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Student Name: _________________________________ Pharmaceutical Chemistry (PMY 331 & PMY 332) Laboratory Manual Prepared by: Lungwani T.M. Muungo, PhD 2003
Pharmaceutical Chemistry Laboratory Manual Pharmaceutics Principles and Evaluation Pharmaceutical Chemistry (PMY 331 & PMY 332) Department of Pharmacy School of Medicine University of Zambia Programme Coordinator: Dr. Lungwani T. Muungo Lungwani.muungo@unza.zm
This manual was prepared for use by students enrolled in the undergraduate Pharmacy Program at the Department of Pharmacy, University of Zambia. Permission is granted to copy the manual provided no charge is made beyond reasonable reimbursement for duplication and handling costs, and provided that this notice is retained in all such copies. This manual was compiled the HOD for official use in 2003. Special thanks to staff members at the Department of Chemistry that actively participated in the laboratory work when pharmacy students were allocated. Specific contributions included optimizing the experimental procedure to better detect phase inversion, ensuring a robust incoming voltage by introducing and modifying the 9V DC adaptors, developing and testing the metal electrodes, and adopting the pharmaceutical lab protocol. Lungwani T.M. Muungo, PhD
Preface 1 Table of Contents Preface.....................................................................................................................................................6 Introduction.............................................................................................................................................7 General Information.............................................................................................................................7 Recommended Textbooks................................................................................................................7 Teaching Staff ......................................................................................................................................7 Role of Teaching Assistants ..............................................................................................................8 Attendance......................................................................................................................................8 Lateness Policy.....................................................................................................................................9 Laboratory and Lecture Schedule ....................................................................................................... 10 Locker Check-In / Check-Out .............................................................................................................. 12 Recording Data, Analysis, and Results................................................................................................. 14 Plagiarism and Falsification ............................................................................................................ 14 Clean-up Check-List............................................................................................................................ 15 Assignment of Grades ........................................................................................................................ 15 Guidelines for Writing Pre-Labs, Worksheets and Individual Laboratory Reports................................. 15 Laboratory Safety............................................................................................................................... 18 Chemical Inventory ........................................................................................................................ 18 Labeling of Preparations................................................................................................................. 18 Chemical Disposal .......................................................................................................................... 19 Dress Code..................................................................................................................................... 19 Dress Code Rationale ..................................................................................................................... 19 Working with Hazardous Chemicals................................................................................................ 19 Emergency Response ..................................................................................................................... 20 In Case of Personal Injury ............................................................................................................... 20 In Case of Spills .............................................................................................................................. 21 In Case of Fire ................................................................................................................................ 21 If the Fire Alarm Sounds................................................................................................................. 22 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve.......................................23 Introduction....................................................................................................................................... 23 Background........................................................................................................................................ 23 Experiment Protocol .......................................................................................................................... 25 Part A. Preparing a Calibration Curve.............................................................................................. 25 Part B. Plotting Your Calibration Curve............................................................................................ 27 Questions .......................................................................................................................................... 27 Lab 2: Preparation of pH Buffers............................................................................................................28 Introduction....................................................................................................................................... 28 Background........................................................................................................................................ 28 Definition of pH and pKa................................................................................................................. 29 Buffer Capacity............................................................................................................................... 34 Experiment Protocol .......................................................................................................................... 36 Part A. Preparing Sorensen’s Buffer................................................................................................ 36 Part B. Preparing McIlvaines’s Buffer.............................................................................................. 37
2 Preface Questions .......................................................................................................................................... 38 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid.....................................39 Introduction....................................................................................................................................... 39 Background........................................................................................................................................ 39 Experiment Protocol .......................................................................................................................... 43 Part A. UV Absorbance Standard Curve of Sodium Salicylate 44 Part B. Determination of the Partition Coefficient 45 Part C. Direct Measurement of the Partition Coefficient 46 Questions .......................................................................................................................................... 47 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa ......................................48 Introduction....................................................................................................................................... 48 Background........................................................................................................................................ 49 pKa and Intrinsic Solubility 49 Calculation of pHp 51 Polymorphism 55 Experiment Protocol .......................................................................................................................... 