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Student Name: _________________________________

Lab Group: _______ Locker #: _______

Pharmaceutical Chemistry
Laboratory Manual

PHC 340Y 2016-2017


Laboratory Website: http://phm.utoronto.ca/pharmlab
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Pharmaceutical Chemistry Laboratory Manual

Pharmaceutics Principles and Evaluation

PHC 340Y 2016-17

Leslie Dan Faculty of Pharmacy


University of Toronto

Lab Coordinator: Dr. David Dubins


d.dubins@utoronto.ca

PHC 340Y Lab Manual 2016/17


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
reimbursement 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
editions of the PHM 224Y manual, written by Barry Bowen, Copyright  2002-2009, Charlene
Ng, Copyright  2001, Mike Vachon, Copyright  1995, J. Graham Nairn, Copyright  1991-
1993, and Ping I. Lee, Copyright  1993, all of the University of Toronto.
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 Lutan Liu.
Contributions to the PHC 340Y Pharmaceutics Laboratory Manual were also made by James
Rogers, University of Alberta.
All rights reserved.

This manual is supplemented by notices and information on Blackboard.


David Dubins, Copyright  2016.
Preface 1

Table of Contents

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

Introduction ..........................................................................................................................8
General Information.................................................................................................................... 8
Recommended Textbooks ....................................................................................................... 8
Teaching Staff.............................................................................................................................. 8
Role of Teaching Assistants ..................................................................................................... 9
Attendance .............................................................................................................................. 9
Lateness Policy .......................................................................................................................... 10
Laboratory and Lecture Schedule ............................................................................................. 11
Locker Check-In / Check-Out ..................................................................................................... 13
Recording Data, Analysis, and Results ...................................................................................... 15
Plagiarism and Falsification ................................................................................................... 15
Clean-up Check-List ................................................................................................................... 16
Assignment of Grades ............................................................................................................... 16
Guidelines for Writing Pre-Labs, Worksheets and Individual Laboratory Reports ................... 16
Laboratory Safety ...................................................................................................................... 19
Chemical Inventory................................................................................................................ 19
Labeling of Preparations........................................................................................................ 19
Chemical Disposal.................................................................................................................. 20
Dress Code ............................................................................................................................. 20
Dress Code Rationale ............................................................................................................ 20
Working with Hazardous Chemicals ...................................................................................... 20
Emergency Response ............................................................................................................ 21
In Case of Personal Injury ...................................................................................................... 21
In Case of Spills ...................................................................................................................... 22
In Case of Fire ........................................................................................................................ 22
If the Fire Alarm Sounds ........................................................................................................ 23

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


Introduction .............................................................................................................................. 24
Background ............................................................................................................................... 24
Experiment Protocol ................................................................................................................. 26
Part A. Preparing a Calibration Curve.................................................................................... 26
Part B. Plotting Your Calibration Curve ................................................................................. 28
Questions .................................................................................................................................. 28

Lab 2: Preparation of pH Buffers ........................................................................................... 29


Introduction .............................................................................................................................. 29
Background ............................................................................................................................... 29
Definition of pH and pKa ........................................................................................................ 30
Buffer Capacity ...................................................................................................................... 35

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2 Preface

Experiment Protocol ................................................................................................................. 37


Part A. Preparing Sorensen’s Buffer ...................................................................................... 37
Part B. Preparing McIlvaines’s Buffer .................................................................................... 38
Questions .................................................................................................................................. 39

Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid ...................... 40
Introduction .............................................................................................................................. 40
Background ............................................................................................................................... 40
Experiment Protocol ................................................................................................................. 44
Part A. UV Absorbance Standard Curve of Sodium Salicylate ............................................... 45
Part B. Determination of the Partition Coefficient................................................................ 46
Part C. Direct Measurement of the Partition Coefficient ...................................................... 47
Questions .................................................................................................................................. 48

Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa ....................... 49
Introduction .............................................................................................................................. 49
Background ............................................................................................................................... 50
pKa and Intrinsic Solubility ..................................................................................................... 50
Calculation of pHp .................................................................................................................. 52
Polymorphism ....................................................................................................................... 56
Experiment Protocol ................................................................................................................. 57
Part A. Intrinsic Solubility Determination.............................................................................. 57
Part B. Preparing Different Salts of Sulfathiazole.................................................................. 58
Part C. Preparing Different Polymorphs of Sulfathiazole ...................................................... 58
Part D. pKa Determination ..................................................................................................... 59
Part E. Melting Point Determination ..................................................................................... 60
Part F. Macroscopic Evaluation ............................................................................................. 61
Questions .................................................................................................................................. 61

Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph
Identification ....................................................................................................................... 62
Introduction .............................................................................................................................. 62
Background ............................................................................................................................... 63
Experiment Protocol ................................................................................................................. 64
Part A. Co-solvency................................................................................................................ 64
Part B. Salt Selection ............................................................................................................. 65
Part C. Polymorph Identification ........................................................................................... 65
Questions .................................................................................................................................. 65

Lab 6: Thermodynamics of Mixing – Enthalpy and Volume .................................................... 66


Introduction .............................................................................................................................. 66
Background ............................................................................................................................... 66
Specific Heat Capacity ........................................................................................................... 66
Partial Molar Quantities ........................................................................................................ 69
Experiment Protocol ................................................................................................................. 70

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Preface 3

Part A. Calibration of the Calorimeter................................................................................... 71


Part B. Specific Heat Capacity of Copper Metal .................................................................... 72
Part C. Heat of Reaction and Heat of Hydration ................................................................... 72
Part D. Measurement of Molar Enthalpy of Reaction ........................................................... 72
Part E. Illustration of Partial Molar Volume .......................................................................... 72

Lab 7: Examination of Viscosity and Suspending Agents ........................................................ 74


Introduction .............................................................................................................................. 74
Background ............................................................................................................................... 75
Experiment Protocol ................................................................................................................. 80
Part A. Characteristics of a Polymeric Solution: Intrinsic Viscosity ....................................... 80
Part B. Characteristics of a Polymeric Solution: Fluid Type................................................... 81
Part C. Measurement of the Sedimentation Rate of an Ion Exchange Resin (Glass Beads) . 81
Questions .................................................................................................................................. 82

Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis .................................................................... 83


Introduction .............................................................................................................................. 83
Background ............................................................................................................................... 84
Half-Life and Shelf-Life .......................................................................................................... 85
Temperature dependency of Kinetics: The Arrhenius Equation ........................................... 86
Kinetics of ASA Hydrolysis ..................................................................................................... 87
Calculating the Amount of ASA as a Function of Time .......................................................... 88
Experiment Protocol ................................................................................................................. 89
Part A. Acetylsalicylic Acid Hydrolysis – Effect of Temperature............................................ 90
Part B. Acetylsalicylic Acid Hydrolysis – Effect of Concentration .......................................... 91
Part C. Acetylsalicylic Acid Hydrolysis – Effect of a Suspension ............................................ 91

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

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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
Part B. Determination of the Tonicity of Atropine Sulfate Solutions .................................. 129
Part C. Calculation and Preparation of an Isotonic Solution of Atropine Sulfate................ 129
Part D. Preparation of an Isotonic Phosphate Buffer .......................................................... 129
Part E. Demonstration of the Action of a Hypotonic, Isotonic, and Hypertonic Sodium
Chloride Solution on Erythrocytes ...................................................................................... 130
Questions ................................................................................................................................ 130

Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water ...................... 131
Introduction ............................................................................................................................ 131
Background ............................................................................................................................. 132
Experiment Protocol ............................................................................................................... 138
Part A. Preparing the Solutions ........................................................................................... 138
Part B. Phase Inversion ........................................................................................................ 143
Questions ................................................................................................................................ 145

Lab 13: Optimization of Powder Flow and Particle Size Determination ................................ 147
Introduction ............................................................................................................................ 147
Background ............................................................................................................................. 148
Experiment Protocol ............................................................................................................... 153
Part A. Compounding Powder Blends ................................................................................. 153
Part B. Determining Tapped Density ................................................................................... 154
Part C. Determining the Angle of Repose............................................................................ 155
Part D. Determining Powder Flowability ............................................................................. 155
Part E. Sieve Analysis ........................................................................................................... 158
Questions ................................................................................................................................ 159

Lab 14: Pharmaceutical Granulations .................................................................................. 160


Introduction ............................................................................................................................ 160
Background ............................................................................................................................. 160
Experiment Protocol ............................................................................................................... 162
Part A. Preparing a Standard Curve for Acetaminophen .................................................... 162
Part B. Preparing the Powder Blends and Granulating ....................................................... 163
Part C. Milling and Sizing ..................................................................................................... 164

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Preface 5

Questions ................................................................................................................................ 164

Lab 15: Tableting, Capsuling, and Dissolution Testing .......................................................... 166


Introduction ............................................................................................................................ 166
Background ............................................................................................................................. 167
Tableting Methods .............................................................................................................. 168
Tablet Properties ................................................................................................................. 169
Experiment Protocol ............................................................................................................... 176
Lab Period 1: ........................................................................................................................... 176
Part A. Tableting .................................................................................................................. 176
Part B. Stability (Shelf Life) .................................................................................................. 177
Demonstration: Tablet Coating ........................................................................................... 180
Lab Period 2: ........................................................................................................................... 181
Part C. Tablet Dissolution .................................................................................................... 181
Part D. Formulating Capsules .............................................................................................. 182
Part E. Content Uniformity: Tablets and Capsules .............................................................. 183
Lab Period 3: ........................................................................................................................... 183
Part F. Capsule Dissolution .................................................................................................. 183
Part G. Detection of Degradation Products / Decomposition: Thin Layer Chromatography
............................................................................................................................................. 183
Summary of Formulation Testing ........................................................................................ 185
Questions ................................................................................................................................ 186

Lab 17: Synthesis and Examination of Colloids .................................................................... 187


Introduction ............................................................................................................................ 187
Background ............................................................................................................................. 187
Experiment Protocol ............................................................................................................... 188
Part A. Yttrium Citrate Colloid ............................................................................................. 189
Part B. Rhenium Heptasulphide Colloid, Method 1 ............................................................ 190
Part C. Rhenium Heptasulphide Colloid, Method 2 (Performed as a Demonstration only) 190
Part D. Analysis of Colloids .................................................................................................. 191
Questions ................................................................................................................................ 192

Lab 18: Formulating Using Molds ........................................................................................ 193


Introduction ............................................................................................................................ 193
Background ............................................................................................................................. 193
Experiment Protocol ............................................................................................................... 200
Part A. Formulating 325 mg Acetaminophen Suppositories (Calibrated Batch Volume
Method)............................................................................................................................... 201
Part B. Formulating 20 mg Benzocaine Lollipops (Mass of Drug Negligible) ...................... 203
Part C. Formulating 20 mg Hydrocortisone Troches (Displacement Factor Method)......... 205
Part D. Double Casting Method: 70 mg Hydrocortisone/150 mg Lidocaine Lip Balm ........ 206
Questions ................................................................................................................................ 209

APPENDIX .......................................................................................................................... 210

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6 Preface

U.S. Standard Sieve Sizes and Lab Sieve Inventory ................................................................. 210
Quadro Comil Meshes............................................................................................................. 211
Methocel and Avicel Grades ................................................................................................... 212
Avicel Grade Usage Chart........................................................................................................ 214
Capsule Properties .................................................................................................................. 215
Working Ranges of Typical Granulating Fluids ........................................................................ 216
Viscosities of Typical Fluids ..................................................................................................... 216
Powder Flowability Indices ..................................................................................................... 216
Average HLB Values of Some Surface Active Agents .............................................................. 217
General Physical Properties of Spans and Tweens ................................................................. 219
HLB Requirement for Some Common Oil Components .......................................................... 221
Buffer Solution Preparation: Polyprotonic Acids and Bases ................................................... 222
Dissociation Constants of Acids in Aqueous Solutions at 25°C ............................................... 223
Dissociation Constants of Bases in Aqueous Solutions at 25°C .............................................. 223
Sorensen Phosphate Buffers ................................................................................................... 224
Fundamental Lab Calculations ................................................................................................ 224
Preparing a Known Molar Concentration............................................................................ 224
Weight-Volume Percent (%w/v).......................................................................................... 225
Dilution Equation................................................................................................................. 225
How to Use a Syringe Filter ................................................................................................. 227
Capsule Filling: Quality Control ........................................................................................... 228
YOUR NOTES ........................................................................................................................... 230

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.

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Preface 7

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 2016/17


8 Introduction

Introduction
General Information
Check-in for the laboratory will be on September 15, 2016, 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 d.dubins@utoronto.ca

PHM340Y Teaching Assistants E-mail Address


Noor Al-Saden n.alsaden@mail.utoronto.ca
Giovanna Schver giovanna.medeiros@mail.utoronto.ca

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Introduction 9

Intro
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

PHC 340Y Lab Manual 2016/17


10 Introduction

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:

Raw Score _____


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

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Introduction 11

Intro
Laboratory and Lecture Schedule
All pre-labs and labs will take place in PB860.

Labs Lectures
(PB860) (PB255)
Fall Term 2016 Thursday Monday
9am-1pm 12am-1pm
Lecture 1 – Acid/Base Equilibria (Rob Macgregor) 12-Sep-16
Lab 1* – Safety Lecture, Locker Check-in, Examination of UV 15-Sep-16
Spectroscopy and Preparation of a Standard Curve
Lecture 2 – Phase Partitioning (Rob Macgregor) 19-Sep-16
Lab 2 – Preparation of pH Buffers 22-Sep-16
Lecture 3 – Mixing (Rob Macgregor) 26-Sep-16

Lab 3 – Effect of pH on the Partition Coefficient of a Slightly Soluble 29-Sep-16
Weak Acid
Lecture 4 – Polymorph and Salt (Ping Lee) 03-Oct-16
Lab 4* - Characterization of Drug Candidates (I) – Measuring Solubility 06-Oct-16
and pKa
Lab 5* – Characterization of Drug Candidates (II) – Co-solvency, Salt 13-Oct-16
Selection and Polymorph Identification
Lab 6* – Thermodynamics of Mixing – Enthalpy and Volume 20-Oct-16
Workshop: Writing Formal Lab Reports for PHC 340 (Heather Sanguins)
Lecture 5 – Chemical Kinetics & Stability (Ping Lee) 17-Oct-16
Lecture 6 – Rheology (Rob Macgregor) 24-Oct-16
Lab 7* – Examination of Viscosity and Suspending Agents 27-Oct-16
Lecture 7 – Diffusion and Membrane Transport 1 (Ping Lee) 02-Nov-15 31-Oct-16

Lab 8 – Kinetics of Acetylsalicylic Acid Hydrolysis 03-Nov-16
Lab 9 – Diffusion and Membrane Transport 1: Permeation Measurement 10-Nov-16
Lecture 9 – Colligative Properties (Rob Macgregor) (Note: this lecture 14-Nov-16
intentionally out of order)
Lab 11* – Tonicity and Pharmaceutics (Note: this lab intentionally out of 17-Nov-16
order)
Lecture 8 – Diffusion and Membrane Transport 2 (Ping Lee) 23-Nov-15 21-Nov-16

Lab 10 – Diffusion and Membrane Transport 2: Drug Release from 24-Nov-16
Ointment Bases
[Exercise 2: Quassignment for Lab 11]
Lecture 10 – Molecules at Interfaces (Rob Macgregor) 28-Nov-16
Lab 12* – Estimation of Critical Micelle Concentration (CMC) of a 01-Dec-16
Surfactant in Water
Lecture 11 – Particle Size and Powder Flow (Ping Lee) 05-Dec-17
Labs Lectures
Winter Term 2017 Thursday Wednesday
1pm-5pm 11a-12p
Lab 13* – Optimization of Powder Flow and Particle Size Determination 05-Jan-17
Lecture 12 – Pharmaceutical Granulation (Ping Lee) 11-Jan-17
Lab 14 – Pharmaceutical Granulations, Part 1 12-Jan-17
PHC340 Midterm 18-Jan-17

Lab 14 – Pharmaceutical Granulations, Part 2 19-Jan-17
Lecture 13 – Tabeting and Dissolution Testing (Ping Lee) 25-Jan-17
Lab 15 – Tableting and Dissolution Testing, Part 1 26-Jan-17
Lecture 14 – Measurement, Part 1 (Rob Macgregor) 01-Feb-17
Lab 15 – Tableting and Dissolution Testing, Part 2 02-Feb-17
Lecture 15 – Measurement, Part 2 (Rob Macgregor) 08-Feb-17
PHC 340Y Lab Manual 2016/17
12 Introduction


Lab 15 – Tableting and Dissolution Testing, Part 3 09-Feb-17
Lecture 16 – TBD (Keith Pardie) 15-Feb-17
Lecture 17 – TBD (Keith Pardie) 01-Mar-17
Lab 16 – Mystery Laboratory 16-Feb-17
Reading Week Feb 21-24

Lab 17 – Synthesis and Examination of Colloids 02-Mar-17
Lecture 18 – Mold Calculations (David Dubins) 08-Mar-17
Lab 18* – Formulating Using Molds 09-Mar-17
Lecture 19 – Ethics & Academic Integrity (Alison Thompson) 15-Mar-17
Lab 19 – Advanced Formulations Project, Part 1 16-Mar-17
Lecture 20 – Ethics & Academic Integrity (Alison Thompson) 22-Mar-17
Industrial Tour (to be confirmed) 23-Mar-17
Forensics Workshop (to be confirmed) 29-Mar-17

Lab 19 – Advanced Formulations Project, Part 2. Lab Check-Out. 30-Mar-17
PHC340 Final Exam Final exam period
* Lab Report to be completed for evaluation

Lab Worksheet to be completed for evaluation

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Introduction 13

Intro
Locker Check-In / Check-Out
Check-In: September 15th, 2016 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, 2017
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.

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.

Keep this and the following page in your laboratory manual.

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14 Introduction

Locker Contents 2016/17


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
Volumetric
Flask 10 mL Graduated Cylinder 1
25 mL Graduated Cylinder 1 Watch
Glass
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
Graduated Beaker
Cylinder
1 mL Bulb Pipette 1
5 mL Bulb Pipette 1
10 mL Bulb Pipette 1
20 mL or 25 mL Bulb Pipette 1 Evaporating
1 mL Graduated Pipette 1 Dish
10 mL Graduated Pipette 1
Thermometer (°C) 1
Watch Glass small 1
Watch Glass large 1
Erlenmeyer 8” Glass Stirring Rod 1
Mortar &
Flask 50 mL Beaker 2 Pestle
150 mL Beaker 2
250 mL Beaker 2
400 mL Beaker 2
600 mL Beaker 2
Glass Slab 1
3” Ceramic Evaporating Dish 1 Funnel
6” Ceramic Evaporating Dish 1 Clamp

Plastic Wash Bottle 1


Ceramic Mortar & Pestle Set (Glass set additional in some cases) 1
Funnel Clamp and Holders 1
Wax Pencil 1
TA Signature - Lab Check-In TA Signature - Lab Check-Out
Bulb Grad.
Pipette Pipette

PHC 340Y Lab Manual 2016/17


Introduction 15

Intro
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.

PHC 340Y Lab Manual 2016/17


16 Introduction

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 2016/17


Introduction 17

Intro
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

PHC 340Y Lab Manual 2016/17


18 Introduction

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
PHC 340Y Lab Manual 2016/17
Introduction 19

Intro
 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
PHC 340Y Lab Manual 2016/17
20 Introduction

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
PHC 340Y Lab Manual 2016/17
Introduction 21

Intro
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.

PHC 340Y Lab Manual 2016/17


22 Introduction

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:

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.

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

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.

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:

PHC 340Y Lab Manual 2016/17


Introduction 23

Intro
Fire Extinguisher Appropriate for: Examples:
Class
Class A Ordinary combustibles (paper, wood,
cardboard)
Class B Flammable/Combustible (gasoline, organic
Liquids and Gasses 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 2016/17


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

Lab 1: Examination of UV Spectroscopy and Preparation


of a Standard Curve
Read the introduction and lab protocol completely
Watch the following related lab videos on the laboratory website:
 UV/Vis Spectrophotometry - Determining Absorbance
Preparing for the Lab
(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
Part A: Prepare a calibration curve for hydrochlorothiazide
What You’ll Be Doing 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.

Io
loge  t
IT
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:
I0 
log10  t  Kt
IT 2.3026
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.

PHC 340Y Lab Manual 2016/17


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

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.

Lab 1
The equation is similar:
I0
(1) log 10  k1c
IT
If this is performed in a cell with a uniform thickness then a measure of the length l may be
added:
I0 I0
(2) log 10  k 1cl or log 10 A
IT IT
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.
0 .5

0 .4
E xtin ctio n

0 .3

0 .2

0 .1

0
0 1 2 3 4 5 6 7 8 9

ml Sta nda rd Fe (0. 0 2 mg Fe/ ml)

Figure 1. A Standard Curve for an Iron Solution

PHC 340Y Lab Manual 2016/17


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

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.
12

10

8
E ( 1% , 1 cm )

0
190 200 210 220 230 240 250 260 270 280 290

Wavelength

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 Plastic transfer pipettes Helios UV/Vis
Sodium Hydroxide Solution (0.1 N) UV Cuvettes (Plastic) Spectrophotometer Volumetric
Sodium Hydroxide (40.0 g/mol) Parafilm 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.

PHC 340Y Lab Manual 2016/17


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

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

Lab 1
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.

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.

*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

PHC 340Y Lab Manual 2016/17


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

Beam direction Beam


direction

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?

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Lab 2: Preparation of pH Buffers 29

Lab 2: Preparation of pH Buffers


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)
Preparing for the Lab
 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

Lab 2
Part A: Preparing Phosphate (Sorensen’s) Buffer @ pH 7.4
What You’ll Be Doing Part B: Preparing McIlvane’s Buffer at 2 assigned pH values
Demonstration: Using a pH meter
Spreadsheets You Will Need Not applicable.
Not Applicable. Retain and store buffers prepared in this lab for use
What You’re Handing In
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
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

PHC 340Y Lab Manual 2016/17


30 Lab 2: Preparation of pH Buffers

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:
Ka
(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) [H  ][A  ]
Ka 
[HA]
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:
10  pH [A  ]
(5) 10 -pKa 
[HA]

(6) [HA] 10 pKa



[A  ] 10 pH
[HA]
(7) 
 10 pKa pH
[A ]
According to the Henderson-Hasselbalch buffer relationship, pH, pKa, and the buffer component
concentrations for a weak acid are related as follows:
[acid]
(8)  10 pKa pH
[base]
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
solution.
 If the pKa is less than the pH, there will be more of the base form in the solution.

PHC 340Y Lab Manual 2016/17


Lab 2: Preparation of pH Buffers 31

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:
Kb
(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

Lab 2
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 pKa3 = 12.32
on weak acid buffers paired with salts of their conjugate
OH
bases.
Since it was already stated a buffer is most effective within 1 O P OH pKa2 = 6.86
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
OH pKa1 = 2.15
buffers have more than one acidic group attached, which
vary in affinity to their respective protons. Sorensen’s Buffer Phosphoric Acid
(phosphate buffer) has three acidic groups, each with
different pKa values (see right panel).

PHC 340Y Lab Manual 2016/17


32 Lab 2: Preparation of pH Buffers

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:

OH OH

- +
O P OH O P O Na

- + - +
O Na O Na
Sodium Phosphate Monobasic Sodium Phosphate Dibasic
weak acid salt 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)
OH OH

- +
O P OH O P O + H

- + - +
O Na O Na
sodium phosphate monobasic sodium phosphate dibasic
(acid) (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)
+
OH Na
OH
- +
O P O Na + H2O O P OH
+ HO
-

- + - +
O Na O Na
Sodium Phosphate Dibasic Sodium Phosphate Monobasic
(salt of conjugate base) (weak acid)

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

PHC 340Y Lab Manual 2016/17


Lab 2: Preparation of pH Buffers 33

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:

Lab 2
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):
[acid]
 10 pKa pH
(14) [base]
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) [acid]  [HA]  10 6.867.4  10 0.54  0.2884


[base] [A  ]
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
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

PHC 340Y Lab Manual 2016/17


34 Lab 2: Preparation of pH Buffers

(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 Sodium Phosphate Dibasic
(NaH2PO4*H20) (Na2HPO4*7H20)

m  C  MW  V m  C  MW  V
mol g mol g
m  0.2  137.99  0.250 L m  0.2  268.07  0.250 L
L mol L mol
m  6.900 g m  13.404 g

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 Sodium Phosphate Dibasic
C1 V1  C 2 V2 C1 V1  C 2 V2
C V C V
V1  2 2 V1  2 2
C1 C1
mol mol
0.0224  250 mL 0.0776  250 mL
V1  L V1  L
mol mol
0.2 0.2
L L
V1  28 mL V1  97 mL
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
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

PHC 340Y Lab Manual 2016/17


Lab 2: Preparation of pH Buffers 35

(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

Lab 2
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:

dn K C K [H  ] 
(23) β   2.303  w  [H  ]   buf a  2 
dpH  [H ] (K a  [H ]) 
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:
[H  ]  10  pH  10 7.4  3.9811  10 8
K a  10  K a  10 6.86  1.3804  10 7
K C K [H  ] 
β  2.303  w  [H  ]   buf a  2 
 [H ] (K a  [H ]) 
 1  10 -14
β  2.303   3.9811  10 8 
  
0.1 M   1.3804  10 7  3.9811  10 8  
8
(1.3804  10 7  3.9811  10 8 ) 2 
 3.9811  10 
β  0.04 mol/(L * pH)

PHC 340Y Lab Manual 2016/17


36 Lab 2: Preparation of pH Buffers

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:
Buffer Capacity of McIlvaine's Buffer
(0.2 M Sodium phosphate dibasic + 0.1 M Citric Acid)
120
100

 (mmol/(L*pH)
Buffer Capacity
80
60
40
20
0
2 3 4 5 6 7 8
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.
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”.

PHC 340Y Lab Manual 2016/17


Lab 2: Preparation of pH Buffers 37

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

Lab 2
Sodium Phosphate Monobasic N/A pH Meter
(verify MW on the bottle used) Mixing plate and magnetic stir
Sodium Phosphate Dibasic (verify bar
MW on the bottle used) 100 mL graduated cylinder
Citric Acid (MW 210.14 g/mol) 250 mL volumetric flask
pH Standardizing Buffers 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.
 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.

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38 Lab 2: Preparation of pH Buffers

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
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.

PHC 340Y Lab Manual 2016/17


Lab 2: Preparation of pH Buffers 39

 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, and store it for Lab 3.

Questions
1. Explain using the Henderson-Hasselbalch equation why the pH of the Sorensen’s buffer

Lab 2
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.

HO pKa = 9.24 HO
- OH
+
B OH + H2O
HO
B + H

HO OH

Boric Acid Borate

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?

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

Lab 3: Effect of pH on the Partition Coefficient of a


Slightly Soluble Weak Acid
Read the introduction and lab protocol completely
Watch the following related lab videos on the laboratory website:
Preparing for the Lab
 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)
Part A: Prepare a standard curve for salicylate
Part B: Salicylate partition experiment at 2 pH values (McIlvaine’s
What You’ll Be Doing 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
Individual formal lab report, due at the beginning of the next lab
What You’re Handing In
(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

PHC 340Y Lab Manual 2016/17


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

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) HAW  HA oil


K o/w

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:
[HA] oil
(2) K o/w 
[HA] w

Lab 3
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:

 H   A - w
HAW 
Ka
(3)
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) Na  A   Na     A 
w
-
w

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:
[H  ] w [A  ] w
(5) Ka 
[HA]w

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:

PHC 340Y Lab Manual 2016/17


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

Oil
(HA)o

Partition
Ko/w

(HA)w
Water
Ka
Dissociation - +
(A )w +(H )w

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:
[HA]oil [HA]oil
(8) K'o/w  

[HA]w  [A ]w Cw
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) [HA]oil  Ko/w[HA]w


Substitute (9) into (8):
K o/w[HA]w
(10) K'o/w 
[HA]w  [A  ]w

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

Simplify:
K o/w
(11) K'o/w 
 [A  ] w 
1   

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

[H  ] w [A  ] w
(12) [HA]w 
Ka
Now substitute Equation (12) into (11):
K o/w
(13) K'o/w 
 [A  ] w 
1    

Lab 3
 [H ] w [A ] w /K a 
Simplify:
K o/w
(14) K'o/w 
K
1  a
[H ] w
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:

1 1 K
(15)    a
K'o/w K o/w [H ]w K o/w
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
[HA]oil
Equation (8): K'o/w  . We measure Cw experimentally, using our ferric chloride assay on
Cw
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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44 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid

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 Plastic Cuvettes (at least 2) Spectronic 20
g/mol) Scintillation Vials spectrophotometer or Helios
Sesame Oil Purified 30 mL Syringe & 0.45 µm Syringe UV/Vis Spectrophotometer
Five McIlvaine's standard buffer filter pH Meter
solutions pH 3.5 – 4.5 (from Lab 2) Parafilm Mechanical Agitator
Hydrochloric Acid (2.0 N) Test Tubes, test tube rack 100 mL graduated cylinder
Ferric Chloride TS Class 2B lab goggles 25 mL graduated cylinder
Face Shield 10 mL graduated cylinder
Plastic Droppers

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

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:

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

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.
 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

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

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

Lab 3
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:
[HA]oil
Cw = [HA]w + [A-]w  K'o/w   K o/w
[HA]w

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

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

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
Part A: Determining the solubility of sulfathiazole
Part B: Preparing two different salts of sulfathiazole
Part C: Preparing two different polymorphs of sulfathiazole
What You’ll Be Doing 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

Lab 4
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
th
1. Gennaro AR, ed., Remington: The Science and Practice of Pharmacy, 20 ed., U.S.A.: Mack
Publishing Company, 2000, p. 700-720, 227-245, and 390-395
nd
2. Lachman L, Lieberman H A and Kanig J L, ed., The Theory and Practice of Industrial Pharmacy, 2
ed., U.S.A.: Lea & Febiger, 1976, p. 1-31.
3. Bates RG, Paabo M and Robinson RA, J. Phys. Chem., 67, 1833 (1963).
nd
4. Aulton ME, Pharmaceutics, the science of dosage form design, 2 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).

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

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:

PHC 340Y Lab Manual 2016/17


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

 S  S0  (for weak acids)


log  T   pH  pK a
 S0 
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.
Ka
(1) [HA]  [H+] + [A-]
Acid Proton Conjugate Base

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

Lab 4
[acid]
(3)  10 pKa pH
[base]
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:
Kb
(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:

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.

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.

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

pH Titration Curve
dV/dpH vs pH
12
8
pKa

dV/dpH (mL/pH unit)


10
6
pH

8
4
6 2
4 0
0 1 2 3 4 5 6 7 8 9 10 11 5 6 7 8 9 10 11 12
Volume NaOH Added (mL) pH

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-]:
K a [HA]
(6) [A  ] 
[H  ]
Now substitute (6) into equation (5):
K a [HA]
(7) ST  [HA] 
[H  ]
 K 
(8) ST  [HA] 1  a 
 [H ] 
Using the identities in (2) and (3),
 10 -pKa 
(9) ST  [HA] 1  pH 
 10 
(10) 
ST  [HA] 1  10 pHpKa 

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

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) 
ST  S0 1  10 pHpKa 
Finally, we can re-arrange this equation into a form similar to the Henderson-Hasselbalch
equation:
 S S 
(12) log  T 0   pH  pK a (for weak acids)
 S0 
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

Lab 4
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:

Weak
WeakAcid:
Acid: Weak
WeakAcid:
Acid:
Total
Total Solubilityvs.
Solubility vs.pH
pH %% Ionizedvs.
Ionized vs.pH
pH
% of drug in ionized form
% of drug in ionized form
Total Solubility (mg/mL)

1000 100
Total Solubility (mg/mL)

1000 100

7575
100
100
5050
1010
2525

11
0 0
00 22 44 66 88
0 0 2 2 4 4 6 6 8 8
0.10.1
pH
pH pH
pH

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:

 S  S0 
(13) log  T   pK a  pH (for weak bases)
 S0 
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

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

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:

Weak
WeakBase:
Base: Weak
WeakBase:
Base:
Total
TotalSolubility
Solubilityvs.
vs.pH
pH %
%Ionized
Ionizedvs.
vs.pH
pH

% of drug in ionized form


% of drug in ionized form
10000
10000 100
100
Total Solubility (mg/mL)
Total Solubility (mg/mL)

1000
1000 7575

100
100 5050

1010 2525

11 00
55 66 77 88 9 9 1010 1111 1212 1313 1414 55 66 77 88 9 9 1010 1111 1212 1313 1414
0.1
0.1
pH
pH pH
pH

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

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

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

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

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 The “official” start of the melt: liquid clearly appears in equilibrium
Point 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 / The substance becomes completely liquid. There are no longer any
Liquefaction Point 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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Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa 57

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 metasable 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 Glass Cover Slips pH Meter
g/mol) Capillary Tubes Graduated Cylinder
Ethanol 95% Grid Slide 25 °C Water Bath
1-Propanol Scintillation Vials 80 °C Water Bath
Sorenson’s Phosphate Buffer pH 7.4 Light Microscope

Lab 4
(from Lab 2) Melting Point Apparatus
Potassium acetate (MW 98.14
g/mol)
Calcium acetate (MW 158.17 g/mol)
Sodium Hydroxide (MW 40.0 g/mol)

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

 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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Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa 59

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
10% 10% 20% 20% 30% 30%
volume*
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.

Lab 4
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

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

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.
Notes:
 only use a small amount (overfilling will result in uneven heating, broader ranges)
 Pack the sample well (loose samples will heat unevenly)

 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)

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Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa 61

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.

Lab 4
Hint 1: Fraction of drug ionized = [A  ]
[HA]  [A  ]

[HA]
Hint 2: For a weak acid,  10 pKa pH
[A - ]

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

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
You will be working in groups of 3 students
Group Allocation
(work in the same groups as Lab 4)
Part A: Determining the solubility of benzocaine in different
concentrations of ethanol
What You’ll Be Doing
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

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Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph 63
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).
nd
2. Lachman L, Lieberman H A and Kanig J L, ed., The Theory and Practice of Industrial Pharmacy, 2
ed., U.S.A.: Lea & Febiger, 1976, p. 32-77, 541-566.

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

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) Capillary Tubes Burette
Benzocaine Burette Clamp
Ethanol 95% 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
 Record the following results:

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Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph 65
Identification

- 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.

Lab 5
 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.

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66 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

Lab 6: Thermodynamics of Mixing – Enthalpy and Volume


Read the introduction and lab protocol completely
Preparing for the Lab
Calculate the mass of potassium hydroxide required for Part C
Group Allocation You will be working in groups of 2 students
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
What You’ll Be Doing 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,
nd
2 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,

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Lab 6: Thermodynamics of Mixing – Enthalpy and Volume 67

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 system loses heat:


(exothermic)
Q = mcp(Tfinal – Tinitial)
Since Tfinal < Tinitial,
Q < 0.

Lab 6
If system gains heat:
(endothermic)
Q = mcp(Tfinal – Tinitial)
Since Tfinal > Tinitial,
Q > 0.

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)

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68 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

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 Measured Tfinal Qhot = mhotCp,w(Tfinal-Thot)
hot water, ~60°C) Observed temperature
at equilibrium
Cold Water Measured Tcold ~ambient Measured Tfinal Qcold = mcoldCp,w(Tfinal-Tcold)
Observed temperature
at equilibrium
Calorimeter Measured Tcold ~ambient Measured Tfinal Qcal = Cp,cal(Tfinal-Tcold)
Observed temperature
at equilibrium
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:
Q cal
(7) C p,cal 
(Tfinal  Tcold )
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 Measured Tfinal QCu= mCuCp,Cu(Tfinal-TCu)
boiling water, ~100°C) Observed temperature
at equilibrium
Cold Water Measured Tcold Measured Tfinal Qw = mwCp,w(Tfinal-Tcold)
~ambient Observed temperature
at equilibrium

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Lab 6: Thermodynamics of Mixing – Enthalpy and Volume 69

Calorimeter Measured Tcold Measured Tfinal Qcal = Cp,cal(Tfinal-Tcold)


~ambient Observed temperature
at equilibrium
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.

Lab 6
 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) Y  n1 Y 1  n2 Y 2  .....  ni Y i  .......
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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70 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

_  Y 
(10) Y i   
 ni  T , P ,n1 ,n2 .....
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) V  n1 V1  n2 V2

And just like Equation (10), the partial molar volumes of a two component system can be
defined as:
_  V 
(12) V1   
 n 1 T , P, n2
_  V 
(13) V 2   
 n 2 T , P, n1

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 Styrofoam cup Hot plate
56.11 g/mol) N95 Mask Thermometer
Sodium Hydroxide Pellets (MW Thermos
40.00 g/mol) Copper tube
Ethanol (95%) Tongs
Hydrochloric Acid (1.0 N)

The following solutions are prepared or provided by the TA:


 Hydrochloric Acid (1.0 N)

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Lab 6: Thermodynamics of Mixing – Enthalpy and Volume 71

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 200 mL 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.

Lab 6
 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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72 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume

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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Lab 6: Thermodynamics of Mixing – Enthalpy and Volume 73

 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.
 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?
 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.

Lab 6

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74 Lab 7: Examination of Viscosity and Suspending Agents

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
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.
What You’ll Be Doing 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
th
1. Schott H, “Rheology”, in Gennaro AR ed., Remington: The Science and Practice of Pharmacy, 20
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.

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Lab 7: Examination of Viscosity and Suspending Agents 75

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 dynes/cm2 or g/(cms), 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 Newtons/m2 or Pascals, which equals 10 P. As the value of viscosity,  ,
decreases, less and less stress, σ, is required to maintain the shear rate, γ.
σ
(1)  = γ
The unit of viscosity is pascal-second (Pas), or 10 P.
1 Pas = 10 P = 10 dynes/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).

Lab 7
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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76 Lab 7: Examination of Viscosity and Suspending Agents

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 A B
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

4.
arm B, and stop at G (when the upper bulb is filled).
Cover the hole in arm A with your thumb, and remove the
G
pipette bulb from arm B. Get a stopwatch ready to start
timing. F
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. 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 D
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
C
called relative viscosity, ηrel.

Relative viscosity:

η1 η test ρ t
(2) η rel    test test
η 2 η water ρ watert water
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:

 ρ t 
(3) η test  η water  test test 
 ρ watert water 
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

PHC 340Y Lab Manual 2016/17


Lab 7: Examination of Viscosity and Suspending Agents 77

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:

liquid sample

h Rb

Rc

The viscosity is calculated by means of the Margules equation


(1/Rb2 – 1/Rc2) T
(4)  = 4h 
= 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

Lab 7
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

PHC 340Y Lab Manual 2016/17


78 Lab 7: Examination of Viscosity and Suspending Agents

at infinite dilution. The intrinsic viscosity is a characteristic of a particular temperature-polymer-


solvent system.

Intrinsic viscosity:
 sp
(7)    lim
c 0
 red  lim
c 0 c
Moreover, the intrinsic viscosity of a polymer solution is proportional to its viscosity-averaged
molecular weight, Mv, according the Mark-Houwink equation:

[  ] = K×Mv
a
(8)
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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Lab 7: Examination of Viscosity and Suspending Agents 79

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:

Lab 7
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:

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80 Lab 7: Examination of Viscosity and Suspending Agents

2r 2 (ρ s  ρ L )g
V 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) Glass beads Size 300 Ostwald Viscometers
Methylcellulose stock solution (1.0% (100 too narrow for this lab)
Methylcellulose USP 1500 cps in de- Brookfield Viscometer
ionized water) Anton Paar Handheld Density
Hydrochloric Acid (0.1 N) 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
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.

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Lab 7: Examination of Viscosity and Suspending Agents 81

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.

 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 2% methylcellulose solution at
each speed of the viscometer (ranging from approx. 3-60 rpm, depending on the model
used).
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

Lab 7
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.

 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.
 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.

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82 Lab 7: Examination of Viscosity and Suspending Agents

 Place the cap on the top of the cylinder and mix thoroughly until homogeneous.
 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
Hint: See
Stoke’s Law, Equation (9) in the Background section, assume L = 1 g/cm3.

PHC 340Y Lab Manual 2016/17


Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis 83

Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis


Read the introduction and lab protocol completely
Watch the following related lab videos on the laboratory website:
Preparing for the Lab
 Measuring pH
(http://phm.utoronto.ca/~ddubins/DL/pH.wmv)
Group Allocation You will be working in groups of 3 students
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
What You’ll Be Doing
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
Individual formal lab report, due at the beginning of the next lab
What You’re Handing In
(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). Lab 8
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)

PHC 340Y Lab Manual 2016/17


84 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

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:

A  B
drug degradation
products

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.

dC A (t)
(1) Reaction Rate =  k
dt
Integrating equation (1):
CA (t) t
(2)  dC
CA (0)
A (t)   k  dt
0

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) CA (t)  const  CC (t)(0)  kt  const  0t
A

(4) C A (t)  C A (0)  k(t  0)


(5) C A (t)  C A (0)  kt Zero-Order Degradation
From equation (5), we can see that the drug 120

starts out at a concentration of CA(0), and 100


degrades linearly with time, with a slope equal y = -1.5x + 100
C A (mg/mL)

80
to k. Here is a zero-order degradation plot of a 60
drug with k=1.5 mg/(mL×h), and CA(0) = 100 40
mg/mL (right panel).
20
For a zero-order reaction, a plot of the 0
concentration of A versus time is a straight line, 0 10 20 30 40 50 60
with a slope equal to k. Time (h)

PHC 340Y Lab Manual 2016/17


Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis 85

First-order reaction: The reaction rate is dependent on the first power of concentration of a
single reactant.
dC A (t)
(6) Reaction Rate =  kC A (t)
dt
Integrating equation (6):
CA (t) t
dC A (t)
(7)

CA (0)
C A (t)
  k  dt
0

(8) ln(C A (t))  const  CC (t)(0)  kt  const  0t


A

(9) ln(C A (t))  ln(C A (0))  k(t  0)

 C (t) 
(10) ln  A    kt
 C A (0) 
Equation (10) can be re-arranged to solve for CA(t), the concentration of “A” as a function of
time:
 C (t) 
ln A 
(11) e  C A (0) 
 e kt
C A (t)
(12)  e  kt
C A (0)

(13) CA (t)  CA (0)ekt


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:

First-Order
First-Order
Degradation
Degradation First-Order
First-Order
Degradation
Degradation
120120 (semi-log
(semi-log
plot)
plot)
100100 100100
y = y100e -0.08x
= 100e -0.08x
C A (mg/mL)
C A (mg/mL)

80 80
C A (mg/mL)
C A (mg/mL)

60 60 -0.08x
-0.08x
y = y100e
= 100e 10 10
40 40

20 20

Lab 8
0 0 1 1
0 0 10 10 20 20 30 30 40 40 50 50 60 60 0 0 10 10 20 20 30 30 40 40 50 50 60 60

Time
Time
(h)(h) Time
Time
(h)(h)

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.

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

PHC 340Y Lab Manual 2016/17


86 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

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):

 0.5  C A (0) 
(14) ln     k  t1/2
 C A (0) 
(15) ln 0.5  k  t1/2
Solving for t1/2:
- ln 0.5 ln(2)
(16) t 1/2  
k k
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:

 0.9  C A (0) 
(17) ln     k  t 0.9
 C A (0) 
(18) ln 0.9  k  t 0.9
- ln 0.9 
(19) t 0.9 
k
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:
  Ea 
 
(20) k  Ae  RT 

where:
A: frequency factor
Ea: activation energy
R: gas constant, 8.314 J/(mol*K), or 1.987 cal/(mol*K)
T: temperature (in Kelvin)

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Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis 87

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:
Ea
(21) ln(k)  ln( A) -
RT
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):
- ln(0.9)
(22) t 90%   Ea 
- 
Ae  R(294.15K) 

Kinetics of ASA Hydrolysis


The reaction of interest in this lab is the degradation of acetylsalicylic acid to salicylic acid in
water:
(23)
O
O
OH
OH
OH
+ H2O +
O O CH3
OH
Salicylic Acid Acetic Acid
O CH3
(SA)
Acetylsalicylic Acid
(ASA)

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.
Lab 8
The rate of hydrolysis which accounts for these factors is:
d([ASA]  [ASA  ])
 k 1 [H 3 O  ][ASA]  k 2 [H 2 O][ASA]  k 3 [OH  ][ASA]
(24) dt
 k 4 [H 3 O  ][ASA - ]  k 5 [H 2 O][ASA - ]  k 6 [OH  ][ASA - ]
Simplifying Equation (2):

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88 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

d([ASA]  [ASA  ])
(25) dt

 [ ASA] k 1 [H 3 O  ]  k 2 [H 2 O]  k 3 [OH  ] 

 [ASA - ] k 4 [H 3 O  ]  k 5 [H 2 O]  k 6 [OH  ]
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:
d[ASA T ]
(26)  k[ASA T ]
dt
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) m ASA  m drug  mSA

Finally, the initial concentration of ASA ([ASAT]t=0) in mmol/L is:


m ASA
(29) [ASA T ]t 0 
180.16 mg/mmol  0.050 L
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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Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis 89

Experiment Protocol
Chemicals Supplies Special Equipment
Acetylsalicylic acid (MW 180.16 g/mol) 0.45 µm syringe Helios UV/VIS
Sodium Salicylate (MW 160.11 g/mol) filters spectrophotometer and
Hydrochloric acid (0.1 N) 3 cc plastic syringe cuvettes
Ethanol 95% Plastic test tubes Three constant temperature
Sodium Hydroxide Pellets (MW 40.00 g/mol) baths (40 °C, 50 °C, 60 °C)
Sodium Phosphate Dibasic (MW 268.07 g/mol)
Ferric Nitrate (2% Fe(NO3)3 in de-ionized
water)
Prepared buffers (pH 2, 4, 6, 8)

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.)
Volume of Stock Volume of Stock q.s. to Final
Buffer Type pH
Solution A Solution B Volume
Clark Lubs Buffer KCl/ HCl 125 mL of 0.2 N KCl 26.5 mL of 0.2 N HCl 500 mL 2
192.5 mL of
McIlvaine’s Buffer 307.5 mL of
Citric Acid/ Phosphate 0.1 M citric acid
0.2 M sodium Do not q.s. 4 Lab 8
phosphate dibasic
428.6 mL of
Potassium Phosphate 71.4 mL of
0.2 M potassium Do not q.s. 6
Buffer 0.2 M NaOH
phosphate monobasic
250 mL of
Clark Lubs / 13 mL of
0.1 M boric acid in 500 mL 8
Borate Buffer 0. 1 N NaOH
0.1 M KCl
250 mL of
Clark Lubs / 220 mL of
0.1 M boric acid in 0.1 500 mL 10
Borate Buffer 0.1 N NaOH
M KCl

PHC 340Y Lab Manual 2016/17


90 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

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. Cover the container with aluminum foil.
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.

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 [ASA]T ln[ASA]
Salicylate ASA
Concentration Concentration
(mM) (mM) [corrected
[from the for salicylate
standard curve] present at t=0]

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

PHC 340Y Lab Manual 2016/17


Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis 91

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
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.
 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 Lab 8
absorbance at 525 nm quickly (since the ASA will continue to hydrolyze).
 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.

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92 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis

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
 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?

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Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement 93

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
Part A: Preparing a standard curve for salicylate
What You’ll Be Doing 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).
nd
4. Diffusion: Mass Transfer In Fluid Systems, 2 Ed. E.L. Cussler, 1996, Chapter 17.
nd
5. Physicochemical Principles of Pharmacy, 3 Ed., A.T. Florence and D. Attwood, 1998, PP. Chapters 3
and 8.
6. Riviere, J.E. Comparative Pharmacokinetics: 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
Lab 9

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:

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94 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

1  dn 
J(t)   
A  dt 
(1)
 mol   1   mol 
 2   2  
 cm sec   cm   sec 
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:
dC(t)
J(t)  D 
dx
(2)
 mol   cm 2   mol 
 2     3 
 cm sec   sec   cm  cm 
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.

Flux

C
C1m
C1
V1 C m2 V2

C2
Fluid 1 Fluid 2
Compartment 1 Membrane Compartment 2
x
0 h
Figure 1. Schematic Diagram of a Membrane Diffusion System.

PHC 340Y Lab Manual 2016/17


Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement 95

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) C10 V1  C1 (t)  V1  C2 (t)  V2


Thus, at any point in time we can re-arrange Equation (4) to solve for C1:

C10 V1  C 2 (t)V2 V 
(5) C1 (t)   C10  C 2 (t) 2 
V1  V1 

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:

 dC(t) 
(6) J(t)  D 
 dx 
In our model, we assume Compartments 1 and 2 are well stirred, and the volumes V1 and V2 do
not change throughout diffusion.
h Cm
2

(7)  J(t)dx  D  dC(t)


0 C1m

(8)  
J(t) x 0  DC(t) C2m
h Cm
1

(9) 
J(t) h  0  D Cm2 (t) - C1m (t) 
(10) J(t) 
D m
h

C1 (t)  C m2 (t) 
Rapid Partitioning into the Membrane
Lab 9

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

PHC 340Y Lab Manual 2016/17


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:
C1m Cm
(11) K1  ; K2  2 ;
C1 C2

Making this assumption of rapid equilibration allows us to substitute the membrane equilibrium
concentrations (K1C1 and K2C2) into Equation (10):

(12) J
D
K1C1  K 2 C 2 
h
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) J
DK
C1  C 2 
h
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.

C C C
C 10 C1m C1 C1m
C1m C m2
C m2
C1 C2
C2
C m2 C02  0
x x x
0 h 0 h 0 h
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.

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Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement 97

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
collecting all of the coefficients into one, and calling it the bulk membrane permeability
parameter, Pm:
(14) J  Pm C1  C 2 

DK
Where Pm 
h
Going back to Equation (1), the flux (the number of moles per second×cm2) leaving
Compartment 1 can be expressed as:
1  dn  V1  dn/V1   V1  dC1 
(15) J1       
A  dt  A  dt  A  dt 
Similarly, the flux entering Compartment 2 can be expressed as:
1  dn  V2  dn/V2   V2  dC 2 
(16) J2       
A  dt  A  dt  A  dt 
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:
V2  dC 2 
(17)    Pm C1  C2 
A  dt 
We can use Equation (5) to express C1 in terms of C2 in Equation (17):

V2  dC 2   0 V  
(18)    Pm   C1  C 2 2   C 2 
A  dt   V1  
Simplifying:

 dC 2   C0 1 1 
(19)    Pm A 1  C2    
 dt   V2  V1 V2  
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:
C2 t
1
(20)   C10  
dC 2  Pm A  dt
C02 0
  C2  1  1   0
V  V V 
 2  1 2 
Lab 9

C2

(21)
 1
1
1  V
 C0  1 1 
ln  1  C 2      
t
 Pm A t 0
     2  V1 V2  
 V1 V2  0

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98 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

 C0  1 1    C0   1 1 
(22) ln  1  C 2     ln  1   Pm A 

(t - 0)
 V2  V1 V2    V2   V1 V2 

 C10  1 1 
  C2    
(23) V
ln  2  V1 V2    P A 1  1 t
C10  m  
   V1 V2 
 V2 
 
 C V  1 1 
(24) ln 1  02  2  1   Pm A   t
 C1  V1    V1 V2 

 C2  V2 
Experimentally, by plotting ln 1    1  vs. time, the solute permeability Pm can be
 C10
  V1 
evaluated from the slope of the linear plot according to:
 1 1 
(25) slope  Pm A  
 V1 V2 
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):
 1 1 
C  V2   Pm A    t
(26) 1  02   1  e  V1 V2 
C1  V1 
 1 1 
C2  V2   Pm A    t
(27)  
 1  1  e  V1 V2 
C10  V1 
 V1   Pm A    t 
 1 1 
(28)  V1 V2  
C 2 (t)  C10   1  e
  
 1
V V2 
 
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:

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Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement 99

 V1 
(29) C 2 (t  )  C10  
 V1  V2 
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) Plastic UV cuvettes UV/VIS spectrophotometer
Sodium Salicylate Stock Solution (1.0 M) Pipettes (1 and 5 mL) 2 L beaker
Dialysis tubing (7-8 cm) with Volumetric Flasks (50, 100, 500,
closures 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):

Fill line (fill to at


least here)

Spectrophotometer beam
travels this way
Lab 9

 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

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100 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement

(e.g. OD210).

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:
 Filling Volume (mL) 
Tubing length    Length of Closures  Length of Protruding Ends   1.5
 Volume Capacity (mL/cm) 
(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:
 5 mL 
Tubing length    1 cm  2  1 cm  2 1.5  8.3 cm
 3.3 mL/cm 
 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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Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement 101

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.
  C  V 
Based on the measured C2, calculate and tabulate 1   2 1  2  as a function of time (t in
 0  
  C1  V1 
sec.).
 
 Plot ln 1   C 2 1  V2  versus time, and draw a best-fit line through all data points.
 0  
  C1  V1 

 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
Lab 9

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:

 V1   Pm A    t 
 1 1 

C 2 (t)  C 
0
 1  e  V1 V2  
 
 V1  V2 
1


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 103

Lab 10: Diffusion and Membrane Transport (II) – Drug


Release from Ointment Bases
Read the introduction and lab protocol completely
Preparing for the Lab
Calculate the dilution scheme for the standard curve
Group Allocation You will be working in groups of 5 students
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
What You’ll Be Doing
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
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:
What You’re Handing In 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
Lab 10

1. United States Pharmacopoeia XXII, United States Pharmacopoeia Convention, Rockville, MD, 1990.
2. The Pharmaceutical Codex, 11th ed., Pharmaceutical Press, London, England, 1983.

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104 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

th
3. Gennaro AR, ed., Remington’s Pharmaceutical Sciences, 20 ed., Lippincott Publishing Co: U.S.A.
2000.
th
4. Martin AN, Physical Pharmacy, 4 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

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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 105

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
Lab 10

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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106 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

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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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 107

SUMMARY CHART: PROPERTIES OF OINTMENT BASES


Oleaginous
Oil/Water Water-miscible
Ointment Absorption Ointment Water/Oil Emulsion
Property of Emulsion Ointment
Bases Bases Ointment Bases
Base Ointment Bases Bases
(Hydrocarbon)

oleaginous oleaginous base + oleaginous base + oleaginous base Polyethylene


compounds w/o surfactant water (< 45% w/w) + water (> 45% Glycols (PEGs)
Composition + w/o surfactant w/w) + o/w
(HLB <8) surfactant (HLB
>9)
Water anhydrous anhydrous hydrous hydrous anhydrous,
Content hydrous
Affinity for hydrophobic hydrophilic hydrophilic hydrophilic hydrophilic
Water
very little some, forms a w/o will take up more will take up a will only take
emulsion water, eventually great deal of up a small
Ability to the emulsion may water becoming amount of
Take up break (phase fluid water before
Water separation) or form becoming fluid
an o/w emulsion
(phase inversion)
difficult difficult moderate to easy easy moderate to
Spreadability
easy
non-washable non-washable non- or poorly washable washable
Washability
washable
oils poor; oils poor; unstable, especially unstable, stable
hydrocarbons hydrocarbons better alkali soaps and especially alkali
Stability better natural colloids soaps and
natural colloids;
nonionics better
solids or oils solids, oils, and solids, oils, and solid and solid and
Drug
(oil solubles aqueous solutions aqueous solutions aqueous aqueous
Incorporation
only) (small amounts) (small amounts) solutions (small solutions
Potential
amounts)
Occlusiveness yes yes sometimes no no
protectants, protectants, emollients, emollients, drug vehicles
emollients, emollients, vehicles cleansing creams, vehicles for
vehicles for for aqueous vehicles for solid, solid, liquid, or
Uses
hydrolyzable solutions, solids, and liquid, or non- non-
drugs non-hydrolyzable hydrolyzable drugs hydrolyzable
drugs drugs
White Hydrophilic Cold Cream type, Hydrophilic PEG Ointment,
Petrolatum, Petrolatum, Hydrous Lanolin, Ointment, Polybase™
White Anhydrous Lanolin, Rose Water Dermabase™,
Examples
Ointment Aquabase™, Ointment, Velvachol®,
Aquaphor®, Hydrocream™, Unibase®
Polysorb® Eucerin®, Nivea®
Source: http://pharmlabs.unc.edu/labs/ointments/bases.htm
Lab 10

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108 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

Experiment Protocol
Chemicals Supplies Special Equipment
Ointment Bases (see specific procedures Cellophane membrane Dermamill 100 Ointment Mill
for ingredients) (dialysis tubing), 45 mm flat Spectrophotometers
 (Hydrocarbon) Simple Ointment width MWCO 12-14,000 Stopwatch
USP, or white petrolatum Elastic bands Hot plate
 (Absorption) Hydrophilic One-ounce ointment jar Evaporating Dish
Petrolatum USP, Aquaphor Plastic Cuvettes Hard Rubber Spatula (Green or
 (w/o emulsion) Cold Cream USP, Plastic Transfer Pipets Black)
or Nivea Cream Burette, burette clamp, retort
 (o/w emulsion) Hydrophilic stand
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

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)
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.
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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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 109

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
Lab 10

 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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110 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

 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)

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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 111

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 5g
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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112 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

2. Absorption Base

e.g. Hydrophilic Petrolatum USP


1. Prepare hydrophilic petrolatum as follows:

cholesterol 3g
stearyl alcohol 3g
white wax 8g
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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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 113

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 70C; maintain at 70°C for
5 minutes.
6. In a separate beaker, dissolve the sodium tetraborate in the purified water, warmed to
70C.
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 homogeneous 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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114 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

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- 0.025 g 0.025 g 0.025 g
hydroxy benzoate)
propylparaben (n-propyl p- 0.015 g 0.015 g 0.015 g
hydroxy benzoate)
sodium lauryl sulfate 1g 1g 1g
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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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 115

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 65C)
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.
Lab 10

 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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116 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

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:

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.

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

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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 117

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
Lab 10

edges).
 Secure the membrane in place with an elastic band.

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118 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

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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Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases 119

 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
Lab 10

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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120 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases

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 121

Lab 11: Tonicity and Pharmaceutics


Read the introduction and lab protocol completely
Preparing for the Lab 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
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
What You’ll Be Doing
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 Vapro and Advanced Instruments
Osmometers
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,
th
pages 208 – 262, 20 Edition, Lippincott, Williams and Wilkins, Philadelphia, 2000.
th
2. Martin, A , Physical Pharmacy, 5 edition, Chapters 5 and 6, pages 101-142, Lee and Febinger,
Philadelphia, 1993.
3. https://online.epocrates.com/u/10a308/atropine
Lab 11

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122 Lab 11: Tonicity and Pharmaceutics

Background

Tonicity Definitions

This is the net movement of water across a selectively permeable membrane driven by a
Osmosis
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.

This is an expression of osmol concentration and a solution has an osmolol concentration of


Osmolality 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
23
contain 6.02 x 10 molecules/kg of water and a 1 osmol solution of sodium chloride will
23
also contain 6.02 x 10 total ions/kg of water with ½ being sodium and the other ½ being
chlorine.

This is an expression of osmolar concentration and a solution has an osmolar concentration


Osmolarity 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.

This is the effective osmolality equal to the sum of the concentrations of the solutes which
Tonicity have the ability to exert an osmotic force across a membrane.

Any given two solutions are isosmotic if they have the same total osmolarity (or osmolality).
Isosmotic

An isotonic solution is isosmotic with physiological tonicity (i.e. 0.9% NaCl). A net flow of
Isotonic water across cell membranes is not observed in cells placed in an isotonic solution.

A hypertonic solution has a higher osmotic pressure when compared with physiological
Hypertonic tonicity (i.e. 0.9% NaCl). Cells placed in a hypertonic fluid will lose cellular water, and shrink.

A hypotonic solution has a lower osmotic pressure when compared with physiological
Hypotonic 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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Lab 11: Tonicity and Pharmaceutics 123

Appendix A: Sodium Chloride Equivalents, Freezing-Point Depressions, and Hemolytic Effects of Certain Medicinals
in Aqueous Solution
ISO-OSMOTIC
0.5% 1% 2% 3% 5%
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
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
phosphate
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,
0.29 0.168 0.27 0.47 3.33 0.27 0.52 0 9.2
monobasic
Sodium phosphate, dibasic
0.42 0.24 2.23 0.40 0.52 0 9.2
(2 H2O)
Sodium phosphate, dibasic
0.22 0.21 4.45 0.20 0 9.2
(12 H2O)
Potassium phosphate
0.44 0.25 2.18 0.41 0.52 0 4.4
monobasic
Potassium phosphate
0.46 0.27 2.08 0.43 0.52 0 8.4
dibasic

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

 0.1% 
D 0.1%  D 0.5%   
 0.5% 
 0.1% 
D 0.1%  0.05 C   
 0.5% 
D 0.1%  0.01 C
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:
ΔTf, reqd  ΔTf, ref  ΔTf, drug
ΔTf, reqd  0.52 C  0.01 C  0.51 C

Now we calculate the concentration of 0.9% NaCl needed to lower the freezing temperature by
the difference, 0.51 °C:
 ΔTf, reqd 
[NaCl] reqd  [Reference ]   
 ΔTref 
 0.51 C 
[NaCl] reqd  0.9% NaCl     0.883% NaCl
 0.52 C 
Converting this to mass of NaCl required:

0.883 g NaCl
m NaCl   30 mL  0.265 g NaCl
100 mL
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:
 0.51 C 
[Dextrose]reqd  1% Dextrose     5.60 % Dextrose
 0.091 C 
mDextrose  5.60%  30 mL  1.68 g Dextrose
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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Lab 11: Tonicity and Pharmaceutics 125

0.9 g NaCl
 30 mL  0.270 g NaCl
100 mL
 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%:
m NaCl 0.18 g NaCl
E 
mdrug,0.5% 1 g drug

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:
0.18 g NaCl 0.1 g drug
  30 mL  0.0054 g NaCl
1 g drug 100 mL

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:
m difference  m reference  m drug
m difference  0.270 g  0.0054 g  0.265 g
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.
Lab 11

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126 Lab 11: Tonicity and Pharmaceutics

Experiment Protocol
Chemicals Supplies Special Equipment
Atropine Sulfate Erythrocytes Vapour Pressure Osmometer
Sodium Chloride (NaCl MW 58.44 g/mol) Glass Slide (VAPRO 5520)
Sodium Phosphate Monobasic (verify Glass Cover Slip Freezing Point Depression
MW on the bottle used) Test tubes Osmometer (Advanced
Sodium Phosphate Dibasic (verify MW Scintillation Vials Instruments 3250)
on the bottle used) pH meter
5.0% Atropine Sulfate Stock Solution Microscope
0.2 M Sodium Phosphate Monobasic
0.2 M Sodium Phosphate Dibasic

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?

VAPRO 5520 Protocol (Vapour Pressure Osmometer)


The VAPRO 5520 comes with its own micropipette, and only
requires 10 µL per sample.
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:
 Rotate the sample chamber crank on the left to the
“OPEN” position, and slide out the black sample holder
(lower black tab on the front of the machine):

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Lab 11: Tonicity and Pharmaceutics 127

Sample chamber crank Slide sample holder open


 With forceps or tweezers, gently place a paper sample disk in the inner circle of the
sample holder. Don’t touch the sample holder with the forceps. Make sure you have
only picked up one disk with the forceps (they tend to stick together). You can use a
needle tip to separate multiple disks.
NOTE: Avoid touching the disk, or the metal part of the sample
holder with your fingers – it will contaminate the sample.
 Load a plastic pipette tip on the micropipette. Depress the
pipette plunger and submerge the plastic tip just below the
liquid level of the sample. Suck up the sample by releasing
the pipette plunger.
 Align the pipette straight up and down in the V-shaped
groove above the sample holder. Aim right for the centre of
the paper sample disk.
 Push the plunger down on the micropipette to release the
sample on the paper sample disk. Make sure there are no
bubbles (pop them with the micropipette tip if there are
any). Do not get the sample anywhere but on the disk. If you
do, wipe the sample holder clean and start with a new paper
sample disk, and a fresh 10 µL sample.
 Lightly push the paper sample disk downwards with the tip of the pipette, to remove
any air in between the disk and the sample holder.
 Slide in the sample holder, and rotate the sample chamber crank to the “CLOSE”
position.
 The measurement will take approx. 80 seconds, and read osmolality in mmol/kg.
 Record your osmolality measurement.
 Rotate the sample chamber crank to “open”, and slide out the sample holder.
 With a clean, fresh Kimwipe, remove the paper sample disk, discard it, and clean the
metal sample holder of all traces of the sample. Avoid touching this area with your
fingers.
 The VAPRO is now ready for the next sample.
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128 Lab 11: Tonicity and Pharmaceutics

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.

Probe Tip

3250 Plastic Sample Tube

 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.

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Lab 11: Tonicity and Pharmaceutics 129

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:
[Atropine] VStock VTotal
(w/v%) (mL) (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 only one of the osmometers 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.
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130 Lab 11: Tonicity and Pharmaceutics

 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.

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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Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 131

Lab 12
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
Part A: Preparing the precursor solutions, assembling the CMC
apparatus, and determining the CMC of three sets of SDS solutions
What You’ll Be Doing
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.
nd
3. Lachman L, Lieberman HA and Kanig JL, eds., The Theory and Practice of Industrial Pharmacy, 2
ed., U.S.A.: Lea & Febiger, 1976, p. 141-183.
rd
4. Aulton M. Aulton’s Pharmaceutics: The Design and Manufacture of Medicines. 3 ed., U.S.A.:
Churchill Livingstone Elsevier. p. 85-90.

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132 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

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:

Hydrophilic (water-loving) head

Lipophillic (oil-loving) tail

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:

CH3 CH2OOC17H35
oil (CH2)10 CH3 CHOOC17H35
CH2 (CH2)16 CH2OOC17H35
interface
CH2OH O CH2OC=O
water
CHOH SO3- Na+ CHOH
CH2OH CH2OH
glycerin sodium glyceryl glyceryl
lauryl monostearate tristearate
sulfate

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.

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Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 133

Lab 12
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:
4 4
Vi = 3 πr3 = 3 π(0.5 x 10-4 cm)3 = 5.24 x 10-13 cm3

The number of droplets in 100 mL is:


Total Volume 100 cm3 14
Volume of a Globule 5.24 x 10 -13 cm3 /droplet = 1.91 x 10 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 6  10 6
Surface Area per Molecule = 16
= 2.72 × 1021 molecules
22  10

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134 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

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 2.72  10 21 -3
Number of Molecules per Mole 6.02  10 23 = 4.5 × 10 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:
wt.% hydrophile
(1) HLB = 5
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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Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 135

Lab 12
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.
Hexagonal Phase
Lamellar Phase
(Cylindrical)
Cubic Phase (Sheets)
(Spherical)

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.

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136 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

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 The square wave produced
weak salt solution is in the dish. by the oscillator, shown on
an oscilloscope.

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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Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 137

Lab 12
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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138 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

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 N/A DC Oscillator
g/mol) 4 connecting wires (with alligator clips)
Sodium Chloride (NaCl MW 58.44 g/mol) Digital Multimeter (DMM)
Heavy Mineral Oil 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 Volume Solution B Volume
(in 150 mL Required (in burette) Required
beaker)
Experiment 1 Solution 1A: 100 mL Solution 1B: 100 mL
(De-ionized 0.04 M SDS
Water)
Experiment 2 Solution 2A: 100 mL Solution 2B: 100 mL
0.01 M NaCl 0.04 M SDS with 0.01 M NaCl
Experiment 3 Solution 3A: 100 mL Solution 3B: 100 mL
0.02 M NaCl 0.04 M SDS with 0.02 M NaCl
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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Magnet
Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 139

Lab 12
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.

DC Oscillator

Metal
Magnet Electrodes

9V DC adaptor

 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).

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140 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

MASTECH®
Multimeter

RadioShack®
Multimeter
 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.

Power
switch To metal
electrode 

To multimeter

To metal
electrode 

The 9V DC Oscillator: Component Diagram

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Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 141

Lab 12
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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142 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

 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:

Submerged electrodes: under liquid level,


not touching sides of beaker or magnetic
stir bar.

 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):

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Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 143

Lab 12
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 homogenous
(approx. 2 minutes)

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144 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

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).

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Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water 145

Lab 12
 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?

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146 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water

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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Lab 13: Optimization of Powder Flow and Particle Size Determination 147

Lab 13: Optimization of Powder Flow and Particle Size


Determination

Lab 13
Preparing for the Lab Read the introduction and lab protocol completely
Group Allocation You will be working in groups of 3 students
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
What You’ll Be Doing
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
http://phm.utoronto.ca/~ddubins/DL/tapdensity2.xls
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 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

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148 Lab 13: Optimization of Powder Flow and Particle Size Determination

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>.
th
5. O’Connor, RE, and Schwartz, JB, Remington: The Science and Practice of Pharmacy, 20 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/U
CM218825.pdf

Background
Types of Additive in a Powder Formulation Examples
Diluents dicalcium phosphate, calcium sulfate, lactose, cellulose,
– increase the bulk to a practical size for kaolin, mannitol, inositol, sodium chloride, dry starch,
handling powdered sugar, hydroxypropylmethylcellulose

Binders or Granulators starch, gelatin, sucrose, glucose, dextrose, molasses,


– impart cohesive qualities to the lactose, acacia, sodium alginate, carboxymethylcellulose,
powder methylcellulose, polyvinylpyrrolidone
– can be in solution or in a dry form (occasionally, polyethylene glycol, waxes, water, alcohol)
Lubricants talc, magnesium sterate, calcium stearate, stearic acid,
- prevent the adhesion of material to the hydrogenated vegetable oils and polyethylene glycol
surface of equipment, reduce
interparticle friction, may improve the
flow of granulation
Glidants colloidal silicon dioxide, talc
- improve the flow characteristics of a
powder mixture
- usually added in the dry state just prior
to compression
Disintegrants starches, clays, celluloses, aligns, gums, and cross-linked
- facilitate the disintegration of a polymers
tablet
Colourants FD & C red, FD & C yellow, FD & C green, FD & C blue, iron
- make the product identifiable oxide, titanium dioxide

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Lab 13: Optimization of Powder Flow and Particle Size Determination 149

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,

Lab 13
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):
 Vtap 
Compressibility Index (or, Consolidation Index) = 1 – V  x 100
 bulk 

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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150 Lab 13: Optimization of Powder Flow and Particle Size Determination

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.
V0  Vn
log(n)  100
V0  V50

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:

 h 
h
Angle of Repose (θ)  tan -1   
 0.5  D 

D
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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Lab 13: Optimization of Powder Flow and Particle Size Determination 151

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

Lab 13
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)

Core cylinder
of powder

2r
The above equation can be simplified to:
𝑔
𝐾( ⁄ )
𝑐𝑚∙𝑠2
(2) 𝑟≥ 𝑐𝑚 𝑔
490.5 ( ⁄ 2 )×𝑑( ⁄ 3 )
𝑠 𝑐𝑚

Solving for K (powder viscosity):


𝑐𝑚 𝑔
(3) 𝐾(𝑃𝑜𝑖𝑠𝑒) ≤ 490.5 ( 𝑠2 ) × 𝑟(𝑐𝑚) × 𝑑 (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.

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152 Lab 13: Optimization of Powder Flow and Particle Size Determination

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 Sieve Opening Nominal Designation Sieve Opening
Designation No. (µm) No. (mesh #) (µm)
(mesh #)
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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Lab 13: Optimization of Powder Flow and Particle Size Determination 153

The particles weighed on the #45 sieve will have an arithmetic mean diameter of:
600  355
 477.5 μm
2

Lab 13
The particles weighed on the #45 sieve will have a geometric mean diameter of:
 log(600)log(355)
 
10  2 
 461.5 μm
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 Parafilm Powder Funnel
Lactose Regular Cardboard Standard Sieves
Avicel PH101 Scintillation Vials #20 Sieve
Dibasic Calcium Phosphate (CaHPO4)

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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154 Lab 13: Optimization of Powder Flow and Particle Size Determination

 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.

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Lab 13: Optimization of Powder Flow and Particle Size Determination 155

Lab 13
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.

Powder flow disks

Powder flow apparatus

 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.

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156 Lab 13: Optimization of Powder Flow and Particle Size Determination

 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:

Funnel on rubber
Powder flow ring clamp
assembly

Powder collection
Trap door
bowl
release hinge

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.

16
16

 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:

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Lab 13: Optimization of Powder Flow and Particle Size Determination 157

Lab 13
Release hinge Trap door (closed)

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)

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158 Lab 13: Optimization of Powder Flow and Particle Size Determination

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 # Mean Weight Fraction Cumulative Arithmetic Geometric
(Passed / Size, Retained Retained, Fraction Weighted Weighted Size, n x
Retained) d* (g) n Retained (cfr) Size, n x d log d
(µm) (frequency
distribution)

⋮ ⋮ ⋮ ⋮ ⋮ ⋮ ⋮
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

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Lab 13: Optimization of Powder Flow and Particle Size Determination 159

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:

Lab 13
 nd  d  
2

σ
av

(2) n
 Determine the geometric mean diameter, dgeo:

log d geo  
 n log d 
(3) n
   n logd  
 
d geo  10  n 

 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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160 Lab 14: Pharmaceutical Granulations

Lab 14: Pharmaceutical Granulations


Preparing for the Lab Read the introduction and lab protocol completely
You will be working in groups of 5 students. This lab will take two
Group Allocation lab periods to complete. Stay in the same groups for both lab
periods.
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
What You’ll Be Doing
formulation.
Part C: Dry-milling granulates, determining powder characteristics,
testing powder potency
Demonstration: High and Low Shear Granulation
http://phm.utoronto.ca/~ddubins/DL/probit.xls
Spreadsheets You Will Need
http://phm.utoronto.ca/~ddubins/DL/probit.pdf
Retain and store your powder blends for second part of Lab 14,
and for Lab 15. Do not throw away your powder blends.
What You’re Handing In
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
nd
1. Pharmaceutics: The Science of Dosage Form Design, 2 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.

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Lab 14: Pharmaceutical Granulations 161

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

Lab 14
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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162 Lab 14: Pharmaceutical Granulations

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 Aluminum trays (reusable – Planetary mixer (Erweka AR 402
Lactose do not discard) with mixer attachment)
Microcrystalline cellulose (Avicel High shear granulator (4M8
PH101) Granulator; Pro-C-epT)
Polyvinlypyrrolidone (Povidone) 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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Lab 14: Pharmaceutical Granulations 163

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.

Lab 14
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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164 Lab 14: Pharmaceutical Granulations

 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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Lab 14: Pharmaceutical Granulations 165

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?

Lab 14

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166 Lab 15: Tableting, Capsuling, and Dissolution Testing

Lab 15: Tableting, Capsuling, and Dissolution Testing


Read the introduction and lab protocol completely
Watch the following related lab videos on the laboratory website:
 Capsule Making (Cap-M-Quik Method)
Preparing for the Lab
(http://phm.utoronto.ca/~ddubins/DL/Capsule_Making.wmv)
 Capsule Making (Hand-Filling Technique)
(http://pharmlabs.unc.edu/video1.php?pack_capsule.flv)
You will be working in groups of 5 students, and combining data
Group Allocation
with another group for the other formulation (A or B).
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
Demonstration: Tablet hardness, friability, tablet coating
Lab Period 2
Part C: Perform a tablet dissolution test on your formulation (A or
What You’ll Be Doing 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)
http://phm.utoronto.ca/~ddubins/DL/Capsule_Filling.xls
Spreadsheets You Will Need
http://phm.utoronto.ca/~ddubins/DL/shelflife.xls
Individual formal lab report, due at the beginning of Lab 16 (see
What You’re Handing In
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 homogeneity, 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.

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Lab 15: Tableting, Capsuling, and Dissolution Testing 167

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.

Lab 15
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

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
subjected to accelerated stability conditions (elevated temperature and humidity). The

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168 Lab 15: Tableting, Capsuling, and Dissolution Testing

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

Diluents  Used as bulking agents which are sometimes microcrystalline cellulose,


called fillers. lactose, dicalcium phosphate,
mannitol, starch

Binders  Added to facilitate the granulation process and polyvinyl pyrrolidone (PVP),
produce a stronger granule. microcrystalline cellulose,
hydroxypropyl
methylcellulose (HPMC),
starch, gelatin

 For immediate-release tablets which are starch, croscarmellose


Disintegrants
intended to disintegrate upon entering the sodium, crospovidone,
stomach, a disintegrant is added to overcome the sodium starch glycolate,
cohesive strength introduced by the compression microcrystalline cellulose,
and by any binder present. Most disintegrants starch, Act-D-Sol
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).

Lubricant  Added to reduce friction at the tablet die wall magnesium stearate, calcium
and adhesion to punch faces. stearate, stearic acid
 Usually mixed with the granulation as the last
step prior to tableting.

Glidant  Used to promote powder flow by reducing colloidal silicon dioxide, talc
interparticle friction and cohesion.
 Used in combination with lubricants as they
have no ability to reduce die wall friction.

Tableting Methods
There are three methods of making compressed tablets: direct compression, wet granulation,
and dry granulation:

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Lab 15: Tableting, Capsuling, and Dissolution Testing 169

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

Lab 15
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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170 Lab 15: Tableting, Capsuling, and Dissolution Testing

Formulation N kg (kilogram) or Sc lb or lbf


(Newton) kp (kilopond) (Strong- (pounds)
Cobb)
Fast-Disintegrating (e.g. 20-70 2-7 2.9-10.0 4.5-15.7
sublingual) Tablet

Chewable Tablet 30 3 4.3 6.7


Small compressed tablet 50 5 7.1 11.2
(100 mg), uncoated

Large compressed tablet 150-200 15-20 21.4-28.6 33.7-45.0


(1000 mg), uncoated

Small compressed tablet, 105 10.7 15.0 23.6


coated
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:

 W
%Friability  100  1  
 W0 
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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Lab 15: Tableting, Capsuling, and Dissolution Testing 171

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-

Lab 15
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:

Weigh 10 tablets Weigh each tablet Calculate the Calculate the


together separately Standard Relative Standard
weight x1 , x2 , x3 ,..., x10 Deviation Deviation
x
10
SD 
xi  x 2 %RSD 
SD
x
n 1

A large %RSD is indicative of processing problems (e.g. >5%).

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172 Lab 15: Tableting, Capsuling, and Dissolution Testing

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 Dissolution Apparatus- USP/NF
Apparatus 1 (basket)
basket method (USP/NF Apparatus 1) and the paddle method

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Lab 15: Tableting, Capsuling, and Dissolution Testing 173

(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.

Lab 15
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

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
Paddle
 Pepsin may be added Basket
 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

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174 Lab 15: Tableting, Capsuling, and Dissolution Testing

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:
  Ea 
 
k  Ae RT 
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:
−ln(0.9)
𝑡90% =
𝑘 𝑇=21°𝐶

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Lab 15: Tableting, Capsuling, and Dissolution Testing 175

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:
Height
or
Outer Locked Actual
Diameter Length Volume
Size (mm) (mm) (mL)

Lab 15
Su07 23.4 88.5 28
7 23.4 78.0 24
Veterinary

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
000 10.0 26.1 1.37
00 8.5 23.3 0.95
0 7.7 21.7 0.68
Human

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 Lactose Avicel Starch Methocel Kaolin Methocel Calcium
Size PH-105 E4M K100M 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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176 Lab 15: Tableting, Capsuling, and Dissolution Testing

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
Chemicals Supplies Special Equipment
Acetaminophen granules (from Lab 14) Size 0 Capsules Planetary Mixer (Erweka or laboratory
Plastic UV cuvettes blender)
Rotary Tablet Press
Hardness Tester
Friability Tester
Glass Slab
Nessler Tube (for tablet disintegration)
USP Dissolution Apparatus
UV-Vis Spectrophotometer

The following solutions are prepared or provided by the TA:


 Hydrochloric Acid (0.1 N)
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 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

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Lab 15: Tableting, Capsuling, and Dissolution Testing 177

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.

Lab 15
 Weigh the appropriate number of tablets to obtain the starting weight (W0) and place
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)

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178 Lab 15: Tableting, Capsuling, and Dissolution Testing

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. Choose the assay
method most appropriate for your sample.

METHOD 1 - 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.

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:
𝑉𝑎𝑠𝑠𝑎𝑦
% 𝐿𝐶 = 𝐶𝑎𝑠𝑠𝑎𝑦 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 × × 100%
𝐿𝐶
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).

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Lab 15: Tableting, Capsuling, and Dissolution Testing 179

METHOD 2: USP XXII Colourimetric Assay Method (Optional)

Standard Preparation
Dissolve an accurately weighed quantity of USP Acetaminophen RS in water (target: 100 mg),
and dilute quantitatively with water in a 100 mL volumetric flask to obtain a solution having a
known exact concentration of 1 mg/mL. Using a syringe equipped with a 0.45 µm syringe filter,
transfer 10 mL of the solution to a second 100 mL volumetric flask, and dilute to the mark, to
obtain the final target concentration of 0.1 mg/mL. Calculate the actual concentration based on
the weight of acetaminophen.

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

Lab 15
means for 20 minutes. Dilute with water to volume and mix. Using a 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.

Procedure
Transfer 10.0 mL each of the Standard preparation, the Assay preparation and water (as a
blank) to three separate 50 mL volumetric flasks and treat each as follows:
1. Add 2.0 mL of 6N HCl and mix. Use caution when pipetting this concentrated HCl
solution.
2. Add 5.0 mL of sodium nitrite solution (10g in 100 mL water), mix and allow to stand
for 15 minutes.
3. Add 5.0 mL of ammonium sulfamate solution (15g in 100g water), swirl gently
allowing the solution to cool to room temperature.
4. Add 15.0 mL of 2.5N sodium hydroxide, allow to cool to room temperature. Dilute to
volume with water and mix.
5. Concomitantly determine the absorbance of the solutions obtained from the Standard
preparation and the Assay preparation in 1 cm plastic cuvettes relative to the water
blank at the wavelength of maximum absorbance at about 430 nm using a
spectrophotometer.
Calculate the % of label claim of the quantity of acetaminophen in each tablet or
capsule:

where is the absorbance of the sample Assay preparation at 430 nm,


is the absorbance of the Standard preparation at 430 nm, mSTD is the amount of
acetaminophen RS used in the Standard preparation in mg, and LC is the label claim of
the amount (strength) of acetaminophen in mg in one dosage form (100 mg).

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180 Lab 15: Tableting, Capsuling, and Dissolution Testing

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 30.00 g
Sorbic Acid 0.50 g
95% Ethanol 10.00 mL
PEG-400 (plasticizer) 10.00 g
PEG-8000 (plasticizer) 10.00 g
Titanium dioxide (opaquant) 5.00 g
Food colouring As desired
Flavourant As desired
Formulating the Coating (performed by Instructor or TA)
1. In a 600 mL beaker, heat 250 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.
4. When the cellulose has been dispersed, add the PEG-400. Continue stirring until
dispersion is homogenous, 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 250 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 homogenous (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.

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Lab 15: Tableting, Capsuling, and Dissolution Testing 181

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.

Lab 15
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.

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182 Lab 15: Tableting, Capsuling, and Dissolution Testing

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.
 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,

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Lab 15: Tableting, Capsuling, and Dissolution Testing 183

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.

Lab 15
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
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. Load
the capsule into a dissolution anchor. 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. You will be
performing TLC analysis on each of your tablet stability samples (room temperature, 40 °C, and
60 °C).

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184 Lab 15: Tableting, Capsuling, and Dissolution Testing

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-methanol-ammonium hydroxide (85-10-5).
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 mark a line along the bottom of the TLC plate 1.5 cm from the bottom edge as noted in
Figure 1. 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.

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.

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Lab 15: Tableting, Capsuling, and Dissolution Testing 185

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
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:
𝑉𝑒𝑟𝑡𝑖𝑐𝑎𝑙 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑠𝑝𝑜𝑡 𝑚𝑖𝑔𝑟𝑎𝑡𝑒𝑑 𝑓𝑟𝑜𝑚 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑙𝑖𝑛𝑒
𝑅𝑓 =

Lab 15
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 A High B Low B High Capsule Lab
Tablet* Tablet* Tablet* Tablet* Period
Tablet Hardness
10     - 1
(mean ±%RSD)
Weight Uniformity
10      1
(mean ±%RSD)
Tablet Thickness
10      1
(mean ±%RSD)
Friability
10     - 1
(mean ±%RSD)
Disintegration Time 1     - 1
Stability – t90%
1     - 1
(@room temp)
Stability – t90% 1 @7days,
    2, 3
(@40°C) 1 @14 days
Stability – t90% 1 @7days,
    2, 3
(@60°C) 1 @14 days
Content Uniformity
3      2
(mean ±%RSD)
Drug Dissolution 2 (tabs)
1     
3 (caps)
Drug degradation
(TLC performed on
the prepared
reference, and 2 1     - 3
week samples
stored at room
temp, 40 °C, 60 °C)
*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.

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186 Lab 15: Tableting, Capsuling, and Dissolution Testing

Questions
1. Identify the function of each of the ingredients in the tablet formulations of this
laboratory exercise.
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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Lab 17: Synthesis and Examination of Colloids 187

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
Part A: Preparing a Yttrium Citrate colloid
Part B: Preparing a Rhenium Heptasulphide colloid, method 1
What You’ll Be Doing 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.
Individual formal lab report, due at the beginning of the next lab
What You’re Handing In
(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

Lab 17
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,

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188 Lab 17: Synthesis and Examination of Colloids

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 Sterlitech filters (pore sizes: Mixing plate and magnetic
Sodium Citrate 5.0, 1.2, 0.8, 0.45 µm) stir bar
Sodium Hydroxide Pellets (MW 40.00 Plastic UV Cuvettes 10 mL Volumetric Flask
g/mol) 50 mL Falcon® tube pH meter
Gelatin Water bath
Sodium Thiosulfate Carey UV/VIS
Phosphate buffer pH 7.4: Spectrophotometer (in
Sodium Phosphate Monobasic Room 819)
(MW 137.99 g/mol) Laser Pointer
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)

The following solutions are prepared or provided by the TA:


 pH Meter Standardizing Buffers (pH 4, 7)

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Lab 17: Synthesis and Examination of Colloids 189

Volume Vstock Mass


Stock Con'n (mL) (mL) (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 25 sec 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

Lab 17
 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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190 Lab 17: Synthesis and Examination of Colloids

Part B. Rhenium Heptasulphide Colloid, Method 1


 Prepare the following solutions, in de-ionized water:
Solution Ingredients Volume
Solution 2A Sodium thiosulfate (30 mg) 10 mL (use a
Potassium Perrhenate (5 mg) 10 mL
Gelatin (50 mg) 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 Obtain 5 mL
(Sorensen’s buffer from previous labs may from TA
be used)
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 10 mL
Gelatin (400 mg)
Solution 3C Gelatin 7.5 mg/mL in ~70 C deionized 10 mL
water
Solution 3D Gelatin 1 mg/mL 100 mL
0.5% Sodium Benzoate in ~70 C deionized
water
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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Lab 17: Synthesis and Examination of Colloids 191

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

Lab 17
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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192 Lab 17: Synthesis and Examination of Colloids

Colloid
???
5 µm 0.45 µm
syringe syringe
filter filter

1.2 µm 0.8 µm
syringe syringe
filter filter

<5 µm <1.2 µm <0.8 µm <0.45 µm


particles particles particles particles

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?

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Lab 18: Formulating Using Molds 193

Lab 18: Formulating Using Molds


Read the introduction and lab protocol completely
Watch the following related lab videos on the laboratory website:
Preparing for the Lab
 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
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
What You’ll Be Doing 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

Lab 18
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

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194 Lab 18: Formulating Using Molds

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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Lab 18: Formulating Using Molds 195

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-

Lab 18
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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196 Lab 18: Formulating Using Molds

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:

𝐷𝑜𝑠𝑒
𝐷𝐹 =
(𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑃𝑙𝑎𝑐𝑒𝑏𝑜 𝑊𝑒𝑖𝑔ℎ𝑡) −(𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑀𝑒𝑑𝑖𝑐𝑎𝑡𝑒𝑑 𝑊𝑒𝑖𝑔ℎ𝑡)+𝐷𝑜𝑠𝑒

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.

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Lab 18: Formulating Using Molds 197

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

Lab 18
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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198 Lab 18: Formulating Using Molds

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 vagina. 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 Examples Pros Cons
Vehicle Type
Oleaginous  Cocoa butter  Easy to prepare  Anal leakage (rectal)
 Hydrogenated vegetable  Self-preserving  Poor systemic absorption
oils  Better for of hydrophobic drugs
e.g. Theobroma oil inflammation/irritation  Difficult polymorphs of
 Synthetic triglycerides (emollient) cocoa butter (overheating +
(Witepsol H-15)  most common cooling too quickly results in
 “Base F” (PCCA) wrong polymorph being
formed, and base won’t
solidify)

Hydrophilic  Glycerinated gelatin  Dissolves slowly at 37°C  Not self-preserving


 Hydrogels (PVA, (doesn’t melt)  Hygroscopic (pain)
hydroxyethyl  Doesn’t melt as quickly  Wet before using
methacrylate, polyacrylic as oleaginous bases  Poor/sensitive mechanical
acid, polyoxyethylene  Well suited to vaginal properties
 PEG (Macrogols) prolonged release  Glycinerated gelatin
 High MW or mixtures  Chemically stable, non- requires mold lubrication
 “Base A” (PCCA) irritating
 PEG base is varying  Miscible with
mixtures of PEG (e.g. 40% water/mucous secretions
PEG 400 and 60% PEG
3350)

Water-  W/O emulsions  Can make an oleaginous  Surfactants can irritate the
Dispersible  Polyoxyl 40 stearate base more hydrophyllic anal mucosa and cause
Bases (surfactant) expulsion
 Fatty bases + surfactant
 “Base MBK” (PCCA) –
PEG 400-Stearate +
Hydrogenated Vegetable
Oil

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Lab 18: Formulating Using Molds 199

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.
Typical Suppository Bases
Ingredient 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

Lab 18

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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200 Lab 18: Formulating Using Molds

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,
homosalate, 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) Non-Stick Cooking Spray Hot Plate
Benzocaine USP (MW 165.19) 2 × Suppository Molds Thermometer
Hydrocortisone USP (MW 362.46) 2 × Lollipop Molds Retort Stand, Vinyl Retort
Lidocaine USP (MW 234.34) 2 × Lip Balm Molds Clamp
Excipients as indicated in each section 12 × Lollipop Sticks 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)

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Lab 18: Formulating Using Molds 201

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


PEG Suppository Base
Weight Percent
Ingredients Mass (g)
(%w/w)
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
homogenous.
3. Turn a test-tube rack upside down, and use it to
Step 2. Melt the PEG in a ceramic
support the suppository mold. In a slow, continuous stream, pour the homogenous
dish and stir with a glass rod.

Lab 18
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. Step 3. Overfill the mold cavities
5. with meltedremove
Remove mold from the stability chamber. Using a metal microspatula, base the
excess of base by scraping firmly across the top of the
mold.
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. Step 5. Scrape off excess base with
a microspatula

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202 Lab 18: Formulating Using Molds

Medicated Suppositories
8. Weigh out 3.90 g of acetaminophen in a small
weighing boat.
NOTE: 12 suppositories x 325 mg acetaminophen/suppository
= 3900 mg acetaminophen. 12 units are planned to
compensate for losses in compounding, in order to prepare 8
suppositories.
9. Set the hot plate to medium. Re-melt all 12
suppositories (whether or not they were removed in Step 9. Mark volume of melted
placebos with wax pencil
tact) in a 50 mL beaker (“Beaker A”), directly on the
hot plate. Mark the volume level of melted vehicle with a wax pencil directly on Beaker
A.
10. Pour approximately half of the melted PEG mixture in a
second, clean 50 mL beaker (“Beaker B”) and place on
the same hot plate.
11. Remove Beaker A from the hot plate. Transfer the 3.90
g of acetaminophen into Beaker A. Mix well with a
glass rod until a uniform, homogenous mixture is
produced.
12. Bring the mixture up to the volume mark using the
remaining melted vehicle in Beaker B, and stir with a
glass rod until homogenous.
13. In a slow, continuous stream, pour the homogenous
mixture from Beaker A into a clean, dry suppository
mold, and over-fill the mold cavities (to avoid Step 12. Bringing it up to volume
formation of holes that could take place due to with remaining base
contraction of the base on cooling).
NOTE: You will 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.

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Lab 18: Formulating Using Molds 203

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
Weight Percent
Ingredients Mass (g)
(%w/w)
Sorbitol (Powder), NF 64.7 %w/w
Polyethylene Glycol 3350, NF 32.3 %w/w

Lab 18
Silica Gel (Micronized) 2.0 %w/w
Total amount of flavourant(s) (target ~1-3%
1.0 %w/w
for water based, ~0.1% for oil based)
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

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204 Lab 18: Formulating Using Molds

temperature of 100-110°C to form a homogeneous 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 homogeneous 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.

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.

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Lab 18: Formulating Using Molds 205

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

Troche Gelatin Base

Weight Percent Mass


Ingredients
(%w/w) (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 8.0
powder.
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

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

Lab 18
alcohol soluble, heat stable, and has a working range of 0.1-0.8% (above which it becomes bitter).

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.

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206 Lab 18: Formulating Using Molds

5. When the gelatin appears smooth and homogenous, 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.
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

Weight Percent Mass


Ingredients
(%w/w) (g)
Petrolatum (occlusive/moisturizing) 48.9
Beeswax (stiffener) 20.2
Polyethylene Glycol 400 (hydrophilic)
Note: PEG 400 is liquid at room temperature, 18.6
and is used to levigate the drugs.
Lemon Oil or Orange Oil (fragrance) 12.3
Total: 100 %w/w 16 g

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Lab 18: Formulating Using Molds 207

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 2
hot plate on high, and set a small ceramic dish on 1
each.
2. Remove the caps from the lip balm casings.
Ensure each lip balm casing is ready by twisting
the bottom ring (called the “knurl”) counter-
clockwise all the way. 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.
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, Fill elevator at
petrolatum, and lemon oil (or orange oil), in bottom of casing
that order. Tare the scale between
measurements.
Knurl
Note: For the beeswax, break off tiny pieces (counter-clockwise = down)
(break or shave larger pieces with a metal

Lab 18
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
plastic a transfer pipette.
5. Place dish 1 back on the hot water bath. Stir the
mixture with a metal spatula until clear, and
homogenous. 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 a smooth paste is obtained. Add the
remaining amount of PEG 400 from the small
beaker to dish 1.

PHC 340Y Lab Manual 2016/17


208 Lab 18: Formulating Using Molds

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.
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
(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 homogenous.
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.
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

PHC 340Y Lab Manual 2016/17


Lab 18: Formulating Using Molds 209

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.

Lab 18

PHC 340Y Lab Manual 2016/17


210 APPENDIX

APPENDIX
U.S. Standard Sieve Sizes and Lab Sieve Inventory
U.S. Standard Sieves
Nominal Sieve Opening Nominal Designation Sieve Opening
Designation No. (µm) No. (mesh #) (µm)
(mesh #)
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

PB860 Opening Opening


Sieve Inventory µm 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

PHC 340Y Lab Manual 2016/17


APPENDIX 211

Quadro Comil Meshes


Particle US Uses
Inches/ Size US Mesh
Serial Number Hole Microns (µm) Mesh# (µm)
7B250Q03750* Square 0.25/ 3175 6 3350
(6350) 2006-11 6350

7B187Q03472* Square 0.187/ 2375 8 2360


Square holes are used for wet
(4750) 4750
granulation, dispersion, moist
food reclaim, cheeses, de-
wrapping candy

7B156Q03746* Square 0.156/ 1981 10 2000


(3962) 3962

7B125G03123* Cheese Grater 0.125/ 1587.5 12 1700 Grater screen is used for
(3175) 3175 harder products or more
ductile, powderizing cream
filled cookies, grating cheese,
chopping nuts, sizing
compressed slugs, cereal
flakes
7B083R03472* Round 0.083/ 1053.8 18 1000
0601 2107.7

Round holes are used for dry


granulation, powders, fat

APPENDIX
dispersion, bulk density
7B045R03137* Round 0.045/ 571.9 30 600 tuning, de-agglomeration
0601 1143.8

PHC 340Y Lab Manual 2016/17


212 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


1
METHOCEL™ Product Chemical Type Methoxyl Hydroxypropoxyl Viscosity of 2%
Content, Content, % 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
2
METHOCEL™ E4M Premium 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
2
METHOCEL™ K100 Premium LV Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 80 - 120
2
METHOCEL™ K4M Premium Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 2663 - 4970
2
METHOCEL™ K15M Premium Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 13275 - 24780
2
METHOCEL™ K100M Premium Hypromellose 2208 19.0 - 24.0 7.0 - 12.0 75000 - 140000
1
USP XXII
2
Also 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


1
METHOCEL™ Product Chemical Type Methoxyl Hydroxy- Viscosity of Viscosity of
Content, propoxyl 2% solution 2% solution
% Content, in water, in water,
% cPs (USP) 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
1
USP XXII
Source: http://www.dow.com/dowexcipients/products/methocel.htm

PHC 340Y Lab Manual 2016/17


APPENDIX 213

AVICEL™ Products
Product Grades Nominal Moisture, % Loose
Particle Size, µm 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 ***Not More


**Special Coarse Grade Than
Source: http://www.fmcbiopolymer.com/Pharmaceutical/Products/Avicelforsoliddoseforms.aspx

APPENDIX

PHC 340Y Lab Manual 2016/17


214 APPENDIX

Avicel Grade Usage Chart


Tablets
Method Desirable Properties Recommended
Product
Roller Compaction -Increase tablet hardness Avicel DG in intra-
-Provide good flow and a strong ribbon during granular phase
compaction No extra-granular
-Reduce number of excipients binders required.
-Improved yields
Direct Compression -Improve flow Avicel PH-102 SCG
-Better compressibility Avicel HFE-102
-Accomodation of moisture-sensitive actives Avicel PH-200
Avicel PH-302
Wet Granulation -Rapid, even wetting as a result of the wicking Avicel PH-101
action of microcrystalline cellulose Avicel PH-301
-Reduced sensitivity of the wet masss to over-
wetting
-Faster drying
-Fewer screen blockages or case hardenings
-Reduce dye migration
-Faster disintegration
Disintegration -Enhance drug dissolution by speeding tablet Ac-Di-Sol
disintegration
-Provide the highest level of disintegration force
at low use levels
-Utilize dual disintegration mechanisms of
wicking and swelling for more rapid
disintegration
Liquids and Suspensions
Suspending Agent -Maintain suspension uniformity by preventing Avicel RC-591
settling Avicel CL-611
-Impart thixotropic viscosity profile
Source:
http://www.fmcbiopolymer.com/Pharmaceutical/Applications/ImmediateRelease/Tablets.aspx

PHC 340Y Lab Manual 2016/17


APPENDIX 215

Capsule Properties

Empty Hard Gelatin Capsule Dimensions


Height
or
Outer Locked Actual
Diameter Length Volume
Size (mm) (mm) (mL)
Su07 23.4 88.5 28
7 23.4 78.0 24
Veterinary

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
000 10.0 26.1 1.37
00 8.5 23.3 0.95
0 7.7 21.7 0.68
Human

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

APPENDIX

PHC 340Y Lab Manual 2016/17


216 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 Viscosity (poise)
o
( C)
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

PHC 340Y Lab Manual 2016/17


APPENDIX 217

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

APPENDIX
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.

PHC 340Y Lab Manual 2016/17


218 APPENDIX

Surfactant Type Examples Applications


Nonionic: Esters: (oil-soluble) -Emulsifiers
-Least irritating -Ethylene Glycol Esters and Propylene Glycol Esters -Most widely used in topical preparations (O/W, W/O
-Compatible with other -Glyceryl Esters (e.g. Glyceryl monostearate) emulsions)
three surfactant classes -Polyglyceryl esters, Sorbitan esters, Sucrose esters, and -Oil soluble, not water soluble
-Compatible over a broad Ethoxylated esters
range of pH values Ethers: (generally derived from PEG and PPG) -Solubilizing agents for oil, detergents, wetting agents,
-Calcium tolerant -e.g. PEG-20 cetyl ether / cetomacrogol 1000 emulsifying agents
-Inert, lower toxicity, -more stable than esters
some suitable for oral Ether-Esters: (water soluble) -Useful for solubilization and emulsification
administration, some -Spans and Tweens (e.g. Span 80, Tween 80) -Viscous liquids, bitter taste
have better palatability -Ester linkage subject to hydrolysis
-May hydrogen bond to preservatives and undesirably
reduce antimicrobial activity
-Viscous liquids, bitter taste
Fatty alcohols: (C8-C18) -Only marginal surfactant effect, useful as co-surfactant
- +
Anionic: Alkali Soaps: R COO M (M=Na, K, NH4) -External use only (laxative effect, poor taste, hemolytic
-Personal care products, -12-18 carbon atoms properties)
industrial purposes -Sensitive to/precipitate in hard water (Ca2+ intolerant) -irritating to the eye
-Cleansing and -Gives an alkaline pH (~10), pH sensitive -useful in enemas/liniments as a counter-irritant
detergency -Incompatible with acidic drugs -good O/W emulsifiers at 5-10%
-Not for oral use -Micelles break up at elevated temperatures -good solubilizers at 20%
Alkaline Soaps: (R COO)2 M (M = Ca, Mg, Zn) -form W/O emulsions (e.g. calamine lotion BP)
-12-18 carbon atoms
-Oil soluble
-Similar disadvantages to Alkali soaps
Amine Soaps: (e.g. triethanolamine stearate) -Hair creams, lotions, cosmetic creams
-Lack calcium intolerance -Better O/W emulsions due to HLB
-Less alkaline, pH ~8
-Less hydrophilic, not as temperature dependent
Sulfuric Acid Esters: -Forms O/W emulsiions
- +
e.g. Sulfated fatty alcohols R OSO3 M -Preparation of creams, emulsifying wax, emulsifying
e.g. Sodium Lauryl Sulfate (SLS, SDS) ointment
-Salt of strong acid and base -Can be used with neutral or negatively charged drugs
-Highly water soluble, ionized at low pH -Generally requires an auxiliary substance (e.g. cetyl
-Does not precipitate with Ca2+ alcohol)
-May precipitate with large positive drugs
Sulfonic Acid Derivatives: (R SO3Na) -Emulsifying agent in creams and ointments
e.g. Dioctyl sodium sulfosuccinate (aerosol OT) -Wetting agent, solubilizer
-Similar to sulfated alcohols
2+
-Less prone to hydrolysis, Ca tolerant
-Hydrophilic
Cationic: Simple Amine Salts (R NH2 HCl) -Wetting, foaming, detergent properties
-External preparations e.g. Octadecylamine HCl
-pH sensitive
+ -
Quaternary Ammonium Salts (NR1R2R3R4 X ) -Emulsifier
e.g. Cetyltrimethyl ammonium bromide -Not for oral or parenteral administration
-Soluble over wide pH range -In creams or emulsions containing nonionic or cationic
-Requires secondary emulsifier/stabilizer drugs for external application
-Incompatible with large anionic compounds -Bactericidal activity
Zwitterionic/ e.g. Alkyl Betaine -Personal care and household cleaning products
Amphoteric: -Positive charge almost always ammonium, negative -Excellent dermatological properties
charge carboxylate, sulfate, sulphonate -Frequently used in shampoos and cosmetics products,
-pH sensitive – charge can change depending on pH and also in hand dishwashing liquids because of their
-Compatible with other surfactant classes high foaming properties
-soluble and effective in the presence of high
concentrations of electrolytes, acids and alkalis

PHC 340Y Lab Manual 2016/17


APPENDIX 219

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.

APPENDIX

PHC 340Y Lab Manual 2016/17


220 APPENDIX

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

PHC 340Y Lab Manual 2016/17


APPENDIX 221

HLB Requirement for Some Common Oil Components


Required
Oil Component 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

APPENDIX
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

PHC 340Y Lab Manual 2016/17


222 APPENDIX

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:

HO O HO O

pKa,2
+
O
-
OH OH O
-
OH O
- + H

O O O O

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

PHC 340Y Lab Manual 2016/17


APPENDIX 223

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

APPENDIX
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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224 APPENDIX

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 mL M/15
Na2HPO4 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:

n  CV
mol  1L 
n  0.2  500 mL     0.1 mol
L  1000 mL 
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:
m  n  MW
m  0.1 mol  268.07 g/mol
m  26.81 g
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:
2g
m  CV   200 mL  2%  200 mL
100 mL
m  4g
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:
m  CV  2%  200 g
m 4g

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

APPENDIX
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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226 APPENDIX

C2 = 2 mM
C1 = 1 M V2 = 100 mL
V1=?

Solution 1 Solution 2
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:
(1) m1  m 2
(2) C1 V1  C 2 V2
C 
(3) V1  V2   2 
 C1 
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:
C 
V1  V2   2 
 C1 
  1M  
 2 mM   
V1  100 mL    1000 mM  
 1M 
 
 
V1  0.2 mL

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:

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APPENDIX 227

C 
V1  V2   2
 C1
 0.1 M 
V1  100 mL   
 1M 
V1  10 mL

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:
C 
V1  V2   2 
 C1 
  1M  
 2 mM   
V1  100 mL    1000 mM  
 0.1 M 
 
 
V1  2 mL

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

APPENDIX

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228 APPENDIX

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 NOTE: For successive
Empty Capsule 2 g measurements, you do not need
to remove the previous capsule(s)
Empty Capsule 3 g from the scale. Simply re-tare the
Empty Capsule 4 g scale, and measure the next
capsule.
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

This method was adapted from PCCA C3 Formula Pack - Comprehensive Compounding Course 2010©.

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APPENDIX 229

Excerpt from USP 795:

Source: http://www.pharmacopeia.cn/v29240/usp29nf24s0_c795.html

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230 APPENDIX

YOUR NOTES
Use these pages as a notepad if you need a little extra space.

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APPENDIX 233

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