e.g. sublingual) Tablet

20-70 2-7 2.9-10.0 4.5-15.7

Chewable Tablet 30 3 4.3 6.7

Small compressed tablet (100 mg), uncoated

50 5 7.1 11.2

Large compressed tablet (1000 mg), uncoated

150-200 15-20 21.4-28.6 33.7-45.0

Small compressed tablet, coated

105 10.7 15.0 23.6

Unit conversion factor 1 0.101971 0.142812 0.224737

Friability

Tablet friability is related to hardness. A friability tester measures the ability of the tablet to resist abrasion which is important to packaging, shipping and handling. Friability tests the strength of tablets against wear.

For tablets weighing <= 650 mg each, the minimum number of tablets to reach 6.5 grams are used for the test. For tablets > 650 mg each, 10 tablets are used in the test. The total initial weight of the tablets is determined (W0).

The tablets are loaded into a friability tester, where they are subjected to controlled falls. The test involves rotating the drum exactly 100 times, over 4 minutes (25 rpm). The intact tablets are then removed and weighed again (W).

The measure of abrasion resistance or % Friability is expressed as a percentage loss in tablet weight:

1100%W

WFriability

A tablet weight loss of 1% is the USP-defined upper limit for friability; however, a target of less than 0.3% is desirable for tablet processing and resilience.

Disintegration Time

When a powdered drug is granulated and compressed into a tablet, the effective surface area of the medicinal compound is decreased. An immediate-release tablet must break up or disintegrate in the gastrointestinal fluids into granules, which then must disintegrate into primary particles. The drug needs to dissolve from the primary particles before the molecules or

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ions of the medicinal compound can be absorbed by the gastrointestinal mucosa. Improperly formulated and improperly processed compressed tablets may retard drug release with a decrease in bioavailability. If a tablet does not disintegrate, the surface available for dissolution is restricted only to the surface area of the tablet.

Disintegration time specification is a useful tool for quality control, but disintegration of a tablet does not imply that the drug has dissolved. A tablet may pass a disintegration test and yet the drug may be biologically unavailable. The disintegration time is a rapid indicator of the effect caused by changes in formulation parameters or stability of the final dosage form.

In the lab, we use Nesler Tubes to determine the disintegration time. It is an empirical measure as the end-point is the visual confirmation of the largest piece of tablet breaking into smaller pieces. In industry, a more robust measure is used. A basket travels up and down in a 37 °C water bath. The basket holds six vertical glass tubes. At the bottom of each glass tube is a 10-mesh screen. A tablet is placed in each tube to start the test. The disintegration time is expressed as the time it takes for the last piece of tablet to fall through the 10-mesh screen.

Disintegration Tester Nessler Tubes

Weight Uniformity

Variation in processing and powder flow can lead to a variation in tablet weight. Assessing weight uniformity provides a measure of the variability in the tabletting process. The test is performed as follows:

A large %RSD is indicative of processing problems (e.g. >5%).

Calculate the Relative Standard

Deviation

SDRSD %

Calculate the Standard Deviation

xxSD i

Weigh each tablet separately

10321 ,...,,, xxxx

Weigh 10 tablets together

weightx

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Dissolution Apparatus- USP/NF Apparatus 1 (basket)

Content Uniformity

A content uniformity test evaluates if the strength of the drug is within acceptable limits of the label claim. It is determined either by weight variation or by assay of individual units. Acceptance limits vary depending on formulation, and the label claim on the product monograph of the drug.5 The following table provides acceptance criteria for different dosage forms.

Dissolution

Although disintegration time of a tablet may influence the rate of drug release to the body, the dissolution rate of the drug from the primary particle is fundamentally important because dissolution of the drug is essential for subsequent absorption to occur. Dissolution testing is a critical test for measuring the in vitro performance of a drug product. It is a quality control tool and an effective aid to formulation development. Dissolution testing can detect changes on stability, and is used to establish an in-vitro and in-vivo correlation for modified release products. When release at a single pH is desired (e.g. for a quick-release formulation), commonly used dissolution methods include the basket method (USP/NF Apparatus 1) and the paddle method

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(USP/NF Apparatus 2). For controlled-release drugs that may release slowly over time through the entire GI tract, more complicated models are required, where the pH during drug dissolution can be varied (USP/NF Apparatus 4). Only the paddle method will be employed in this laboratory exercise.

The sample is withdrawn midway through the dissolution vial. Varying the sample withdrawal location can very strongly influence the dissolution profile, so care must be taken when manually withdrawing samples to do so in the same location. Measurements are taken spectrophotometrically, and a standard curve is used to convert absorbance at a given wavelength into concentration. Concentration is then converted to mass by multiplying the known volume remaining of the dissolution vessel. This mass is then converted to % released by dividing by label claim of the dosage form. Typically this test is conducted 6 times for a given formulation. In this laboratory due to resource limitations, you will only need to conduct one dissolution test per formulation.

Typical Dissolution Test Parameters:

Volume: 500, 900, or 1000 mL

Release Medium:

To Simulate Intestinal Fluid (SIF): pH 6.8

Pancreatin may be added

To Simulate Gastric Fluid (SGF): pH 1.2

Pepsin may be added

SDS may be added (must be justified)

Temperature: For all IR dosage forms, 37±0.5°C

Agitation Speed: Basket: 50-100 rpm, Paddle: 50—75 rpm

Sampling Interval: typically 15 minute intervals, 5 minute intervals for quick dissolving.

Last time point: 60 min.

Stability Testing

Stability testing methods will depend on the formulation (e.g. tablet, capsule, suspension, etc.), anticipated storage conditions, and type of drug. Simply speaking, a group of drug products are

Uses of Dissolution Testing in Industry Single-point specifications:

• As a routine quality control test. (For highly soluble and rapidly dissolving drug products.) • “Q”=NLT 75%/30 minutes, means that a “pass” is that not less than 75% of the dose will be

released into the dissolution medium by 30 minutes. • Different Q values for different drugs and formulations (USP defined).

Two-point specifications: 1. For characterizing the quality of the drug product. 2. As a routine quality control test for certain types of drug products (e.g., slow dissolving or poorly water soluble drug product like carbamazepine). Dissolution profile comparison: (Different methods used: Similarity factors, model independent, model dependent) 1. For accepting product sameness under SUPAC-related changes. 2. To waive bioequivalence requirements for lower strengths of a dosage form. 3. To support waivers for other bioequivalence requirements. IV/IVC: In Vitro/In Vivo Correlations -Only really works if PK is absorption-limited

Paddle

Basket

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stored in different controlled environments for a certain period of time. Typical storage conditions are:

Source: Guidance for Industry: Stability Testing of Existing Drug Substances and Products, Health Products and Food Branch Document, Health Canada 2006

At planned time points, samples are retrieved for testing. Timepoints such as 1M(month), 2M, 3M, 6M, 9M, 12M, 18M and 24M are typically used.

The tests of the products at each time point must include physical appearance, assay (drug content), relative substances (impurities) and other product specific properties such as hardness, disintegration time for tablets, and pH/flow properties for liquids.

The stability test will determine the resilience and shelf life of the formulation, and identify the degradation products.

Accelerated stability tests involve incubating the formulation at a higher temperature to increase degradative processes that lead to drug decomposition, such as chemical incompatibilities with excipients, or drug hydrolysis. Briefly, k, the first-order rate constant for decomposition, can be estimated at different temperatures (in this lab, 40°C and 60 °C). The k values for decomposition over time at a given temperature can be calculated by plotting ln(C) vs. time, using the drug content determined from the assay at room temperature for time=0. The slope of the graph at that temperature will be equal to –k.

As temperature rises, decomposition of the drug occurs more rapidly. As described in Lab 8, the temperature dependence of k can be described using the Arrhenius equation:

Aek

Where A is the pre-exponential frequency factor, Ea is the activation energy, and R is the universal gas constant. An Arrhenius plot can be constructed for ln(k) vs. 1/T. The slope of this graph is –Ea/R, and the intercept is ln(A). Once the Arrhenius constants are determined from the Arrhenius plot, the decomposition rate may be estimated at room temperature using the above relationship. Then, the shelf life may be calculated as the time to 10% degradation of the drug (or 90% of the drug remaining, t90%) using this k value:

𝑡90% =−ln (0.9)

𝑘𝑇=21°𝐶

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174 Lab 15: Tableting, Capsuling, and Dissolution Testing

Capsules

Capsules are generally made of gelatin, which is formed via the hydrolysis of collagen. Unlike tablets, where the tablet weight is a process parameter of the tableting press, capsules are filled by volume. Capsules are simpler to prepare than tablets as hardness and friability are no longer concerns; consequently, capsules are very commonly used in early clinical trials. Capsules are manufactured in a variety of standard sizes. A smaller size number denotes a larger volume:

Size

Outer Diameter

(mm)

Height or

Locked Length (mm)

Actual Volume

(mL)

Vet

erin

ary

Su07 23.4 88.5 28

7 23.4 78.0 24

10 23.4 64.0 18

11 20.9 47.5 10

12el 15.5 57.0 7.5

12 15.3 40.5 5

13 15.3 30.0 3.2

man

000 10.0 26.1 1.37

00 8.5 23.3 0.95

0 7.7 21.7 0.68

1 6.9 19.4 0.50

2 6.4 18.0 0.37

3 5.8 15.9 0.30

4 5.3 14.3 0.21

5 4.9 11.1 0.13

Knowledge of fill weights and powder density is required to properly formulate a capsule with the proper potency. Tapped powder density and fill weights can be measured in the lab. Tables are also available for common excipients:

Capsule Size

Lactose Avicel PH-105

Starch Methocel E4M

Kaolin Methocel K100M

Calcium Carbonate

4 190 110 180 100 165 100 220

3 225 130 205 150 250 150 275

2 340 160 285 188 375 185 350

1 450 230 375 250 540 250 460

0 550 320 510 350 600 350 640

00 775 450 700 475 765 475 925

000 1260 725 1180 620 1700 620 1450

If the powder density of an excipient is known, it can simply be converted into a capsule fill weight by multiplying the density by the known fill volume for that capsule size in the capsule

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175 Lab 15: Tableting, Capsuling, and Dissolution Testing

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size chart. Or, 5 empty capsules may be tarred, then filled with the excipient and weighed again, and the average fill weight calculated directly.

Experiment Protocol

Lab Period Chemicals Supplies Special Equipment Part 1 Acetaminophen granules

(from Lab 14) Acetaminophen USP Corn Starch Magnesium Stearate Polyvinylpyrrolidone Avicel PH101 Lactose USP Methocel E15 Premium LV Sorbic Acid 95% Ethanol PEG-400 (plasticizer) PEG-8000 (plasticizer) Titanium dioxide (opaquant) Food colouring Flavourant

Size 0 Capsules Plastic UV cuvettes Scintillation Vials (for stability testing) Hand blenders Plastic UV cuvettes Resealable plastic powder bags 10 mL syringe 0.45 µm syringe filters

Planetary Mixer Rotary Tablet Press Hardness Tester Friability Tester Glass Slab Nessler Tube (for tablet disintegration) Hand blender UV-Vis Spectrophotometer Erweka Tablet Coating Pan Magnets (for coating pan) Heat gun

Part 2 Lactose Plastic UV cuvettes Size 0 capsules

USP Dissolution Apparatus UV-Vis Spectrophotometer Capsule Filling Machines USP Dissolution Apparatus UV Visualization Chamber

Part 3 Acetaminophen USP Ethyl acetate Acetic Acid Methanol

Plastic UV cuvettes 5 µL glass capillary micropipettes pencil ruler TLC plate Silicon grease Filter paper

UV-Vis Spectrophotometer TLC chamber 10 mL volumetric flasks

The following solutions are prepared or provided by the TA:

Lab Period 1:

Prepare Hydrochloric Acid (0.1 N)

Turn on lab ovens for stability trials (set to 40 °C and 60 °C)

Turn on UV-Vis Spectrophotometers

You may use your standard curve from Lab 14 for this laboratory for your drug assay and dissolution runs. Please ensure that you use the same spectrophotometer, and that you use the same wavelength as the calibration curve.

Lab Periods 2 and 3:

Prepare Hydrochloric Acid (0.1 N)

Set up and warm up USP Dissolution Apparatus

Turn on UV-Vis Spectrophotometers

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176 Lab 15: Tableting, Capsuling, and Dissolution Testing

Lab Period 3:

Prepare 0.05% acetaminophen reference solution

Prepare ethyl acetate:acetic acid TLC solvent (95:5)

Locate and set up TLC supplies

Lab Period 1:

NOTE: Divide each formulation approximately into 2 separate bags. Label 1 bag “For Tableting” and the other bag “For Capsuling”. Store the Capsuling bag in your locker for Period 2.

Part A. Tableting

For only the formulations intended for tableting, weigh out the required amount of starch and magnesium stearate as listed in the following table for the two tablet formulations using the low and high shear granules you made in Lab 14.

Tablet Compositions Formulation A Formulation B

Ingredients weight (g) % w/w weight (g) % w/w

Granulation (high or low shear): 100.00 94.50% 100.00 94.50%

• Acetaminophen (in granulate) 30.23 28.57% 30.23 28.57%

• Lactose (in granulate) 39.31 37.15% 25.23 23.84%

• Avicel PH101 (in granulate) 25.23 23.84% 39.31 37.15%

• Polyvinylpyrrolidone (in granulate) 5.23 4.94% 5.23 4.94%

Corn starch 5.29 5.00% 5.29 5.00%

Magnesium stearate 0.529 0.50% 0.529 0.50%

Total (g): 105.82 100% 105.82 100%

Add the post-granulation excipients (corn starch and magnesium stearate) to the acetaminophen granules obtained from the previous experiment, and shake the formulation in a bag for 10 minutes by inverting the bag continuously and repeatedly.

Using the Sanchez single punch press or the Globe Pharma rotary press, compress 100 mg acetaminophen tablets (350 mg tablets weight) as assisted by your instructor.