56 Part A. Intrinsic Solubility Determination 56 Part B. Preparing Different Salts of Sulfathiazole 57 Part C. Preparing Different Polymorphs of Sulfathiazole 57 Part D. pKa Determination 58 Part E. Melting Point Determination 59 Part F. Macroscopic Evaluation 60 Questions .......................................................................................................................................... 60 Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification .........................................................................................................................................61 Introduction....................................................................................................................................... 61 Background........................................................................................................................................ 62 Experiment Protocol .......................................................................................................................... 63 Part A. Co-solvency 63 Part B. Salt Selection 64 Part C. Polymorph Identification 64 Questions .......................................................................................................................................... 64 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume....................................................................65 Introduction....................................................................................................................................... 65 Background........................................................................................................................................ 65 Specific Heat Capacity 65 Partial Molar Quantities 68 Experiment Protocol .......................................................................................................................... 69 Part A. Calibration of the Calorimeter 70 Part B. Specific Heat Capacity of Copper Metal 71 Part C. Heat of Reaction and Heat of Hydration 71 Part D. Measurement of Molar Enthalpy of Reaction 71 Part E. Illustration of Partial Molar Volume 71 Lab 7: Examination of Viscosity and Suspending Agents ........................................................................73 PHC 340Y Lab Manual 2017-2018
Preface 3 Introduction....................................................................................................................................... 73 Background........................................................................................................................................ 74 Experiment Protocol .......................................................................................................................... 79 Part A. Characteristics of a Polymeric Solution: Intrinsic Viscosity ................................................... 79 Part B. Characteristics of a Polymeric Solution: Fluid Type .............................................................. 80 Part C. Measurement of the Sedimentation Rate of an Ion Exchange Resin (Glass Beads)................ 80 Questions .......................................................................................................................................... 81 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis ....................................................................................82 Introduction....................................................................................................................................... 82 Background........................................................................................................................................ 83 Half-Life and Shelf-Life ................................................................................................................... 85 Temperature dependency of Kinetics: The Arrhenius Equation ....................................................... 85 Kinetics of ASA Hydrolysis............................................................................................................... 86 Calculating the Amount of ASA as a Function of Time ..................................................................... 87 Experiment Protocol .......................................................................................................................... 88 Part A. Acetylsalicylic Acid Hydrolysis – Effect of Temperature........................................................ 89 Part B. Acetylsalicylic Acid Hydrolysis – Effect of Concentration ...................................................... 90 Part C. Acetylsalicylic Acid Hydrolysis – Effect of a Suspension ........................................................ 90 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement ...........................................93 Introduction....................................................................................................................................... 93 Background........................................................................................................................................ 93 Experiment Protocol .......................................................................................................................... 99 Part A. Standard Curve: Salicylate................................................................................................... 99 Part B. The Diffusion Experiment..................................................................................................100 Questions ........................................................................................................................................101 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases.........................103 Introduction.....................................................................................................................................103 Background......................................................................................................................................104 Experiment Protocol ........................................................................................................................108 Part A. UV Absorbance Standard Curve of Salicylic Acid ................................................................108 Part B. Ointment Base Preparation...............................................................................................110 1. Hydrocarbon Base........................................................................................................................111 2. Absorption Base...........................................................................................................................112 3. Emulsion Bases W/O Type............................................................................................................113 4a. Emulsion Bases O/W Type ..........................................................................................................114 4b. Emulsion Bases O/W Type..........................................................................................................115 5. Hydrophillic/Water Soluble Bases.................................................................................................115 6. Poloxamer Gel/Cream ..................................................................................................................116 Part C. Salicylic Acid Base Compounding and Drug Release ...........................................................117 Part D. Using an Ointment Mill .....................................................................................................119 Results & Questions.........................................................................................................................119 Lab 11: Tonicity and Pharmaceutics.....................................................................................................121 Introduction.....................................................................................................................................121 Background......................................................................................................................................122 Experiment Protocol ........................................................................................................................126 Part A. Determination of the Tonicity of Sodium Chloride Solutions..............................................126 PHC 340Y Lab Manual 2017-2018
4 Preface Part B. Determination of the Tonicity of Atropine Sulfate Solutions 127 Part C. Calculation and Preparation of an Isotonic Solution of Atropine Sulfate 128 Part D. Preparation of an Isotonic Phosphate Buffer 128 Part E. Demonstration of the Action of a Hypotonic, