Professional Documents
Culture Documents
Pharmaceutical Chemistry
Laboratory Manual
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 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 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
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
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.
The following icons are used in the margins throughout this manual:
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
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:
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
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:
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
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:
Fire Extinguishers
Fire Alarm
Safety Shower
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.
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.
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.
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.
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.
5. Experimental (10%)
This section should be no more than 2 pages long, but depending on the
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.
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
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
Emergency Response
The University Emergency phone number is 416-978-2222.
In Case of Spills
Chemicals spilled in the laboratory must be cleaned up immediately to reduce and
eliminate hazards. The Chemical Spill Cart is located in the laboratory outside the
entrance of Room 865.
In the event of a localized, minor spill, use the following procedure:
In the event of a major spill that exceeds the clean-up capabilities of the laboratory, the
following procedure is to be followed:
In Case of Fire
If the fire is contained in beakers or flasks, smother the fire simply by covering the
vessels so that no oxygen can enter.
If electrical equipment is on fire, unplug it quickly or cut the power if possible.
If your clothing is on fire, do not run. Stop, drop, and roll. If the clothing of someone
next to you is on fire, help him to the floor and use your lab coat or fire blanket, or
whatever is available to smother the fire. Once the fire is extinguished, help the person
away from the general fire area.
If the fire is small and contained, a qualified person should attempt to use a fire
extinguisher to eliminate the fire. Many fire extinguishers handle multiple types of fires.
There are 4 major classes:
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.
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.
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
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
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
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):
Spectrophotometer beam
travels this way
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?
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
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.
(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]
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.
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.
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.
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.
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:
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
(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)
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.
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
Source: http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer
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
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
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:
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
HAW
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:
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):
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!
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
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.
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).
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.
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.
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
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?
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).
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.
[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.
pH Titration Curve
dV/dpH vs pH
12
8
pKa
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 pHpKa
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 pHpKa
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
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
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
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:
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
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)
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.
The crystals produced in this exercise will be examined during the second part of this exercise
one week from now.
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
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)
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 - ]
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?
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.
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.
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
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:
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.
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
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.
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.
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).
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.
_ 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)
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.
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.
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.
Lab 6
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.
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
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
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
liquid sample
h Rb
Rc
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
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.
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.
Lab 7
http://ww.colorcon.com/pharma/excipients/methocel
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:
2r 2 (ρ s ρ L )g
V NOTE: Watch your units!
9η
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
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.
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.
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)
Background
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) kt const 0t
A
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)
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
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)
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, 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.
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)
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
-
Ae R(294.15K)
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):
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.
We added the initial mass of drug mdrug = 50 mg. By subtraction, the initial mass of ASA is:
(28) m ASA m drug mSA
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)
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]
Interpretation of Data
Plot the logarithm of concentration of ASA remaining against time. A straight line indicates a
pseudo first-order reaction (Fig. 1 - Title - Pseudo first-order hydrolysis of ASA at specified pH
values and temperatures). The observed rate constants and the half-lives, and t90 can be
evaluated from the line.
Using the two different rate constants (k) obtained at the same pH value the apparent energy of
activation is calculated by means of the Arrhenius equation - plot log k against 1/T, T being the
absolute temperature. Now calculate k25°C. Predict the amount of ASA remaining in your
buffered solution at room temperature after 30 days.
The influence of pH on the hydrolysis of ASA may be determined by using the buffer solutions at
different pH values. Use the results provided by your section to plot a pH profile at the
temperature given by the instructor (Fig.2. Plot the pseudo first-order rate constant and half-life
against the pH values. Title of graph - observed rate constant and half-life for ASA hydrolysis
at____°C as a function of pH).
Depending upon the experimental conditions selected, various graphs can be prepared.
a) Log or concentration of ASA remaining against time, different lines representing
different temperatures.
b) Log of concentration of ASA remaining against time, different lines representing
different pH values.
c) Log of concentration of ASA remaining against time, different lines representing
different original concentrations of ASA.
d) What is the value of the rate constants? Does this properly hold for zero, first and
second order reactions?
e) Plot of concentration of ASA remaining or of concentration of salicylic acid formed
against time for a zero order process. Interpret both the significance of the curved
portion and linear portion of the line. Explain the phenomenon observed.
NOTE: For Part A, you will analyze data from the whole class in your final report. You will not be
sharing data for Parts B and C.
Plot log [ASAT] vs. time
Calculate k from the slope
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?
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.
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:
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.
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:
C10 V1 C 2 (t)V2 V
(5) C1 (t) C10 C 2 (t) 2
V1 V1
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
(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
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.
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 dn/V1 V1 dC1
(15) J1
A dt A dt A dt
Similarly, the flux entering Compartment 2 can be expressed as:
1 dn V2 dn/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
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.
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
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
(e.g. OD210).
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
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?
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.
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
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.
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.
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
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.
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
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.
1. Hydrocarbon Base
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.
Lab 10
2. Absorption Base
cholesterol 3g
stearyl alcohol 3g
white wax 8g
white petrolatum 86 g
to make: 100 g
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.
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.
Lab 10
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.
The PEGs are characterized by their number referring to their approximate molecular
weight. Smaller numbers indicate the number of ethylene glycol repeat units.
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:
edges).
Secure the membrane in place with an elastic band.
For the final report, plot the concentration of salicylic acid released vs. time for the six
bases on the same graph.