NOTE: 350 mg * 28.6 % w/w acetaminophen = 100 mg acetaminophen

Take 10 tablets from each formulation and determine the tablet hardness (mean ± %RSD) using the Hardness Tester and record in tabular form. The tablet should be placed FLAT in the hardness tester. The piston squeezes the tablet on the edges.

Determine the weight uniformity of 10 tablets by weighing each tablet separately. Report the weight of each tablet in tabular form, and report the mean ± %RSD.

Weigh the appropriate number of tablets to obtain the starting weight (W0) and place

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the tablets in the friabilator. After running the friabilator for 4 minutes (4 min x 25 rpm = 100 revolutions), weigh the intact tablets to obtain the final weight. Report the %friability of each tablet formulation.

Determine the disintegration time by placing one tablet in a Nessler tube. Fill the tube with about 50 mL of 0.1 N HCl and invert the tube repeatedly until the tablet disintegrates. This is defined as the point at which the largest visible tablet piece reduces to a particle size not visibly discernible from the other particles present (in other words, you can no longer see the remaining tablet). Record the time it takes for the tablet to disintegrate. If disintegration takes longer than 1 hour, you may report >1hr for the disintegration time.

NOTE: The particles do not need to dissolve.

Part B. Stability (Shelf Life)

For the purpose of this laboratory, 30 tablets will be required. Set aside 3 properly labeled prescription vials containing 10 tablets each. The vials should be labeled with the Product name, Strength, Group name, Storage condition, and Date. One vial is kept in your locker (at room temperature), the second vial is placed in a 40 °C oven, and the third vial is placed in a 60 °C oven, all for stability analysis. Assay procedures are described below. The first assay is conducted at room temperature during the first lab period. The assay is repeated during each lab period at accelerated temperatures, for each formulation. The following information will be collected:

Lab Period 1 Lab Period 2 Lab Period 3

Content @ Room Temp (1 tablet) - -

- Content @ 40°C (1 tablet) Content @ 40°C (1 tablet)

- Content @ 60°C (1 tablet) Content @ 60°C (1 tablet)

Drug Assay: How much drug is really in my dosage form?

(Used for content uniformity and stability testing)

The purpose of drug assay is to determine the acetaminophen content of a solid formulation by using colourimetry or UV absorption. In the UV analysis, the drug absorbs ultra violet light at a wavelength of approximately 242-245 nm. This is measured in the UV range of the spectrophotometer. In the colourimetric analysis, the drug is conjugated with other chemicals to yield an orange solution, the intensity of which is measured by the spectrophotometer. The absorbance value at 430 nm should vary linearly with the concentration of acetaminophen in the solution and is measured in the visible range of the spectrophotometer.

UV determination of Acetaminophen

Obtain a pair of plastic UV cuvettes from your TA or instructor.

Standard Curve Preparation

For this method, you may use the same standard curve you prepared in Lab 14.

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178 Lab 15: Tableting, Capsuling, and Dissolution Testing

Assay Preparation

Crush and transfer one dosage form, equivalent to about 100 mg of acetaminophen, to a 100 mL volumetric flask.

Add 60 mL of water, insert the stopper, and shake vigorously by mechanical means for 20 minutes.

Dilute with water to volume and mix.

Using a 10 mL syringe equipped with a 0.45 µm syringe filter*, transfer 10.0 mL of this solution to a 100 mL volumetric flask, dilute with water to volume, and mix.

NOTE: You may also perform this step by filtering 5 mL and diluting in a 50 mL volumetric flask.

*See the Appendix of this manual for instructions on how to use a syringe filter.

Procedure

Measure the absorbance of your diluted assay solution at the same UV wavelength as your standard curve. Calculate the concentration of the sample using your standard curve. The percent label claim can be calculated using:

% 𝐿𝐶 = 𝐶𝑎𝑠𝑠𝑎𝑦 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 ×𝑉𝑎𝑠𝑠𝑎𝑦

𝐿𝐶× 100%

Where Cassay is the concentration of the assay in mg/mL determined using the standard curve, Dilution Factor is the factor the original assay solution was diluted by (in this case 10X), Vassay is the original volume of the assay solution (100 mL), and LC is the label claim of the amount of acetaminophen in mg in one dosage form (100 mg).

Demonstration: Tablet Coating

Coating Pan Formulation (makes 500 mL)

You will be coating a batch of placebo tablets (Avicel PH102) using the following protocol:

General Formula for Spray Gun Coating Formulation

Material Name Quantity

Methocel E15 Premium LV 15.00 g

Sorbic Acid 0.25 g

95% Ethanol 125.0 mL

Deionozed Water 125.0 mL

PEG-400 (plasticizer) 5.00 g

PEG-8000 (plasticizer) 5.00 g

Titanium dioxide (opaquant) 2.50 g

Food colouring As desired

Flavourant As desired

Formulating the Coating (performed by Instructor or TA)

1. In a 600 mL beaker, heat 125 mL of water to 65-70 C.

2. Add the PEG-8000 to the hot water and dissolve.

3. While gently agitating, slowly sprinkle the Methocel E15 Premium onto the surface of

the hot water solution. Avoid making bubbles.

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4. When the cellulose has been dispersed, add the PEG-400. Continue stirring until

dispersion is hom*ogenous, although solution of cellulose may not be complete.

5. Add the titanium dioxide.

6. In a 10 mL graduated cylinder, dissolve sorbic acid in 10 mL of ethanol, and ensure the

solution is complete.

7. Add 115 mL of ethanol to the step 4 solution, mix well, and add the sorbic acid solution

from step 6.

8. Add colourant and flavourant of choice.

9. Hand blend the coating solution until hom*ogenous (1-2 minutes). Ensure the hand

blender head does not rise above the liquid level in order to avoid bubble formation.

Using the Erweka Coating Pan Attachment

10. Ensure the coating pan and spray gun are clean and dry.

11. With the help of a TA or instructor, load ~250 mL of coating solution into the spray gun.

12. Load 400-500 tablets into the coating pan.

13. Attach the coating pan to the Erweka AR 402 base. Optional: add magnets to inside of

the pan to help agitate while pan is rotating.

14. Turn on pan speed to 250 rpm.

15. One person turns on the heat gun and sets the temperature to 750 (“High” setting). Aim

the heat gun at the tablets and preheat them for 2 minutes.

16. Another person holds the spray gun and in very short bursts with ample heating in

between, slowly sprays the tablets.

Note: Coating using the coating pan is a balance between heating and dosing. If the coating

material is applied too quickly, the tablets will stick together. The strategy is to have the heat

gun dry the coating material very quickly after it is applied, so that a big clump of fused

tablets is avoided.

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180 Lab 15: Tableting, Capsuling, and Dissolution Testing

Lab Period 2:

Demonstrations: Dissolution, Capsule Hand-Filling, Capsule Machine

Part C. Tablet Dissolution

Add 900 mL of 0.1 N HCl dissolution medium to the dissolution vessel (prepare additional 0.1 N HCl if required) and allow it to equilibrate at 37 °C. Although six (6) dissolution vessels are generally used for each sample, in this laboratory one vessel for each sample will be sufficient so as to allow all groups access to the equipment. Introduce a tablet sample to the dissolution vessel. Immediately start the rotation of the paddles at 100 rpm as time zero. Remove aliquot samples of the dissolution medium at several time intervals (5, 10, 15, 20, 30, 40, 50, 60 minutes) for analysis. Filter the samples using a 0.45 μm syringe filter into a plastic UV cuvette for reading if necessary.

The spectrophotometer should be set to the wavelength you selected for your standard curve in Lab 14. Return the samples to the dissolution vessel after each reading in order to maintain the same dissolution volume.

From the cumulative amount of drug released, calculate the percentage of labeled amount of drug dissolved and plot it as a function of time to obtain the dissolution profile for each formulation and process. These can be plotted on a single set of axes to facilitate comparison.

Stability (continued from Lab Period 1)

Perform the drug assay on one tablet of each formulation, at each temperature (room temperature, 40 °C, and 60 °C.

Part D. Formulating Capsules

Select one formulation (either high or low shear) for preparing 50 capsules. The capsule hand-filling and capsule machine techniques will be demonstrated during the lab. The following steps are followed:

1) Using the sensitive scales, pre-weigh 5 x empty Size 0 gelatin capsules. 2) Fill 5 x Size 0 Capsules using the Hand-Filling Method or the Capsule Machines

Capsule Hand-Filling

NOTE: A video on capsule compounding - Capsule Making (Hand-Filling Technique) is available in the “VIDEOS” section of the laboratory website.

Clean and dry the glass slab in your locker.

Put on latex or nitrile gloves.

Use a plastic spatula to evenly spread a thick layer of powder about half the length of the capsule body onto the glass slab.

Remove the cap from the capsule

Gently push the open end of the capsule body into the powder repeatedly with a slight rotating motion, until the capsule body is filled.

NOTE: if the powder falls out, you may scoop the powder into the capsule body with a spatula.

http://pharmlabs.unc.edu/video1.php?pack_capsule.flv

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Replace the cap, then snap shut until a firm click is felt.

3) Determine the fill weight of your granulate:

Using the sensitive scales, weigh the 5 filled capsules together. Subtract the weight of the empty capsules from Step 1, then divide your answer by 5. This is the average fill weight of your granulate for Size 0 capsules.

4) Determine how much diluent to add to compound 100 mg capsules:

Use the Capsule_Filling.xls spreadsheet on Blackboard for calculations on how much diluent to add to each formulation. The capsule machine accommodates 50 capsules. Design a batch size of 60 capsules per formulation. An excess number of capsules is used to account for powder losses.

Mix the calculated amount of lactose and granulate together in a mortar and pestle, in the batch amounts calculated by Capsule_Filling.xls.

5) Capsule Filling Using a Capsule Machine

Use of the capsule machine will be demonstrated during the lab.

Obtain a capsule machine from your instructor or TA, and compound 50 capsules using the capsule machine.

NOTE: A video on capsule compounding - Capsule Making (Cap-M-Quik Method) is available in the “VIDEOS” section of the laboratory website.

6) QC of Compounded Capsules

Determine the weight uniformity of 10 capsules by using the “Capsule Filling: Quality Control” sheet in the appendix of this manual. You can also use the electronic version on the “Capsule QC” worksheet in Capsule_Filling.xls, available in the downloads section of the laboratory website.

Determine whether or not your capsule batch passed or failed.

NOTE: failing a batch will not affect your final mark.

Part E. Content Uniformity: Tablets and Capsules

NOTE: Perform Content Uniformity tests of all tablet and capsule formulations in parallel, to save time.

Following the same assay procedure in stability testing, determine the quantity of drug in 3 doses for each formulation (your formulation of high and low shear, and your capsule formulation) at room temperature. “Content uniformity” answers the question “how uniform is my potency across doses?” The parameter of interest is %RSD.

Report the mean ± %RSD of drug content for tablets and capsules.

At the end of the lab, transfer your capsules to prescription bottles, properly label them, and store them in your lockers.

Lab Period 3:

Part F. Capsule Dissolution

Add 900 mL of 0.1 N HCl dissolution medium to the dissolution vessel (prepare

http://phm.utoronto.ca/~ddubins/DL/Capsule_Filling.xls

http://phm.utoronto.ca/~ddubins/DL/Capsule_Making.wmv

http://phm.utoronto.ca/~ddubins/DL/Capsule_Filling.xls

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182 Lab 15: Tableting, Capsuling, and Dissolution Testing

additional 0.1 N HCl if required) and allow it to equilibrate at 37 °C. Although six (6) dissolution vessels are generally used for each sample, in this laboratory one vessel for each sample will be sufficient so as to allow all groups access to the equipment. Introduce the capsule to the dissolution vessel. Immediately start the rotation of the paddles at 100 rpm as time zero. Remove aliquot samples of the dissolution medium at several time intervals (5, 10, 15, 20, 30, 40, 50, 60 minutes) for analysis. Filter the samples using a 0.45 μm syringe filter into a plastic UV cuvette for reading if necessary.

The spectrophotometer should be set to the same wavelength you selected for your standard curve in Lab 14. Return the samples to the dissolution vessel after each reading in order to maintain the same dissolution volume.

Stability (continued from Lab Period 2)

Perform the drug assay on one tablet of your high and low shear formulation, at each temperature (room temperature, 40 °C, and 60 °C).

You may download and use shelflife.xls from the laboratory website to perform the shelf-life analysis.

Part G. Detection of Degradation Products / Decomposition: Thin Layer Chromatography

Thin layer chromatography (TLC) is a quick and convenient method to evaluate the extent of decomposition that may be occurring in samples under study at various storage conditions. Decomposed fragments migrate at a different rate from the drug on a TLC plate. Briefly, the stationary phase (silica gel) is porous, and hydrophobic. A more hydrophobic drug will want to “cling” more to the stationary phase (and not elute as far up the plate). A mobile phase with a higher dielectric constant (more hydrophilic) will also favour the drug eluting more slowly up the plate. By varying the polarity of the mobile phase, travel up the plate can be fine-tuned to separate out drugs (or drug fragments) of different hydrophilicities. The following is a typical list of TLC solvents:

Solvent Notation Dielectric Constant

Hexane H 1.9

Petroleum Ether PE 2.0

Cyclohexane Cy 2.0

Carbon Tetrachloride 2.2

Benzene B 2.3

Tolune 2.4

Diethyl Ether DE 3.4

Chloroform Ch 4.8

Ethyl Acetate EA 6.0

Acetic Acid AA 6.2

Isopropyl Alcohol 18.3

Acetone 20.7

Ethanol 24.3

Methanol M 32.6

Water 78.5

Source: https://infohost.nmt.edu/~jaltig/TLC.pdf

http://phm.utoronto.ca/~ddubins/DL/shelflife.xls

https://infohost.nmt.edu/~jaltig/TLC.pdf

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183 Lab 15: Tableting, Capsuling, and Dissolution Testing

Lab

You will be performing TLC analysis on each of your tablet stability samples (room temperature, 40 °C, and 60 °C).

1. Sample Preparation

Crush one unit of your dosage forms in a mortar and pestle. Transfer to a 10 mL volumetric flask (located on the TA cart). Fill the first 5 mL of the flask with methanol. Stopper the flask, and shake. Dilute to the final volume (10 mL) with methanol, and mix. Now, using a 5 mL syringe equipped with a 0.45 µm syringe filter*, filter 2.5 mL of this solution into a 50 mL volumetric flask, then dilute to volume with methanol.

2. Reference Preparation (to be provided by the TA)

The TA will provide the reference solution (0.05% acetaminophen). This is produced by starting with 100 mg of acetaminophen powder, and using the same dilution scheme as above.

3. Preparation of the TLC Chamber (TO BE DONE IN THE FUME HOOD)

Freshly prepare approximately 20 mL of the developing solvent with the following composition expressed in parts not percentages; ethyl acetate-acetic acid (95:5). Transfer the developing solvent to a TLC chamber. Place a saturation pad (a piece of filter paper) against one of the inner walls of the chamber so that it makes contact with the developing solvent. Place the cover on the TLC tank and allow the chamber to equilibrate. (A small amount of silicon grease along the top edge of the tank will aid in sealing the cover.)

4. Preparation of the TLC Plate

Gently (without scraping off the silica coating) mark a line along the bottom of the TLC plate 1.5 cm from the bottom edge as noted in the figure. Use a dull pencil and do not press hard enough to mark the silica surface. Mark another line across the plate 1 cm down from the top edge.

NOTE: Do not use marker or pen to mark your TLC plates. Only use a soft (non-mechanical) pencil. Ink will elute with the solvent front and contaminate your results.

Using a 5 μL glass capillary micropipette, spot 5 μL aliquots of the Sample preparation on the starting line drawn 1.5 cm from the bottom. Best results will be obtained if the spot is kept as small as possible. This can be achieved by releasing 1 μL portions from the micropipette and air dying between successive applications. Spot the 5% reference solution on either side using a 5 μL volume respectively. Allow the spots to air dry for 5 minutes before development to ensure that no solvent remains. You may spot 4-6 samples on the same plate.

5. Development and Visualization of Spots

Remove the cover of the TLC chamber and carefully place the TLC plate into the chamber taking care not to splash the developing solvent onto the plate. If there is enough room, you may put two plates in the same chamber. The plate should be leaning against the inner wall of the

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184 Lab 15: Tableting, Capsuling, and Dissolution Testing

chamber with the glass side touching the tank wall. Replace the cover of the chamber. The developing solvent will begin to migrate and will continue to do so until the upper line on the TLC plate is reached. Remove the TLC plate when the solvent has ceased to migrate and allow to air dry completely in the fume hood. Once the plate is dried, you can visualize the spots under the UV chamber. Mark all spots on the TLC plate lightly with a pencil. Visually compare the intensity of the spots of the sample with those obtained from the reference solutions. A table of the spots observed for the Sample preparation can be made by recording the Rf of each spot such that:

𝑅𝑓 =𝑉𝑒𝑟𝑡𝑖𝑐𝑎𝑙 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑠𝑝𝑜𝑡 𝑚𝑖𝑔𝑟𝑎𝑡𝑒𝑑 𝑓𝑟𝑜𝑚 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑙𝑖𝑛𝑒

𝑉𝑒𝑟𝑡𝑖𝑐𝑎𝑙 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑚𝑖𝑔𝑟𝑎𝑡𝑒𝑑 𝑓𝑟𝑜𝑚 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑙𝑖𝑛𝑒

Report the number of spots for each sample and the respective Rf value. You may keep your TLC plates, and photograph them for inclusion in your final lab report.

Summary of Formulation Testing

Formulation # Units A Low Tablet*

A High Tablet*

B Low Tablet*

B High Tablet*

Capsule Lab Period

Tablet Hardness (mean ±%RSD)

10 - 1

Weight Uniformity (mean ±%RSD)

10 1

Tablet Thickness (mean ±%RSD)

10 1

Friability (mean ±%RSD)

10 - 1

Disintegration Time 1 - 1

Stability – t90% (@room temp)

1 - 1

Stability – t90% (@40°C)

1 @7days, 1 @14 days

2, 3

Stability – t90% (@60°C)

1 @7days, 1 @14 days

2, 3

Content Uniformity (mean ±%RSD)

3 2

Drug Dissolution 1

2 (tabs) 3 (caps)

Drug degradation (TLC performed on the prepared reference, and 2 week samples stored at room temp, 40 °C, 60 °C)

1 - 3

*Your group of 5 will be performing these tests for your formulations only (A or B). Combine data with another lab group to obtain a full data set for the final report.

Questions

1. Identify the function of each of the ingredients in the tablet formulations of this laboratory exercise.

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2. Identify and discuss any effect of the granulation process on tablet hardness, friability, disintegration time, and dissolution.

3. Identify and discuss any impact of the formulation on tablet hardness, friability, disintegration time, and dissolution.

4. How did tablet dissolution compare with capsule dissolution? Were the same trends observed?

5. How did tablet content uniformity (average and %RSD) compare with capsule content uniformity for the formulation you selected?

6. Were there any differences in potency (the average content uniformity, a measure of accuracy) and %RSD of content uniformity (a measure of precision) in comparing the different tablet formulations? Can you think of any reasons that might explain these differences?

7. What is the difference between a lubricant, glidant, and anti-adherent?

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186 Lab 17: Synthesis and Examination of Colloids

Lab 17: Synthesis and Examination of Colloids Preparing for the Lab Read the introduction and lab protocol completely

Group Allocation You will be working in groups of 4 students

What You’ll Be Doing

Part A: Preparing a Yttrium Citrate colloid Part B: Preparing a Rhenium Heptasulphide colloid, method 1 Part C: Analyzing colloids (Observing Tyndall effect, performing turbidometric analysis and particle size analysis) Demonstration: Rhenium Heptasulphide colloid, method 2

Spreadsheets You Will Need Not applicable.

What You’re Handing In Individual formal lab report, due at the beginning of the next lab (see Guidelines for Writing Individual Laboratory Reports for details)

Introduction

Some small volume colloids suitable for injection may be prepared by carefully controlled inorganic synthesis. These reactions are designed to produce a consistent concentration of small particles all within the colloidal particle size range. All three of the colloids localize in their respective body compartments by phagocytosis.

Three colloids will be examined:

The first will involve the titration of a citrate solution combined with yttrium from an acidic environment to a plateau near neutral pH. As this plateau is reached, the reaction steps necessary to produce the colloidal yttrium citrate will be observed.

The second colloid will produce rhenium heptasulphide by the reaction of potassium perrhenate with sodium thiosulfate in the presence of HCl. Careful heating, cooling and stabilization with phosphate buffer results in a straw coloured colloid. In this colloid, the rhenium heptasulphide is used as a carrier or co-precipitant for 99mTc2S7.

The third colloid uses Re as the “active ingredient” and as a result the reaction producing the Re2S7 must go to completion. All of the Re must be in the colloidal form in order to undergo phagocytosis in the body and as complete a removal of the colloid from the fluid compartment as possible. This synthesis uses H2S in the presence of HCl to accomplish this.

The Tyndall effect will be observed in each colloid. The particle size of the colloids will be measured using differential filtration.

References

1. Bardy A et al. International Journal of Applied Radiation and Isotopes (1973); vol 24, pp 57-60 [French].

2. J. Liu, H. Wong, J. Moselhy, B. Bowen, X. Wu, M. Johnston. Targeting colloidal particulates to thoracic lymph nodes. Lung Cancer, 51(3); 377-386.

3. http://en.wikipedia.org/wiki/Rayleigh_Scattering

4. http://en.wikipedia.org/wiki/John_Tyndall

Background

In the late 1850s, John Tyndall, an Irish scientist, studied radiant energy in the atmosphere. He was the first to show that water vapour in the Earth’s atmosphere led to a Greenhouse Effect, as it absorbs the most energy. His work also led to the development of absorption spectroscopy,

http://en.wikipedia.org/wiki/Rayleigh_Scattering

http://en.wikipedia.org/wiki/John_Tyndall

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which has been prevalent in these labs. He intensely studied the way heat and light flow through the air in both the presence and absence of particles. The Tyndall Effect is the property of microscopic particles to scatter light visible to the naked eye. What would laser light shows at rock concerts be without the Tyndall Effect? They would be boring dots on the ceilings and walls. The Tyndall Effect was later examined by Rayleigh, who coined the term Rayleigh Scattering. Rayleigh scattering is used to explain why the sky is blue.

Why is the sky blue?

Rayleigh scattering is inversely proportional to the fourth power of wavelength. This means that as the wavelength increases, scattering drastically reduces. Blue wavelengths are shorter in length than the yellows and reds:

Color Wavelength

Violet 380–450 nm

Blue 450–495 nm

Green 495–570 nm

Yellow 570–590 nm

Orange 590–620 nm

Red 620–750 nm

The particles suspended in the atmosphere scatter more blue light than red light, which is why on a clear day when the sun is high in the sky, the sky will appear blue.

Experiment Protocol

Chemicals Supplies Special Equipment Yttrium(III) nitrate hexahydrate Sodium Citrate Sodium Hydroxide Pellets (MW 40.00 g/mol) Gelatin Sodium Thiosulfate Phosphate buffer pH 7.4:

Sodium Phosphate Monobasic (MW 137.99 g/mol)

Sodium Phosphate Dibasic (MW 268.07 g/mol) Hydrogen Sulphide (gas) Potassium Perrhenate Nitrogen (gas) Lead acetate impregnated filter paper Sodium Chloride (NaCl MW 58.44 g/mol)

Sterlitech filters (pore sizes: 5.0, 1.2, 0.8, 0.45 µm) Plastic UV Cuvettes 50 mL Falcon® tube

Mixing plate and magnetic stir bar 10 mL Volumetric Flask pH meter Water bath Carey UV/VIS Spectrophotometer (in Room 819) Laser Pointer

The following solutions are prepared or provided by the TA:

pH Meter Standardizing Buffers (pH 4, 7)

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188 Lab 17: Synthesis and Examination of Colloids

Stock Con'n Volume

(mL) Vstock

(mL) Mass

(g)

HCl (1N): (dilute from 6N HCl) 1 N 100 16.7

3A: Don't bother. It never works.

3B: 10

HCl Stock 6 N 6.7

Gelatin 0.40

3C: Gelatin (heat in microwave for 25 seconds to dissolve) 7.5 mg/mL 100 0.75

3D: 100

Gelatin 1 mg/mL 0.10

Sodium Benzoate 0.5% 0.50

3E: NaOH (for demo, and adjusting buffer) 2 N 100 8.00

1-Shot Sorensen's Phosphate Buffer (pH 7.4) 1000

Sodium Phosphate Monobasic 13.58

Sodium Phosphate Dibasic 3.04

Part A. Yttrium Citrate Colloid

Prepare the following solutions, in de-ionized water:

Solution Ingredients Volume

Solution 1A 35 mg/mL sodium citrate 10 mL

Solution 1B 0.1 N NaOH 50 mL

In a 140 mL beaker, dissolve 50 mg of yttrium nitrate in a small volume of de-ionized water (5 mL should be sufficient).

Add 80 mL of de-ionized water to the beaker.

Add 0.2 mL of Solution 1A (35 mg/mL sodium citrate) into the beaker.

Place the beaker on a stir plate and put a magnetic stir bar in the beaker. Place the pH electrode into the solution.

Using a dropper (transfer pipette), titrate the solution by 0.05 mL increments with Solution 1B (0.1 N NaOH) drop by drop, until pH 7.0 is attained. After each drop is added, wait until the pH stabilizes so you do not exceed pH 7.0.

Add de-ionized water to a final volume of 90 mL (remember to record your volumes).

Place 30 mL in a 50 mL Falcon® tube using a syringe, withdraw the air from the bottle, tie a noose around the neck with string, and hang it in a boiling water bath for 1 hour.

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Part B. Rhenium Heptasulphide Colloid, Method 1

Prepare the following solutions, in de-ionized water:

Solution Ingredients Volume

Solution 2A Sodium thiosulfate (30 mg) Potassium Perrhenate (5 mg) Gelatin (50 mg)

10 mL (use a 10 mL volumetric flask)

Solution 2B 0.9% w/v NaCl in water 50 mL

Solution 2C 1 N HCl Obtain 5 mL from TA

Solution 2D Sorensen’s Phosphate Buffer, pH 7.4 (Sorensen’s buffer from previous labs may be used)

Obtain 5 mL from TA

To prepare the rhenium heptasulphide colloid:

Place 4 mL of Solution 2A into an Erlenmeyer flask, and add 6 mL of Solution 2B (0.9% NaCl).

Add 1 mL of Solution 2C (1 N HCl) to the flask, and place in a boiling water bath for precisely 3 minutes.

Remove the flask from the bath, place it in a beaker containing cold water, and immediately add 4 mL of Solution 2D (Sorensen’s Buffer, pH 7.4).

Allow the solution to cool.

Part C. Rhenium Heptasulphide Colloid, Method 2 (Performed as a Demonstration only)

Prepare the following solutions, in de-ionized water:

Solution Ingredients Volume

Solution 3A 1 %w/v lead acetate 100 mL

Solution 3B 4 N HCl Gelatin (400 mg)

10 mL

Solution 3C Gelatin 7.5 mg/mL in ~70 C deionized water

10 mL

Solution 3D Gelatin 1 mg/mL

0.5% Sodium Benzoate in ~70 C deionized water

100 mL

Solution 3E 2 N NaOH 50 mL

Pre-soak filter paper in Solution 3A (1%w/v lead acetate). Set out to dry.

In a glass test tube, dissolve 5 mg of potassium perrhenate in 0.5 mL of Solution 3B (4 N HCl with gelatin).

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190 Lab 17: Synthesis and Examination of Colloids

In a fume hood:

Attach a glass Pasteur pipette to the tubing attached to the hydrogen sulphide and place it in another test tube containing water and adjust the flow of gas to 10 bubbles per minute.

Transfer this pipette into the reaction test tube and bubble the gas (faire barboter) through the mixture for 10 minutes. The mixture should be black.

Turn off the gas and remove the pipette and place it in the other test tube containing water.

Allow the reaction mixture to stand for 30 minutes.

After 30 minutes, add 5 mL of Solution 3C (7.5 mg/mL gelatin).

Bubble nitrogen gas through the reaction mixture for 30 minutes or until a drop of the reaction mixture placed on a dry filter paper previously soaked in a 1% lead acetate solution does not produce the black colour of lead sulphide.

Transfer to a 50 mL Falcon tube. Add Solution 3D (1 mg/mL gelatin) to a final volume of 50 mL. Transfer to a 140 mL beaker. Lower a pH electrode into the mixture, and titrate the solution to pH 5.0 using Solution 3E (2 N NaOH).

Part D. Analysis of Colloids

Tyndall Effect

1. Demonstrate the Tyndall effect with all three colloids. Describe how you perform the demonstration and the results of your analysis.

2. Obtain a laser from your TA or instructor. Take a picture of the laser beam shining through each colloid for inclusion in your report.

Turbidity and Particle Size Analysis

Using a 10 mL syringe with each membrane filter (pore sizes of 5.0, 1.2, 0.8, 0.45 µm), perform a particle size analysis by determining the percentage of the colloidal particles in each particle size range. Use the following protocol for filtration:

3. Run a wavelength scan from 350-600 nm on your unfiltered sample, and select an appropriate wavelength for spectroscopic analysis.

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Colloid ???

5 µm syringe

filter

0.45 µm syringe

filter

0.8 µm syringe

filter

1.2 µm syringe

filter

4. Fill a clean 10 mL syringe with the unfiltered colloid.

5. Select a filter size, and screw the corresponding syringe filter onto the 10 mL syringe.

6. Express the fluid through the syringe filter to the fill line of a clean cuvette.

7. Measure the absorbance at your selected wavelength in Step 3.

8. Recover the filtrate from the cuvette. Retain the filtrate, in case a filter clogs.

9. Repeat this process for each filter size. If a filters clogs, it may be necessary to start with a filtrate from a larger pore size sample.

10. Calculate the % retained on each filtrate by dividing each absorbance value by the absorbance of the unfiltered colloid. Then, to calculate particle size ranges, subtract off percent retained values of the next smaller pore size filtrate. The particle size ranges will be:

<0.45 µm, 0.45–0.8 µm, 0.8–1.2 µm, 1.2–5 µm, >5 µm

11. Repeat the experiment for the other colloids that you prepared.

12. Plot the percent retained vs. the particle size ranges for each colloid.

Questions

1. Describe your final preparation procedures and the appearance of your colloids.

2. Is there any difference in the particle size distribution of the three colloids?

3. How would you prepare “kits” for a more simplified production of each of the colloids? What quality control steps would be required?

4. What is the assumption(s) you are making in step 3 of the particle size analysis, in calculating the percent retained? How might your assumption(s) affect your results?

<5 µm particles

<1.2 µm particles

<0.8 µm particles

<0.45 µm particles

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Lab 18: Formulating Using Molds

Preparing for the Lab

Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:

UNC Eshelman School of Pharmacy Suppository Videos (http://pharmlabs.unc.edu/labs/suppository/videos.htm)

Group Allocation You will be working in groups of 2 students

What You’ll Be Doing

Part A: Formulating 325 mg acetaminophen suppositories in PEG base using the Calibrated Batch Volume method

Pick any two of: Part B: Formulating 20 mg benzocaine lollipops in Sorbitol-PEG vehicle, and QC’ing your work Part C: Formulating 20 mg hydrocortisone troches using the Displacement Factor Method Part D: Formulating 70 mg hydrocortisone/150 mg lidocaine lip balm using the Double Casting Method.

Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/moldcalcs.xls

What You’re Handing In Lab 18 Worksheet (due at the beginning of the next lab)

NOTE: Do not write on, cut, or dispose of any of the molds in this lab. They are re-useable.

Introduction

There are many interesting opportunities to design and compound custom medications using molds, both in a compounding pharmacy and within the pharmaceutical industry. In addition to the realm of suppositories, formulations involving molds have found their way into pediatrics (lollipops, gummy bears, and hard candies), adult medicines (troches, rectal rockets, lozenges, and lip balms), veterinary applications, and even custom rapid-dissolve tablets. The calculations for formulating each design share a common theme, and once understood, are applicable to new mold types. A solid understanding of the types of excipients and calculations involved can unlock a myriad of possibilities for the formulation scientist to improve efficacy and compliance.

References

1. Allen, Loyd V. Jr. et al. Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Lippincott Williams & Wilkins; 9 edition (Jan 7, 2010) p. 324

2. http://www.foodproductdesign.com/articles/1998/05/generating-yummy-gummies.aspx 3. http://pharmlabs.unc.edu/labs/suppository/casting.htm 4. http://faculty.ksu.edu.sa/bquadeib/Documents/PHT%20453%20practical%20notes.doc 5. http://www.pharmacopeia.cn/v29240/usp29nf24s0_m54856.html

Background

If the densities of substances did not change when changing from liquid to solid, and if volumes of components were truly additive when combined, then mold calculations would be straightforward. However, we know that not to be the case. Effects such as hydration, electrostriction, contraction upon cooling, and changes in density of substances upon mixing complicate simply measuring out what we think would work when pouring a liquid into a mold to solidify. It is therefore important to somehow take this into account when compounding the formulation. The consequence of not doing so would be an inaccurate dose. Under-potent formulations could result in a lack of efficacy, and over-potency runs the danger of unwanted

http://pharmlabs.unc.edu/labs/suppository/videos.htm

http://phm.utoronto.ca/~ddubins/DL/moldcalcs.xls

http://www.foodproductdesign.com/articles/1998/05/generating-yummy-gummies.aspx

http://pharmlabs.unc.edu/labs/suppository/casting.htm

http://faculty.ksu.edu.sa/bquadeib/Documents/PHT%20453%20practical%20notes.doc

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adverse events. Some molds come “pre-calibrated” to specific base types, however if different bases are used, the calibration provided are not accurate.

The first step in using a mold is therefore calibrating it to the specific vehicle that is selected by the formulator. Two methods will be presented here to deal with the calculations: the displacement factor method, the calibrated batch volume method, and the double casting method. Simplifications may also be made when the mass of drug or a particular excipient is negligible compared with the weight of the overall formulation. As with any mold formulation, always compound extra units to account for losses due to adherence to the glassware, broken/misformed units, and units that fall out of ±10% of their target mass.

Displacement Factor Method

The displacement factor method (also called the Density Factor method) is a popular approach to formulating most mold-type preparations. This method first requires calibration of the mold with vehicle alone. The placebo units are formed, and then weighed. Knowledge (or determination) of a displacement factor is required to determine how much vehicle is required. The following is a published table of the density (displacement) factors for various drugs in cocoa butter vehicle:

The following procedure is typically used:

1. Determine the average weight of 1 unit with vehicle alone (or average placebo weight) by calibrating the mold with an excess of empty melted vehicle.

2. Obtain the displacement factors for the drug (and each excipient) in the selected base (tabular, or provided).

NOTE: If the amount of drug or a particular excipient in the unit dose is small (e.g. 1-5%) and the

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194 Lab 18: Formulating Using Molds

displacement factor is unknown, then the formulator may assume that adding the drug will not significantly affect the total volume or density of the formulation, and a displacement factor of 1 may be used to arbitrarily take into account the volume displacement of the drug. This is a convenient simplification, but may not be as accurate at higher doses. Note that this is different than disregarding the mass of the drug completely, as the mass of the drug is still accounted for.

3. The weight of vehicle required is the total theoretical amount - the mass of each excipient divided by the respective displacement factor in the vehicle.

4. The final formulation is calculated as:

mvehicle = mplacebo – (mdrug/DFdrug in vehicle)

Where: mvehicle is the total batch weight of vehicle required for the medicated batch;

mplacebo is the total weight of vehicle required for a placebo batch;

mdrug is the amount of drug required to produce the right concentration of drug in vehicle for the correct dose in the medicated batch; and

DFdrug in vehicle is the displacement factor for the drug in the vehicle.

NOTE: If excess is compounded to account for losses, mdrug is not simply the dose × # units compounded. This would result in a sub-potent formulation.

Calibrated Batch Volume Method

The calibrated batch volume method for determining the quantities of excipients and drug required is simple, as it does not require advance knowledge or determination of displacement factors. Calibration with empty vehicle is first performed in order to determine the volume of melted vehicle required. Briefly, a first (placebo) batch is made using the vehicle alone. The units are formed, cooled, removed, weighed, and subsequently re-melted in a volume-calibrated vessel (e.g. a small beaker). A second batch is then made in a new vessel, starting with adding the drug itself. A portion of the melted vehicle is added, mixed, and then brought up to the same volume as the melted placebos, with sufficient mixing. The mixture is then poured into molds and cast. The units are cooled, removed, and weighted to check for accuracy.

This method requires that the liquid/solid transition of the vehicle is reversible. Although this is true for suppositories and troches, it is not the case for lollipops or gummy bears. When the dosage form is relatively small and the script is small, it may also be difficult to measure and calibrate the total batch volume reliably. This makes the Calibrated Batch Volume impractical for small batches.

Double Casting Method

One method that circumvents the need for mold calibration is the double casting (or double pour) method. This method involves mixing the required amount of drug with a quantity of melted base that is known to be insufficient for the entire suppository batch. The drug/base mixture is poured into the desired number of mold cavities, incompletely filling them. The empty space at the top of each mold is then overfilled with plain melted base. The suppositories are then trimmed with a hot spatula and removed, and then subsequently re-melted, mixed, and re-cast to ensure uniformity. The same mold must be used in both casting steps. Although calibration is circumvented, the work involved is essentially the same as the other methods, and this method has the added disadvantage of exposing the drug to heat much longer than the

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other methods. Like the calibrated batch volume method, this method also requires that the liquid/solid transition of the vehicle be reversible, which although is true for suppositories, will not be the case for other formulations (e.g. lollipops and gummy bears). It requires a reasonable estimate of the approximate mold volume so the formulator can estimate the amount of base to incorporate with the drug for the first step.

Source: http://pharmlabs.unc.edu/labs/suppository/casting.htm

Determining Your Own Displacement Factor

Provided you know the final weights of the placebo and medicated formulations using any of the methods above, a displacement factor may be calculated for future batches. This will save the pharmacist time for script refills, even if the dose changes. The displacement factor is calculated using the following formula:

You can see in this formula that if the medicated units weigh the same as the blank units, the displacement factor simplifies to (Dose/Dose) = 1, which will be true as the percentage of drug decreases compared to the overall weight of the formulation. Thus, the determined displacement factor for a drug in vehicle will not be reliable (noisy, and highly variable) if the dose is relatively small. In this case, you can disregard the mass contribution of the drug to the overall formulation.

𝐷𝐹 =𝐷𝑜𝑠𝑒

(𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑃𝑙𝑎𝑐𝑒𝑏𝑜 𝑊𝑒𝑖𝑔ℎ𝑡) −(𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑀𝑒𝑑𝑖𝑐𝑎𝑡𝑒𝑑 𝑊𝑒𝑖𝑔ℎ𝑡)+𝐷𝑜𝑠𝑒

http://pharmlabs.unc.edu/labs/suppository/casting.htm

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196 Lab 18: Formulating Using Molds

Disregarding the Mass of a Drug or Excipient

As mentioned above, the displacement factor approaches 1 as the dose decreases. Similarly, the mass of vehicle required for the entire medicated batch:

mvehicle = mplacebo – (mdrug/DFdrug in vehicle)

will approach mplacebo (the mass of vehicle required for a placebo batch) as the dose decreases. At a certain point, subtracting the mass of drug from the vehicle and recalculating the amount of vehicle required will lead to a negligible adjustment. USP 795 specifies that “compounded preparations are to be prepared to ensure that each preparation shall contain not less than 90.0% and not more than 110.0% of the theoretically calculated weight or volume per unit of the preparation.” When the mass of drug is negligible, it allows the pharmacist to ignore subtracting the displaced amount of vehicle while still falling well within USP 795 limits.

The consequence of this is that following mold calibration with empty vehicle, if the mass of drug or excipient is negligible (e.g. <1%), it can simply be added to the vehicle formula without subtracting off the mass of vehicle that would be displaced by it. The compounding steps are then simply to calibrate the mold with empty vehicle, weigh the placebos to obtain the average weight, and then compound the medicated version by adding the drug without adjustment. This simplification will be used in the lollipop formulation of this laboratory.

Formulating Lollipops

Lollipops are a preferred formulation when compliance is an issue for pediatric use, especially when delivery is desired locally to the oral mucosa, teeth, gums, or throat. One drawback of compounding a lollipop is that the drug must be capable of surviving the high temperatures of the melted base – sometimes upwards of 135 °C. For candy lollipops, temperature control is critical – a thermometer is required to know when to stop heating. Too high or too low a temperature will result in an unsatisfactory product.

Lollipops can be formulated for analgesics, antipyretics, antibiotics, antifungals, and many other drug classes. Some examples of drugs that have been compounded as lollipops include:

Drug Indication

Lidocaine, Tetracaine, Benzocaine Dental/Buccal Pain (local)

Fentanyl Systemic pain

Tranexamic acid Excessive Bleeding (local)

Lorazepam Sedation / relaxant / insomnia

Ketoconazole Oral Thrush

Dextramethorphan Cough suppressant

Acetaminophen Fever / pain

A number of different fun mold shapes can be purchased for compounding. As with any mold, the mold must be calibrated prior to use in order to compound the correct dose.

In this lab, you will be compounding benzocaine lollipops in Sorbitol-PEG base. This base takes longer to cool and harden, but is much easier to work with as it requires lower temperatures to melt than a sucrose-corn syrup lollipop. You will first calibrate the mold with empty vehicle (in

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the absence of drug) to determine the average lollipop weight. You will then create a second set of lollipops to contain 20 mg of the model drug (benzocaine).

Formulating Suppositories

A suppository is defined as a small plug of medication, designed to melt or dissolve at body temperature within a body cavity other than the mouth, typically the rectum or vagin*. The word suppository comes from the latin word suppositus, the past participle of supponere, “to put something under or next to something else”. Suppositories offer an alternative pathway to deliver a drug locally (e.g. antifungals, laxatives, antibiotics) and also systemically (e.g. NSAIDs, antiemetics, opioid analgesics). The ideal suppository vehicle does not interact with or degrade the active ingredients, is a solid at room temperature and melts or dissolves at body temperature, is stable under ambient storage conditions, and promotes release and absorption (if applicable) of the active. The first suppositories were compounded in cocoa butter. The formulator now has the flexibility to choose the vehicle type:

Suppository Vehicle Type

Examples Pros Cons

Oleaginous Cocoa butter

Hydrogenated vegetable oils e.g. Theobroma oil

Synthetic triglycerides (Witepsol H-15)

“Base F” (PCCA)

Easy to prepare

Self-preserving

Better for inflammation/irritation (emollient)

most common

Anal leakage (rectal)

Poor systemic absorption of hydrophobic drugs

Difficult polymorphs of cocoa butter (overheating + cooling too quickly results in wrong polymorph being formed, and base won’t solidify)

Hydrophilic Glycerinated gelatin

Hydrogels (PVA, hydroxyethyl methacrylate, polyacrylic acid, polyoxyethylene

PEG (Macrogols)

High MW or mixtures

“Base A” (PCCA)

PEG base is varying mixtures of PEG (e.g. 40% PEG 400 and 60% PEG 3350)

Dissolves slowly at 37°C (doesn’t melt)

Doesn’t melt as quickly as oleaginous bases

Well suited to vagin*l prolonged release

Chemically stable, non-irritating

Miscible with water/mucous secretions

Not self-preserving

Hygroscopic (pain) Wet before using

Poor/sensitive mechanical properties

Glycinerated gelatin requires mold lubrication

Water-Dispersible Bases

W/O emulsions

Polyoxyl 40 stearate (surfactant)

Fatty bases + surfactant

“Base MBK” (PCCA) – PEG 400-Stearate + Hydrogenated Vegetable Oil

Can make an oleaginous base more hydrophyllic

Surfactants can irritate the anal mucosa and cause expulsion

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In general, the guiding principle of formulating a suppository is that if systemic delivery is the goal of the formulation, to select a base that is opposite in hydrophilicity than the drug. In other words, to optimize systemic absorption of a hydrophobic drug, a hydrophilic base is selected, and vice versa.

For PEG suppositories, a low molecular weight PEG is typically mixed with a higher molecular weight PEG to produce a suppository with a tailorable hardness, melting point, and dissolution time. Micronized silica may be used (1-2% of formulation by weight, typically 25- 35 mg in a 2.1 g suppository), to stiffen a formulation that is too soft.

Ingredient

Typical Suppository Bases

1 2 3 4 5 6 7 8

PEG 8000 --- --- --- --- --- 70% --- ---

PEG 6000 47% 47% 47% --- --- --- --- 52%

PEG 4000 33% 33% --- 25% 5% --- 25% ---

PEG 1540 --- --- 33% --- --- 30% 65% ---

PEG 1000 --- --- --- 75% 95% --- --- ---

PEG 400 20% --- --- --- --- --- 10% 48%

Water --- 20% 20% --- --- --- --- ---

Examples of Rectal Suppositories

Source: Allen, Loyd V. Jr. et al. Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Lippincott Williams & Wilkins; 9 edition (Jan 7, 2010) p. 324

Acetaminophen is a very important antipyretic and analgesic. In this lab, you will be preparing 8 x 325 mg acetaminophen suppositories using the calibrated batch volume method, in PEG vehicle. These would be appropriate for an adult patient in need of pain or fever control, who is unable to swallow.

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Formulating Troches

Troches (pronounced troh-keys) are another term for oral lozenges. The troche shape will vary depending on the mold. The two major vehicle types for troches are gelatin-based, and higher molecular weight polyethylene glycol based formulations. Each will have different properties and potential drug-excipient interactions.

Oral hydrocortisone (e.g. 10 and 20 mg Cortef® Hydrocortisone Tablets, Pfizer Canada Inc.) is indicated for a very wide variety of conditions, including endocrine and rheumatic disorders, collagen and dermatologic diseases, allergic states, hematologic disorders, ophthalmic, respiratory, neoplastic, and gastrointestinal diseases, and edematous states.

Compounding the correct dose for a troche requires knowledge of the displacement factor for hydrocortisone in troche vehicle, and proper calibration of the mold. In this lab, you will calibrate a troche mold with empty vehicle to determine the blank troche weight, and then use the displacement factor method to determine the amount of vehicle required for the medicated batch. You will then compound 20 mg hydrocortisone troches.

Source: http://webprod5.hc-sc.gc.ca/dpd-bdpp/item-iteme.do?pm-mp=00023184

Formulating Lip Balms

Lip balms or lip salves are excellent for local delivery of drug to the topical surface of the lips, and can also be useful for other parts of the body (e.g. sunscreen sticks). Although many cosmetic lip balm products are available, there are also medicated formulations listed in the Therapeutic Products Directorate of Health Canada as over-the-counter products, that have a Drug Identification Number (DIN). These include preparations with octinoxate, oxybenzone, hom*osalate, and avobenzone (all used for absorption of ultraviolet light in sunblock).

The lip balm training formulation in this laboratory is an occlusive, hydrophobic preparation of hydrocortisone and lidocaine, which would be useful as a treatment for painful mouth cold sores, associated with Herpes Simplex Virus type I. Almond oil could be substituted for lemon oil, depending on the preference of the patient. We are using this formula to demonstrate the Double Casting Method.

Experiment Protocol

Chemicals Supplies Special Equipment Acetaminophen USP (MW 151.17) Benzocaine USP (MW 165.19) Hydrocortisone USP (MW 362.46) Lidocaine USP (MW 234.34) Excipients as indicated in each section

Non-Stick Cooking Spray 2 × Suppository Molds 2 × Lollipop Molds 2 × Lip Balm Molds 12 × Lollipop Sticks

Hot Plate Thermometer Retort Stand, Vinyl Retort Clamp Test tube rack Stability Chamber (set to 5°C)

The following solutions are prepared or provided by the TA:

Not Applicable (TA to turn on stability chamber to 5 °C for cooling formulations)

http://webprod5.hc-sc.gc.ca/dpd-bdpp/item-iteme.do?pm-mp=00023184

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200 Lab 18: Formulating Using Molds

Part A. Formulating 325 mg Acetaminophen Suppositories (Calibrated Batch Volume Method)

PEG Suppository Base

Ingredients Weight Percent

(%w/w) Mass (g)

Polyethylene Glycol 3350, NF 58.8 %w/w

Polyethylene Glycol 400 (Liquid) 39.2 %w/w

Silica Gel (Micronized) 2 %w/w

Total: 100 %w/w ~35 g

Mold Calibration

1. Fill a 600 mL beaker approximately a third of the way with tap water on a hot plate, set to high. Set up a large porcelain dish on the beaker. This should result in a dish temperature between 70-80 °C when the water boils.

2. Weigh the required amounts of PEG 3350 and PEG 400. Transfer the PEG 3350 to the porcelain dish. An excess amount of vehicle is required for the anticipated amount; in this case 35.0 g will be more than enough base to compound 12 suppositories. Mix with a glass rod until the PEG 3350 melts completely. Add the PEG 400 and silica gel, and mix with a glass rod until hom*ogenous.

NOTE: With any fusion preparation, constant stirring will prevent excipient separation.

3. Turn a test-tube rack upside down, and use it to lightly support the suppository mold (do not press the mold into the rack with too much pressure, or the mold cavities will deform). In a slow, continuous stream, pour the hom*ogenous mixture (Step 2) into the dry mold and overfill each cavity so that there is a thick bead of continuous melted suppository vehicle across the top of the mold. This is to account for contraction of the base during cooling.

4. When the suppositories begin to solidify, place the filled suppository mold in the lab stability chamber (at 5 °C) for at least 15 minutes. Set the hot plate to low and allow the hot plate to cool down.

5. Remove mold from the stability chamber. Using a metal microspatula, remove the excess of base by scraping firmly across the top of the mold.

Step 5. Scrape off excess base with a microspatula

Step 2. Melt the PEG in a ceramic dish and stir with a glass rod.

Step 3. Overfill the mold cavities with melted base

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201 Lab 18: Formulating Using Molds

Lab

Step 12. Bringing it up to volume

with remaining base

6. Smooth the surface, then carefully remove the suppositories from the mold by gently pushing upwards from the bottom.

7. Weigh the well-formed placebo suppositories individually to determine the average placebo (blank) weight.

Medicated Suppositories

8. Weigh out 3.90 g of acetaminophen in a small weighing boat.

NOTE: 12 suppositories x 325 mg acetaminophen/suppository = 3.90 g acetaminophen. 12 units are planned to compensate for losses in compounding, in order to prepare 6 well-formed suppositories. If you were unable to recover and re-melt all 12 suppositories, scale down the mass of drug sin Step 8 to the correct dose (325 mg * # recovered suppositories).

9. Re-melt your well-formed placebo suppositories in a 50 mL beaker (Beaker A), directly on the hot plate on medium heat. Remove the beaker from the hot plate, and mark the liquid level with a wax pencil.

10. Discard the melted placebo suppository mix from Beaker A. Clean Beaker A, being careful not to erase your calibration mark. Weigh out enough ingredients for your medicated batch of suppositories, and repeat step 2, preparing a fresh batch of the same amount of vehicle in a large ceramic dish, on the water bath.

11. Transfer the 3.90 g of acetaminophen into Beaker A. This step is performed without heating. Add the fresh vehicle for the melted mixture, filling Beaker A approximately ¾ of the way to the calibrated mark (including the acetaminophen volume). Stir with a glass rod until an opaque, hom*ogenous mixture is produced.

12. Continue to fill Beaker A to the calibrated mark with vehicle, and stir with a glass rod until hom*ogenous.

13. In a slow, continuous stream, pour the hom*ogenous mixture from Beaker A into a clean, dry suppository mold, and over-fill the mold cavities (to avoid formation of holes that could take place due to contraction of the base on cooling). Do not use the same mold as your placebo batch, as previous vehicle will interfere with suppository ejection.

Step 9. Mark volume of melted placebos with wax pencil

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202 Lab 18: Formulating Using Molds

NOTE: You might not have enough melted suppository mixture to fill every mold cavity. Fill each one sequentially in the same manner as in mold calibration, generously overfilling each cavity, and when you run out of the mixture, leave the remaining cavities empty.

14. When the suppositories begin to solidify, place the filled suppository mold in the lab stability chamber (at 5 °C) for at least 15 minutes.

15. Remove mold from the stability chamber. Using a metal microspatula, remove the excess of base by scraping firmly across the top of the mold.

16. Smooth the surface, then carefully remove the suppositories from the mold by gently pushing upwards from the bottom.

17. Complete a QC spreadsheet for your suppository batch (use the “Mold QC” worksheet in moldcalcs.xls) and hand it in with your final formulation. Use the average placebo and medicated suppository weights to calculate a displacement factor for acetaminophen in this vehicle. You may also use the “Displacement Factor Method” worksheet in moldcalcs.xls to verify your calculations.

How could you use the calculated Displacement Factor to compound more quickly, if you were to prepare this formulation again?

18. Wrap the suppositories individually in aluminum foil, and properly label. To wrap, cut a small aluminum square (approx. 5 cm x 5 cm) and place the suppository in the centre of the square, diagonally. Fold the corners in towards the suppository ends, and then roll the suppository applying a slight pressure during rolling.

19. Rinse the molds in hot water in the lab sinks to clean them and remove any material. Return the molds to your TA or instructor. Do not dispose of the molds, they are reusable.

Part B. Formulating 20 mg Benzocaine Lollipops (Mass of Drug Negligible)

Children find the concept of lozenges difficult, as their cue for candy is to chew and swallow. Lollipops offer a favourable alternative that can be individually flavoured to their liking. This improves both compliance and effectiveness of the topical delivery of anesthetics to the buccal cavity. Drugs may also be delivered systemically in the same manner, provided the drug in question can withstand exposure to heat in the compounding process.

In this procedure you will calibrate your lollipop mold with the base, then formulate 10 x 20 mg benzocaine lollipops in order to compound 6 lollipops (the extra is compounded to account for losses).

Sorbitol – PEG Lollipop Base

Ingredients Weight Percent

(%w/w) Mass (g)

Sorbitol (Powder), NF 64.7 %w/w

Polyethylene Glycol 3350, NF 32.3 %w/w

http://phm.utoronto.ca/~ddubins/DL/moldcalcs.xls

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203 Lab 18: Formulating Using Molds

Lab

Silica Gel (Micronized) 2.0 %w/w

Total amount of flavourant(s) (target ~1-3% for water based, ~0.1% for oil based)

1.0 %w/w

Colourant of Choice 1-2 drops per batch (trace)

Total: 100%w/w 90.0 g

In this procedure, the weight of the drug is small compared to the overall weight of the lollipop (<0.2%). You will be calibrating the mold with an excess amount of empty vehicle (90 g), calculating the batch mass of benzocaine required, and then scaling your final batch to compound 10 x 20 mg benzocaine lollipops. A total of 10 is planned in order to account for breakage and losses, with a target of 6 perfect lollipops.

Mold Calibration

1. Set a 600 mL beaker directly on a hot plate, set to medium. Set up a thermometer inside the beaker, on a retort stand using a vinyl retort clamp.

2. Add the polyethylene glycol 3350 to the 600 mL beaker and stir with a glass rod until completely melted. Add the sorbitol powder, and continuously mix while maintaining a temperature of 100-110°C to form a hom*ogeneous liquid-like dispersion. The mixture will not become clear, but should be smooth.

NOTE: Don’t overheat the mixture, or it will separate into 2 phases. If this happens, remove the mixture from the hot plate and mix vigorously until one phase is obtained.

3. Add the silica gel, colouring, and flavouring to Step 2. Continuously mix until a hom*ogeneous liquid-like dispersion is obtained. Remove the beaker from the hot plate and continue monitoring the temperature.

4. Holding the mold at arm’s length, prepare the lollipop molds by lubricating with a light coating of non-stick cooking spray. There should not be enough oil to pool in the mold cavity. A thin layer is all that is required. Insert the lollipop sticks into the empty molds. Weigh the empty mold with sticks inserted.

5. Stir the mixture until the vehicle begins to thicken (~90°C) and appears uniform. Fill the 6 mold cavities with Step 3 so that the molds are filled flush to the top, and the lollipop sticks are positioned with the tip at the circle centre and submerged. If the mixture starts to solidify while filling, reheat to ~100oC and continue.

6. Place the filled lollipop mold in the lab stability chamber (at 5 °C) or freezer in PB 819 for at least 15 minutes.

7. Carefully remove the placebo lollipops from the mold. Individually weigh the lollipops to determine the average placebo lollipop weight.

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204 Lab 18: Formulating Using Molds

Medicated Lollipops

8. Based on the average weight of vehicle per lollipop, calculate how much benzocaine and vehicle is required to compound 10 lollipops for the final medicated batch.

9. If the benzocaine appears lumpy, triturate the benzocaine to reduce the particle size to a smooth powder in a small glass mortar and pestle.

10. With a clean 600 mL beaker, repeat the above steps for mold calibration (Steps 1-7), adding the benzocaine at the appropriate time (Step 3). Use a fresh mold – you do not need to wait for the calibration batch to cool.

11. At the end of the lab period, remove from mold, and weigh the recovered lollipops.

12. Dispense the lollipops in an appropriate container, and label.

13. Complete a QC spreadsheet for your lollipop batch (in the lab worksheet, or the “Mold QC” tab in moldcalcs.xls) and hand it in with your final formulation. Did the batch pass weight/dosage specifications?

14. Rinse the molds in hot water in the lab sinks to clean them and remove any material. Return the molds to your TA or instructor. Do not dispose of the molds, they are reusable.

Part C. Formulating 20 mg Hydrocortisone Troches (Displacement Factor Method)

Troche Gelatin Base

Ingredients Weight Percent

(%w/w) Mass

(g)

Silica Gel - Micronized (stiffener) 0.9

Stevia Powder (sweetener) 0.2

Acacia NF (thickener) 1.5

Gelatin (gelling agent) 20.0

Glycerin (plasticizer, sweetener) 46.0

70% Sorbitol USP (plasticizer, sweetener) Note: This is sorbitol solution, not powder.

8.0

De-ionized Water 23.4

Flavour of Choice (1-2 drops per batch) (trace)

Colour of Choice (1-2 drops per batch) (trace)

Total: 100 %w/w 50 g

http://phm.utoronto.ca/~ddubins/DL/moldcalcs.xls

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205 Lab 18: Formulating Using Molds

Lab

Mold Calibration

1. Calculate an excess amount of ingredients for at least 45 placebo troches (~50 g total batch weight). Even though you are only making 30 troches, 45 is planned to account for losses in compounding.

2. Spray mold cavity lightly with non-stick cooking spray and allow excess to drain on a paper towel.

3. Set a hot plate on high. Tare a 150 mL beaker on a lab scale, and weigh in the measured amounts of de-ionized water, glycerin and 70% sorbitol solution. Stir with a glass rod until clear. Place the beaker on the hot plate. Set up a thermometer on a retort stand at the middle of the solution (not touching the sides or bottom) to monitor the temperature while heating. Heat mixture to 80°C.

4. Remove the 150 mL beaker from the hot plate. Add the gelatin, and mix with a glass rod. Stir with periodic heating to prevent the gelatin from gelling or burning on the bottom of the beaker.

5. When the gelatin appears smooth and hom*ogenous, remove it from the hot plate. The mixture will not appear completely transparent when the gelatin has melted.

6. Using a glass mortar and pestle, triturate the silica gel, stevia powder, and acacia to a fine powder.

7. Add the powders from Step 6 into the melted gelatin. Stir with a glass rod until evenly dispersed and uniform.

8. Add the colourant and flavourant of choice. Mix well.

9. As soon as the ingredients are properly mixed, pour mixture (Step 8) into mold, ensuring the cavities are full.

NOTE: Do not allow the mixture to cool before filling the mold, or it will thicken. Remove excess vehicle with the back of a warmed metal spatula. Molds should be filled flush to the top with the partitions visible and not covered with vehicle, to allow for proper separation of troches. Avoid overfilling the troche mold. Do not close the lid before the troches have solidified.

10. Allow the troches to cool. Carefully remove and weigh the placebo troches individually to determine the average placebo troche weight.

Medicated Troches

11. Based on the average weight of vehicle per troche, calculate how much hydrocortisone and vehicle is required to compound 45 x 20 mg hydrocortisone troches for the final medicated batch. Use a displacement factor of 1.50 for hydrocortisone in troche gelatin base.

About Stevia: Stevia powder (or stevioside) is a sweetener that is rapidly gaining popularity in North America. It is 250-300 times sweeter than sugar, and does not affect blood sugar metabolism. It is water and alcohol soluble, heat stable, and has a working range of 0.1-0.8% (above which it becomes bitter).

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206 Lab 18: Formulating Using Molds

12. In a small glass mortar and pestle, triturate the required amount of hydrocortisone USP (powder), silica gel, stevia powder, and acacia powder together, and reduce the particle size to a smooth powder.

13. With a clean 140 mL beaker, repeat the above steps for mold calibration (Steps 1-8), adding the medicated powdered mixture at the appropriate time (Step 6).

Part D. Double Casting Method: 70 mg Hydrocortisone/150 mg Lidocaine Lip Balm

Lip Balm Base

Ingredients Weight Percent

(%w/w) Mass

(g)

Petrolatum (occlusive/moisturizing) 48.9

Beeswax (stiffener) 20.2

Polyethylene Glycol 400 (hydrophilic) Note: PEG 400 is liquid at room temperature, and is used to levigate the drugs.

18.6

Lemon Oil or Orange Oil (fragrance) Note: Do not weigh out in weighing dish (the oil will dissolve the dish)

12.3

Total: 100 %w/w 16 g

Lip Balm Preparation

Note: The Double Casting Method does not require calibrating the mold.

1. Set up a hot water bath by filling two x 250 mL beakers with ~100 mL tap water, and placing them on the same hot plate (diagonally). Set the hot plate on high, and set a small ceramic dish on each.

2. Remove the caps from the lip balm casings. Check that each lip balm casing has a fill elevator inside it. Ensure each lip balm casing is ready by twisting the bottom ring (called the “knurl”) counter-clockwise all the way, until it becomes difficult to turn. At this point, the fill elevator will be at the bottom of the casing. If the fill elevator is at the top and does not lower, gently apply downward pressure while turning the knurl. Weigh and record the mass of the casing without the cap.

3. Calculate an excess amount of ingredients for two lip balm sticks (16 g total batch weight). This should be enough material to make two units in excess.

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207 Lab 18: Formulating Using Molds

Lab

4. Place and tare the first ceramic dish (dish 1) on an open-air (2 decimal) lab scale. Directly weigh in the calculated amounts of beeswax, petrolatum, and lemon oil (or orange oil), in that order. Tare the scale between measurements.

Note: For the beeswax, break off tiny pieces (break or shave larger pieces with a metal weighing spatula if required). For the petrolatum, transfer in small amounts on a clean spot of dish 1, with a small metal weighing spatula. For the lemon (or orange) oil, use a plastic transfer pipette.

5. Place dish 1 back on the hot water bath. Stir the mixture with a metal spatula until clear, and hom*ogenous. Do not overheat the mixture.

6. Using the sensitive scales, weigh out enough hydrocortisone USP and lidocaine USP for two lip balm sticks (140 mg hydrocortisone USP and 300 mg lidocaine USP) in separate, small weighing boats. Transfer both powders into the second small ceramic dish (dish 2).

7. Weigh out the required amount of PEG 400 in a small beaker. Levigate the drug in dish 2 with the PEG 400, slowly adding it dropwise, and mixing with a spatula until the drug particles are completely wet. Add the remaining amount of PEG 400 from the small beaker to dish 1.

8. Using an oven mitt, pour off approximately one quarter of the vehicle from dish 1 into dish 2, on the second water bath. This should be less than the amount required to compound two lip balm sticks. Mix dish 2 until uniform. If two phases form, remove dish 2 from the hot plate, and stir continuously while cooling, until uniform (but not solidified).

Note: Before pouring the melted vehicle into the lip balm casing, dab the bottom of the ceramic dish onto a lab diaper or tissue, to remove any condensate from the bottom of the dish.

9. Remove dish 2 from the hot plate. Carefully fill the two lip balm casings with the medicated, melted mixture, dividing it approximately equally between them. Ensure all of the liquid is transferred.

10. Top up the lip balm casings with the remaining empty vehicle (from dish 1). Fill to the very top of the casing, without overfilling. The base will contract slightly as it cools. After the base forms a skin on the top, you may transfer it to the lab stability chamber set at 5 (set at 5 °C) for faster cooling (~10 minutes).

11. After the lip balm sticks have cooled and solidified, remove them from the casings by twisting the knurls clockwise, until completely ejected. You will see a small plastic piston

Fill elevator at bottom of casing

Knurl (counter-clockwise = down)

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208 Lab 18: Formulating Using Molds

(called the “fill elevator”) at the bottom of each casing – remove these as well by continuing to twist the knurls clockwise.

12. Place the solidified formulation back into dish 2, recovering any vehicle stuck on the fill elevator with a small metal weighing spatula. Re-melt the solidified lip balm sticks on dish 2. Stir until hom*ogenous.

13. Reload the fill elevators into the lip balm casings. To accomplish this, push the fill elevator back into the casing, as you twist the knurl counter-clockwise, until the fill elevator starts to descend. Continue twisting the knurl counter-clockwise until the fill elevator reaches the very bottom. At this point, you will no longer be able to twist the knurl counter-clockwise.

14. Carefully refill the lip balm casings with all of the medicated mixture, being careful to transfer as much of the vehicle as possible.

15. Allow the formulations to cool. Weigh the final casings (not including caps) to determine the medicated lip balm weight. Replace the cap, and properly label the formulation.

16. Complete a QC spreadsheet for your lip balm units (in the “Lip Balm QC” tab in moldcalcs.xls) and hand it in with your final formulations. Did the units pass weight/dosage specifications?

Recover Re-melt & mix Reload Re-pour

Questions

1. A patient returns to the pharmacy with a suppository formulation that has grown mold. How would you change the formulation for future batches?

2. You fill the suppository script above and the patient returns to the pharmacy complaining that the medication doesn’t seem to be working. What might be the problem(s) and what follow up steps would you take to improve drug efficacy?

3. What physical state (crystalline or amorphous) will the drug be in the suppositories or lollipops after going through the molten and cooling steps?

4. What effect will the added drug have on the melting or solidification point of the suppository base?

NOTE: Lab 19 will be a group project assigned during the laboratory.

http://phm.utoronto.ca/~ddubins/DL/moldcalcs.xls

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209 APPENDIX

APPENDIX

U.S. Standard Sieve Sizes and Lab Sieve Inventory

U.S. Standard Sieves

Nominal Designation No.

(mesh #)

Sieve Opening (µm)

Nominal Designation No. (mesh #)

Sieve Opening (µm)

2 9500 45 355

4 4750 50 300

6 3350 60 250

8 2360 70 212

10 2000 80 180

12 1700 100 150

14 1400 120 125

16 1180 140 106

18 1000 170 90

20 850 200 75

25 710 230 63

30 600 270 53

35 500 325 45

40 425 400 38

Sieve PB860 Inventory

Opening µm

Opening Inches

400 1 37 0.0015

325 1 44 0.0017

170 2 88 0.0035

140 4 105 0.0041

120 2 125 0.0049

100 3 149 0.0059

80 4 177 0.0070

70 1 212 0.0083

60 3 250 0.0098

50 1 300 0.0117

45 1 354 0.0139

40 2 425 0.0165

35 2 500 0.0197

25 1 707 0.0278

20 4 840 0.0331

18 1 1000 0.0394

16 1 1190 0.0469

12 2 1700 0.0661

Top 2 Bottom 3

Total 41

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210 APPENDIX

Quadro Comil Meshes

Serial Number Hole Inches/ Microns

Particle Size (µm)

US Mesh#

US Mesh (µm)

Uses

7B250Q03750* (6350) 2006-11

Square

0.25/ 6350

3175 6 3350

Square holes are used for wet

granulation, dispersion, moist

food reclaim, cheeses, de-

wrapping candy

7B187Q03472* (4750)

Square

0.187/ 4750

2375 8 2360

7B156Q03746* (3962)

Square

0.156/ 3962

1981 10 2000

7B125G03123* (3175)

Cheese Grater

0.125/ 3175

1587.5 12 1700 Grater screen is used for

harder products or more

ductile, powderizing cream

filled cookies, grating cheese,

chopping nuts, sizing

compressed slugs, cereal

flakes

7B083R03472* 0601

Round

0.083/ 2107.7

1053.8 18 1000

Round holes are used for dry

granulation, powders, fat

dispersion, bulk density

tuning, de-agglomeration

7B045R03137* 0601

Round

0.045/ 1143.8

571.9 30 600

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211 APPENDIX

Methocel and Avicel Grades

METHOCEL™ Premium methylcellulose and hypromellose products are a broad range of water-soluble cellulose ethers. They enable pharmaceutical developers to create formulas for tablet coatings, granulation, controlled release, extrusion, molding, and for controlled viscosity in liquid formulations.

METHOCEL™ Premium Products for Pharmaceutical Applications - Hypromellose Grades METHOCEL™ Product Chemical Type

1 Methoxyl

Content, %

Hydroxypropoxyl Content, %

Viscosity of 2% solution in water, mPa·s (USP/EP/JP)

METHOCEL™ E3 Premium LV Hypromellose 2910 28.0 - 30.0 7.0 - 12.0 2.4 - 3.6

METHOCEL™ E5 Premium LV Hypromellose 2910 28.0 - 30.0 7.0 - 12.0 4.0 - 6.0

METHOCEL™ E6 Premium LV Hypromellose 2910 28.0 - 30.0 7.0 - 12.0 4.8 - 7.2

METHOCEL™ E15 Premium LV Hypromellose 2910 28.0 - 30.0 7.0 - 12.0 12 - 18

METHOCEL™ E50 Premium LV Hypromellose 2910 28.0 - 30.0 7.0 - 12.0 40 - 60

METHOCEL™ E4M Premium2 Hypromellose 2910 28.0 - 30.0 7.0 - 12.0 2663 - 4970

METHOCEL™ E10M Premium CR Hypromellose 2910 28.0 - 30.0 7.0 - 12.0 9525 - 17780

METHOCEL™ F50 Premium Hypromellose 2906 27.0 - 30.0 4.0 - 7.5 40 - 60

METHOCEL™ F4M Premium Hypromellose 2906 27.0 - 30.0 4.0 - 7.5 2663 - 4970

METHOCEL™ K3 Premium LV Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 2.4 - 3.6

METHOCEL™ K100 Premium LV2 Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 80 - 120

METHOCEL™ K4M Premium2 Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 2663 - 4970

METHOCEL™ K15M Premium2 Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 13275 - 24780

METHOCEL™ K100M Premium2 Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 75000 - 140000

1USP XXII

2Also available in faster hydrating CR (controlled release) grade

Source: http://www.dow.com/dowexcipients/products/methocel.htm

METHOCEL™ Cellulose Ethers are the first choice for the formulation of hydrophilic matrix systems, providing a robust mechanism for the slow release of drugs from oral solid dosage forms. With a choice of viscosity grades, METHOCEL™ provides a simple solution to meet a range of drug solubility needs. Tablets are easily manufactured with existing, conventional equipment and processing methods. Source: http://www.colorcon.com/products/core-excipients/extended-controlled-release/methocel-controlled-release/Product%20Overview

METHOCEL™ Premium Products for Pharmaceutical Applications - Methylcellulose Grades METHOCEL™ Product Chemical Type

1 Methoxyl

Content, %

Hydroxy-propoxyl Content, %

Viscosity of 2% solution in water, cPs (USP)

Viscosity of 2% solution in water, mPa·s (EP/JP)

METHOCEL™ A15 Premium LV Methylcellulose, USP 27.5 - 31.5 0 12 - 18 12 - 18

METHOCEL™ A4C Premium Methylcellulose, USP 27.5 - 31.5 0 300 - 560 320 - 480

METHOCEL™ A15C Premium Methylcellulose, USP 27.5 - 31.5 0 1125 - 2100 1298 - 2422

METHOCEL™ A4M Premium Methylcellulose, USP 27.5 - 31.5 0 3000 - 5600 2663 - 4970 1USP XXII Source: http://www.dow.com/dowexcipients/products/methocel.htm

http://www.dow.com/dowexcipients/products/methocel.htm

http://www.colorcon.com/products/core-excipients/extended-controlled-release/methocel-controlled-release/Product%20Overview

http://www.dow.com/dowexcipients/products/methocel.htm

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212 APPENDIX

AVICEL™ Products Product Grades Nominal

Particle Size, µm Moisture, % Loose

Bulk Density, g/cc

Roller Compaction Avicel DG 45 NMT 5.0 0.25 - 0.40

Wet Granulation Avicel PH-101 50 3.0 to 5.0 0.26 - 0.31

Direct Compression Avicel PH-102 100 3.0 to 5.0 0.28 - 0.33

Avicel HFE*-102 100 NMT*** 5.0 0.28 - 0.33

Superior Compactibility Avicel PH-105 20 NMT 5.0 0.20 - 0.30

Superior Flow Avicel PH-102 SCG** 150 3.0 to 5.0 0.28 - 0.34

Avicel PH-200 180 2.0 to 5.0 0.29 - 0.36

High Density Avicel PH-301 50 3.0 to 5.0 0.34 - 0.45

Avicel PH-302 100 3.0 to 5.0 0.35 - 0.46

Low Moisture Avicel PH-103 50 NMT 3 0.26 - 0.31

Avicel PH-113 50 NMT 2 0.27 - 0.34

Avicel PH-112 100 NMT 1.5 0.28 - 0.34

Avicel PH-200 LM 180 NMT 1.5 0.30 - 0.38

Mouthfeel Improvement Avicel CE-15 75 NMT 8 N/A

*High Functionality Excipient

**Special Coarse Grade

***Not More

Than

Source: http://www.fmcbiopolymer.com/Pharmaceutical/Products/Avicelforsoliddoseforms.aspx

http://www.fmcbiopolymer.com/Pharmaceutical/Products/Avicelforsoliddoseforms.aspx

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213 APPENDIX

Avicel Grade Usage Chart

Tablets

Method Desirable Properties Recommended Product

Roller Compaction -Increase tablet hardness -Provide good flow and a strong ribbon during compaction -Reduce number of excipients -Improved yields

Avicel DG in intra-granular phase No extra-granular binders required.

Direct Compression -Improve flow -Better compressibility -Accomodation of moisture-sensitive actives

Avicel PH-102 SCG Avicel HFE-102 Avicel PH-200 Avicel PH-302

Wet Granulation -Rapid, even wetting as a result of the wicking action of microcrystalline cellulose -Reduced sensitivity of the wet masss to over-wetting -Faster drying -Fewer screen blockages or case hardenings -Reduce dye migration -Faster disintegration

Avicel PH-101 Avicel PH-301

Disintegration -Enhance drug dissolution by speeding tablet disintegration -Provide the highest level of disintegration force at low use levels -Utilize dual disintegration mechanisms of wicking and swelling for more rapid disintegration

Ac-Di-Sol

Liquids and Suspensions

Suspending Agent -Maintain suspension uniformity by preventing settling -Impart thixotropic viscosity profile

Avicel RC-591 Avicel CL-611

Source: http://www.fmcbiopolymer.com/Pharmaceutical/Applications/ImmediateRelease/Tablets.aspx

http://www.fmcbiopolymer.com/Pharmaceutical/Applications/ImmediateRelease/Tablets.aspx

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214 APPENDIX

Capsule Properties

Empty Hard Gelatin Capsule Dimensions

Size

Outer Diameter

(mm)

Height or

Locked Length (mm)

Actual Volume

(mL)

Vet

eri

nar

Su07 23.4 88.5 28

7 23.4 78.0 24

10 23.4 64.0 18

11 20.9 47.5 10

12el 15.5 57.0 7.5

12 15.3 40.5 5

13 15.3 30.0 3.2

man

000 10.0 26.1 1.37

00 8.5 23.3 0.95

0 7.7 21.7 0.68

1 6.9 19.4 0.50

2 6.4 18.0 0.37

3 5.8 15.9 0.30

4 5.3 14.3 0.21

5 4.9 11.1 0.13

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215 APPENDIX

Working Ranges of Typical Granulating Fluids

Granulating Fluid Typical Concentration Used (%)

acacia 10-20

alcohol (up to 100%)

cellulose derivatives 5-10

gelatin 10-20

glucose 25-50

polyvinylpyrrolidone 3-15

starch 5-10

sugar 70-85

water (up to 100%)

Viscosities of Typical Fluids

Newtonian Viscosities

Temperature (

oC)

Viscosity (poise)

Water 20 0.0100

Water 50 0.0055

Water 99 0.0028

Ethanol, absolute 20 0.0120

Ethanol, absolute 50 0.0070

Ethanol, 40% w/w 20 0.0291

Ethanol, 40% w/w 50 0.0113

Ethyl ether 20 0.0024

Glycerin, anhydrous 20 15.00

Glycerin, 95% w/w 20 5.45

Castor oil 20 10.3

Powder Flowability Indices

Consolidation Index (%) Flow

5-15 Excellent

12-16 Good

*18-21 Fair to passable

*21-35 Poor

33-38 Very Poor

>40 Extremely Poor

Angle of Repose Flow

<25 Excellent

25-30 Good

*30-40 Passable

>40 Very Poor

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Average HLB Values of Some Surface Active Agents

Common Name Compound HLB Acacia 12.0

Arlacel 80 Sorbitan monooleate 4.3

Arlacel 60 Sorbitan monostearate 4.7

Arlacel 40 Sorbitan monopalmitate 6.7

Arlacel 20 Sorbitan monolaurate 8.6

Brij 30 Polyoxyethylene lauryl ether 9.5

Brij 35 Polyoxyethylene lauryl ether 16.9

Methocel 15 cps Methylcellulose 10.5

Myrj 45 Polyoxyethylene monostearate 11.1

Myrj 49 Polyoxyethylene monostearate 15.0

Myrj 51 Polyoxyethylene monostearate 16.0

Myrj 52 Polyoxyethylene monostearate 16.9

Myrj 53 Polyoxyethylene monostearate 17.9

PEG 400 monooleate Polyoxyethylene monooleate 11.4

PEG 400 monostearate Polyoxyethylene monostearate 11.6

PEG 400 monolaurate Polyoxyethylene monolaurate 13.1

Pharmagel B Gelatin 9.8

SDS (or SLS) Sodium dodecyl sulfate (or Sodium lauryl sulfate) 40

Span 85 Sorbitan trioleate 1.8

Span 65 Sorbitan tristearate 2.1

Span 80 Sorbitan monooleate 4.3

Span 60 Sorbitan monostearate 4.7

Span 40 Sorbitan monopalmitate 6.7

Span 20 Sorbitan monolaurate 8.6

Tween 61 Polyoxyethylene sorbitan monostearate 9.6

Tween 81 Polyoxyethylene sorbitan monooleate 10.0

Tween 65 Polyoxyethylene sorbitan tristearate 10.5

Tween 85 Polyoxyethylene sorbitan trioleate 11.0

Tween 21 Polyoxyethylene sorbitan monolaurate 13.3

Tween 60 Polyoxyethylene sorbitan monostearate 14.9

Tween 80 Polyoxyethylene sorbitan monooleate 15.0

Tween 40 Polyoxyethylene sorbitan monopalmitate 15.6

Tween 20 Polyoxyethylene sorbitan monolaurate 16.7

From: Gennrbinro AR, et al., Remington: The Science and Practice of Pharmacy, 21st ed., USA: Mack Publishing Company, 2006, p. 311-314, 335-6.

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Surfactant Type Examples Applications Nonionic: -Least irritating -Compatible with other three surfactant classes -Compatible over a broad range of pH values -Calcium tolerant -Inert, lower toxicity, some suitable for oral administration, some have better palatability

Esters: (oil-soluble) -Ethylene Glycol Esters and Propylene Glycol Esters -Glyceryl Esters (e.g. Glyceryl monostearate) -Polyglyceryl esters, Sorbitan esters, Sucrose esters, and Ethoxylated esters

-Emulsifiers -Most widely used in topical preparations (O/W, W/O emulsions) -Oil soluble, not water soluble

Ethers: (generally derived from PEG and PPG) -e.g. PEG-20 cetyl ether / cetomacrogol 1000 -more stable than esters

-Solubilizing agents for oil, detergents, wetting agents, emulsifying agents

Ether-Esters: (water soluble) -Spans and Tweens (e.g. Span 80, Tween 80) -Ester linkage subject to hydrolysis -May hydrogen bond to preservatives and undesirably reduce antimicrobial activity -Viscous liquids, bitter taste

-Useful for solubilization and emulsification -Viscous liquids, bitter taste

Fatty alcohols: (C8-C18) -Only marginal surfactant effect, useful as co-surfactant

Anionic: -Personal care products, industrial purposes -Cleansing and detergency -Not for oral use

Alkali Soaps: R COO- M

+ (M=Na, K, NH4)

-12-18 carbon atoms -Sensitive to/precipitate in hard water (Ca2+ intolerant) -Gives an alkaline pH (~10), pH sensitive -Incompatible with acidic drugs -Micelles break up at elevated temperatures

-External use only (laxative effect, poor taste, hemolytic properties) -irritating to the eye -useful in enemas/liniments as a counter-irritant -good O/W emulsifiers at 5-10% -good solubilizers at 20%

Alkaline Soaps: (R COO)2 M (M = Ca, Mg, Zn) -12-18 carbon atoms -Oil soluble -Similar disadvantages to Alkali soaps

-form W/O emulsions (e.g. calamine lotion BP)

Amine Soaps: (e.g. triethanolamine stearate) -Lack calcium intolerance -Less alkaline, pH ~8 -Less hydrophilic, not as temperature dependent

-Hair creams, lotions, cosmetic creams -Better O/W emulsions due to HLB

Sulfuric Acid Esters: e.g. Sulfated fatty alcohols R OSO3

- M

e.g. Sodium Lauryl Sulfate (SLS, SDS) -Salt of strong acid and base -Highly water soluble, ionized at low pH -Does not precipitate with Ca2+ -May precipitate with large positive drugs

-Forms O/W emulsiions -Preparation of creams, emulsifying wax, emulsifying ointment -Can be used with neutral or negatively charged drugs -Generally requires an auxiliary substance (e.g. cetyl alcohol)

Sulfonic Acid Derivatives: (R SO3Na) e.g. Dioctyl sodium sulfosuccinate (aerosol OT) -Similar to sulfated alcohols -Less prone to hydrolysis, Ca

2+ tolerant

-Hydrophilic

-Emulsifying agent in creams and ointments -Wetting agent, solubilizer

Cationic: -External preparations

Simple Amine Salts (R NH2 HCl) e.g. Octadecylamine HCl -pH sensitive

-Wetting, foaming, detergent properties

Quaternary Ammonium Salts (NR1R2R3R4+ X

e.g. Cetyltrimethyl ammonium bromide -Soluble over wide pH range -Requires secondary emulsifier/stabilizer -Incompatible with large anionic compounds

-Emulsifier -Not for oral or parenteral administration -In creams or emulsions containing nonionic or cationic drugs for external application -Bactericidal activity

Zwitterionic/ Amphoteric:

e.g. Alkyl Betaine -Positive charge almost always ammonium, negative charge carboxylate, sulfate, sulphonate -pH sensitive – charge can change depending on pH -Compatible with other surfactant classes -soluble and effective in the presence of high concentrations of electrolytes, acids and alkalis

-Personal care and household cleaning products -Excellent dermatological properties -Frequently used in shampoos and cosmetics products, and also in hand dishwashing liquids because of their high foaming properties

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General Physical Properties of Spans and Tweens

Source: Karsa, David R. Design and Selection of Performance Surfactants, Volume 2. CRC Press LLC. Boca Raton, Florida USA, 1999.

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Surfactant Molecular Formula Molecular Structure

Span 20 C18H34O6

Span 40 C22H42O6

Span 60 C24H46O6

Span 80 C24H44O6

Tween 20

C18H34O6(C2H4O)20

Tween 40 C22H42O6(C2H4O)20

Tween 60 C24H46O6(C2H4O)20

Tween 80 C24H44O6(C2H4O)20

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HLB Requirement for Some Common Oil Components

Oil Component

Required HLB

Almond Oil 6 ± 1

Apricot Kernal Oil 7 ± 1

Avocado (Persea Gratissima) Oil 7 ± 1

Beeswax 12 ± 1

C12-15 Alkyl Benzoate 13 ± 1

Canola Oil 7 ± 1

Caprylic/Capric Triglyceride 5 ± 1

Carnauba (Copernicia Cerifera) Wax 12 ± 1

Carrot (Daucus Carota Sativa) Root Extract 6 ± 1

Carrot (Daucus Carota Sativa) Seed Oil 6 ± 1

Castor (Ricinus Communis) Oil 14 ± 1

Ceresin 8 ± 1

Cetearyl Alcohol 15.5 ± 1

Cetyl Alcohol 15.5 ± 1

Cetyl Esters 10 ± 1

Cetyl Palmitate 10 ± 1

Cocoa (Theobroma Cacao) Butter 6 ± 1

Coconut (Cocos Nucifera) Oil 8 ± 1

Cyclomethicone 7.5 ± 1

Diisopropyl Adipate 9 ± 1

Dimethicone 5 ± 1

Isopropyl Myristate 11.5 ± 1

Isopropyl Palmitate 11.5 ± 1

Jojoba (Buxus Chinensis) Oil 6 ± 1

Kukui Nut (Aleurites Moluccana Seed) Oil 7 ± 1

Lanolin 10 ± 1

Macadamia (Macadamia Ternifolia) Nut Oil 7 ± 1

Mango (Mangifera Indica) Seed Butter 8 ± 1

Mango (Mangifera Indica) Seed Oil 7 ± 1

Mineral Oil 10 ± 1

Myristyl Myristate 8 ± 1

Olive (Olea Europaea) Oil 7 ± 1

Petrolatum 7 ± 1

Retinyl Palmitate 6 ± 1

Safflower (Carthamus Tinctorius) Oil 8 ± 1

Sesame (Sesamum Indicum) Oil 7 ± 1

Shea Butter (Butyrospermum Parkii) 8 ± 1

Soybean (Glycine Soja) Oil 7 ± 1

Soybean Oil 7 ± 1

Stearic Acid 15 ± 1

Stearyl Alcohol 15.5 ± 1

Sunflower (Helianthus Annuus) Seed Oil 7 ± 1

Sweet Almond (Prunus Amygdalus Dulcis) Oil 7 ± 1

Tocopherol 6 ± 1

Wheat Germ (Trictum Vulgare) Oil 7 ± 1

Source: http://www.theherbarie.com/files/resource-center/formulating/Required_HLB_for_Oils_and_Lipids.pdf

http://www.theherbarie.com/files/resourcecenter/formulating/Required_HLB_for_Oils_and_Lipids.pdf

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Buffer Solution Preparation: Polyprotonic Acids and Bases

These buffer problems should be looked upon exactly as those for monoprotic acids and bases. For simplicity, assume that each ionization step goes to completion before the next ionization commences. In these cases, choose the salt/acid pair with pKa close to the desired pH of the buffer. Working buffer concentration are typically on the order of 0.1 M.

For example, citric acid has the following pKa values:

pKa,1 = 3.13 pKa,2 = 4.76 pKa,3 = 6.40

Looking only at the second pKa:

O O

OOH

OH OHO-

O O

OOH

OH O-

O- + H

pKa,2

The buffer capacity is greatest for ± 1.0 pH around the pKa value. Thus, the salt/acid ratio from the Henderson-Hasselbach equation can be calculated:

pH = pKa + log ([conjugated base] / [acid])

In order to have a buffer solution of pH 4.78, one would need an equimolar concentration of the above salt/acid couple. To generate the components of this couple, the addition of HCl to the sodium citrate, or NaOH to the citric acid would be required.

Example:

Say we want a 0.1 M, pH 5 citric buffer and decided to add NaOH to citric acid, the calculation is as follows:

(1) Citric acid is fully protonated (i.e., 3 H+), and we wish to prepare a buffer concentration of 0.1 M, then 0.1 M of NaOH is required for the first ionization.

(2) From the above equation, the salt/acid ratio required is:

5.0 = 4.78 + log [salt] / [acid] [salt] / [acid] = 1.66 i.e., [salt] = 1.66 [acid]

(3) Since we want [salt] + [acid] = 0.1 M, there are 2 equations and 2 unknowns. therefore, 1.66 [acid] + [acid] = 0.1 M [acid] = 0.038 M

[salt] = 1.66 x 0.038 = 0.063 M

(4) Therefore, the total amount of NaOH required is 0.1 + 0.063 = 0.163 M

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Dissociation Constants of Acids in Aqueous Solutions at 25°C

Substance Ka pKa

Acetic 1.75 x 10-5 4.76

Acetylsalicylic 3.27 x 10-4 3.49

Aluminum Hydroxide 6.3 x 10-13 12.20

Benzoic 6.3 x 10-5 4.2

o-Chlorobenzoic Acid 1.20 x 10-3 2.92

Cinnamic (Trans) 1.32 x 10-4 3.88

Formic 1.76 x 10-4 3.75

Fumaric 9.3 x 10-4 3.03

Glycine 1.67 x 10-10 9.78

Lactic 1.39 x 10-4 3.86

Maleic 1.0 x 10-2 2.00

5.5 x 10-7 6.26

Malic 4 x 10-4 3.4

9 x 10-6 5.05

Phenol 1.3 x 10-10 9.89

Phosphoric 7.5 x 10-3 2.12

6.2 x 10-8 7.21

4.8 x 10-13 12.32

Salicylic 1.06 x 10-3 2.97

3.6 x 10-14 13.44

Sulfanilic 6.5 x 10-4 3.19

Tartaric 9.6 x 10-4 3.02

2.9 x 10-5 4.54

Valeric 1.56 x 10-5 4.81

Dissociation Constants of Bases in Aqueous Solutions at 25°C

Substance Kb pKb

Ammonium Hydroxide 1.75 x 10-5 4.74

Atropine 4.47 x 10-5 4.35

Benzocaine 6 x 10-12 11.22

Caffeine 4.1 x 10-14 13.39

Codeine 9 x 10-7 6.05

Ethanolamine 2.77 x 10-5 4.56

Hydroquinine 4.7 x 10-6 5.33

Nicotine 7 x 10-7 6.15

Procaine 7 x 10-6 5.15

Quinine 1 x 10-6 6.0

1.3 x 10-10 9.89

Urea 1.5 x 10-14 13.82

(pKa + pKb = 14)

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Sorensen Phosphate Buffers

Prepare M/15 solutions and mix appropriate quantities to make 100 mL of buffer at the desired pH.

pH mL M/15 Na2HPO4

mL M/15 KH2PO4

5.8 7.8 92.2

5.9 9.9 90.1

6.0 12 88.0

6.1 15.3 84.7

6.2 18.5 8.15

6.3 22.4 77.6

6.4 26.5 73.5

6.5 31.8 68.2

6.6 37.5 62.5

6.7 43.5 56.5

6.8 50.0 50.0

6.9 55.4 44.6

7.0 61.1 38.9

7.1 66.6 33.4

7.2 71.5 28.5

7.3 76.8 23.2

7.4 80.4 19.6

7.5 84.1 15.9

7.6 86.8 13.2

7.7 89.4 10.6

Fundamental Lab Calculations

Preparing a Known Molar Concentration

To prepare 500 mL of a 0.2 M solution of dibasic sodium phosphate, we first need to know the number of moles to add:

mol 1.0mL 1000

L 1 mL 500

mol 0.2n

VCn

Unfortunately the lab scales measure grams, not moles of substance. The molecular weight of dibasic sodium phosphate is 268.07 g/mol. In order to convert moles into grams, a compound’s molecular weight is used in the following equation:

g 26.81m

g/mol 268.07 mol 0.1m

MWnm

In order to prepare 500 mL of a 0.2 M dibasic sodium phosphate solution, dissolve 26.81 g into a 500 mL volumetric flask, then dilute to the mark.

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Weight-Volume Percent (%w/v)

Usually when a preparation is liquid and a percentage is specified, by default it is assumed that it is percent weight-volume (%w/v). Weight volume percent is the easiest concentration unit to work with. It is equivalent to saying “g/100 mL total solution”. Note, this is different than saying g / 100 mL solution added. The total solution includes the volume of the solute added (thus effects like electrostriction are taken into account). The measure %w/v is great to work with, because you don’t have to look up the molecular weight of the solute to perform the dilution. All you have to do is multiply the final desired volume (in mL) by the %w/v, and your answer will be in grams.

For example, to prepare a 2% w/v solution of any solute in a volume of 200 mL:

g 4m

mL 2002%mL 200mL 100

g 2CVm

Weigh out 4 g into a 200 mL volumetric flask, and dilute to the mark.

Weight-Weight Percent (%w/w)

When a preparation is solid and a percentage is specified, by default it is assumed that it is percent weight-weight (%w/w). Similar to %w/v, it means “g/100g”. The mathematics are similar. To prepare a 2% w/w mixture of any compound for a total mass of 200 g:

g 4m

g 200%2CVm

Weigh out 4 g of the compound to be mixed with the remaining 196 g of remaining components. Note that as long as you keep the units consistent, they need not be in grams.

Dilution Equation

Although fundamental, the dilution equation is easy to forget, especially if you haven’t been in a lab in a long time. The equation is really just a mass balance. It states that although the concentration will change when you dilute, the mass of the solute added will remain constant:

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The most common problem in a wet lab is usually, how much of Solution 1 do I take to make Solution 2?

The equation used is:

221

2211

CVV (3)

VCVC (2)

mm (1)

For this example, a 1 M stock solution is to be diluted to a 2 mM solution. How much of that solution you make is up to you, however, it will depend on:

How much of Solution 2 will you need? This will depend on the lab.

How much of Solution 2 can you make? This will depend on the volume of Solution 1.

Is the resulting dilution feasible/realistic/doable given the equipment you have?

Let’s say we need 100 mL of Solution 2, as the example states. We use Equation (3), and calculate the volume of V1 required:

mL 0.2V

M 1

mM 1000

M 1 mM 2

mL 100V

CVV

221

Now take 0.2 mL of Solution 1 and pour into a volumetric flask, then dilute to 100 mL.

The problem is that you look around the lab, and realize the smallest bulb pipette you can find is 1 mL. Here is where dilution in series comes in handy. You can make an intermediate concentration, so that you can use the equipment you have and still get reasonably accurate results. Let’s try making a 100 mL at C = 0.1 M, first:

C1 = 1 M V1=?

Solution 1 Solution 2

C2 = 2 mM V2 = 100 mL

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mL 10V

M 1

M 0.1 mL 100V

CVV

221

10 mL is no problem with the equipment you have. Take 10 mL of Solution 1 and dilute it to 100 mL to get a 0.1 M solution. Now for the second step:

mL 2V

M 0.1

mM 1000

M 1 mM 2

mL 100V

CVV

221

Now take 2 mL of the 0.1 M solution, add it to a 100 mL volumetric flask, and dilute to the mark.

How to Use a Syringe Filter

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Capsule Filling: Quality Control

Preparation Name:

Compounder:

Date/Time:

Theoretical Powder Weight per Capsule

Total batch powder weight for all capsules compounded: g

Number of capsules compounded: capsules

Theoretical Powder Weight per Capsule: g

Average Empty Capsule Weight

Empty Capsule 1 g

Empty Capsule 2 g

Empty Capsule 3 g

Empty Capsule 4 g

Average of 4 Capsules g

Theoretical Total Capsule Weight g

(=Theoretical Powder Weight + Average Empty Capsule Weight)

Weights of 10 Randomly-Selected Compounded Capsules

Final Capsule 1 g Final Capsule 6 g

Final Capsule 2 g Final Capsule 7 g

Final Capsule 3 g Final Capsule 8 g

Final Capsule 4 g Final Capsule 9 g

Final Capsule 5 g Final Capsule 10 g

Average of 10 Capsules g

Minimum Theoretical Capsule Weight: g (=Theoretical Capsule weight * 0.9)

Maximum Theoretical Capsule Weight: g (=Theoretical Capsule weight * 1.1)

QC Pass/Fail?:

(PASS if Average capsule weight within 90-110% of theoretical limits)

Capsule Weight Deviation: (Target: <2%) =100% * (Actual-Theoretical)/Theoretical

Weight Coefficient of Variation: =100% * STDEV/AVG

NOTE: For successive measurements, you do not need to remove the previous capsule(s) from the scale. Simply re-tare the scale, and measure the next capsule.