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
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
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 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.
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
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):
Lab 11
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.
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
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
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
Probe Tip
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.
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.
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.
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:
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.
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
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
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
=
Source: Gennrbinro AR, et al., Remington: The Science and Practice of Pharmacy, 21st ed., USA: Mack
Publishing Company, 2006, p. 311-314, 335-6.
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:
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:
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
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
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.
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).
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
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):
Place a medium-sized magnetic stir bar into the 150 mL beaker and set the speed on
low.
Pour about 40 mL of Solution 1B (0.04 M SDS) into a 50 mL burette.
NOTE: Unclamp the burette to load it, so that Solution 1B does not spill accidentally into
1A.
The final connected apparatus should look like the following picture:
The metal electrodes are protected by a layer of heat shrink tubing, allowing the
internal conductivity of the solution to be measured, not at the surface. Ensure the
exposed metal is completely submerged under the surface of the water, and not
touching the bottom or sides of the beaker, or magnetic stir bar.
On the multimeter, press the SELECT button, to select alternating current (the “~”
symbol will appear on the LCD screen).
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.
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.
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?
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?
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?
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
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
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
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
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:
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
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.
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
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)
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.
Lab 13
NOTE: Use the rubberized ring clamp (shown above) to determine tapped density.
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
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:
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)
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?
⋮ ⋮ ⋮ ⋮ ⋮ ⋮ ⋮
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
Lab 13
nd 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?
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.
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.
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)
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.
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.
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.
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?
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
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.
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
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.
Binders Added to facilitate the granulation process and polyvinyl pyrrolidone (PVP),
produce a stronger granule. microcrystalline cellulose,
hydroxypropyl
methylcellulose (HPMC),
starch, gelatin
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:
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:
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
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.
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:
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
(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
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
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
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
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
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
Lab Period 1:
NOTE: Divide each formulation approximately into 2 separate bags. Label 1 bag “For Tableting”
and the other bag “For Capsuling”. Store the Capsuling bag in your locker for Period 2.
Part A. Tableting
For only the formulations intended for tableting, weigh out the required amount of
starch and magnesium stearate as listed in the following table for the two tablet
formulations using the low and high shear granules you made in Lab 14.
Tablet Compositions
Formulation A Formulation B
Ingredients weight (g) % w/w weight (g) % w/w
Granulation (high or low shear): 100.00 94.50% 100.00 94.50%
• Acetaminophen (in granulate) 30.23 28.57% 30.23 28.57%
• Lactose (in granulate) 39.31 37.15% 25.23 23.84%
• Avicel PH101 (in granulate) 25.23 23.84% 39.31 37.15%
• Polyvinylpyrrolidone (in granulate) 5.23 4.94% 5.23 4.94%
Corn starch 5.29 5.00% 5.29 5.00%
Magnesium stearate 0.529 0.50% 0.529 0.50%
Total (g): 105.82 100% 105.82 100%
Add the post-granulation excipients (corn starch and magnesium stearate) to the
acetaminophen granules obtained from the previous experiment, and shake the
formulation in a bag for 10 minutes by inverting the bag continuously and repeatedly.
Using the Sanchez single punch press or the Globe Pharma rotary press, compress 100
mg acetaminophen tablets (350 mg tablets weight) as assisted by your instructor.
NOTE: 350 mg * 28.6 % w/w acetaminophen = 100 mg acetaminophen
Take 10 tablets from each formulation and
determine the tablet hardness (mean ± %RSD)
using the Hardness Tester and record in tabular
form. The tablet should be placed FLAT in the
hardness tester. The piston squeezes the tablet
on the edges.
Determine the weight uniformity of 10 tablets by
weighing each tablet separately. Report the
weight of each tablet in tabular form, and report the mean ± %RSD.
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.
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).
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:
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
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,
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:
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.
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.
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.
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?
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,
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.
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)
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.
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).
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).
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.
Colloid
???
5 µm 0.45 µm
syringe syringe
filter filter
1.2 µm 0.8 µm
syringe syringe
filter filter
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?
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
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 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.
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.
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
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.
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
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)
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
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% --- --- --- --- ---
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.
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
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)
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
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.
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.
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
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.
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.
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).
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.
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?
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?
Lab 18
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
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
APPENDIX
dispersion, bulk density
7B045R03137* Round 0.045/ 571.9 30 600 tuning, de-agglomeration
0601 1143.8
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
AVICEL™ Products
Product Grades Nominal Moisture, % Loose
Particle Size, µm Bulk Density,
g/cc
Superior Flow Avicel PH-102 SCG** 150 3.0 to 5.0 0.28 - 0.34
APPENDIX
Capsule Properties
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
APPENDIX
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.
Source: Karsa, David R. Design and Selection of Performance Surfactants, Volume 2. CRC Press LLC. Boca Raton,
Florida USA, 1999.
APPENDIX
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
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
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
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)
n CV
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.
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:
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:
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.
APPENDIX
Preparation Name:
Compounder:
Date/Time:
This method was adapted from PCCA C3 Formula Pack - Comprehensive Compounding Course 2010©.
Source: http://www.pharmacopeia.cn/v29240/usp29nf24s0_c795.html
APPENDIX
YOUR NOTES
Use these pages as a notepad if you need a little extra space.
APPENDIX
APPENDIX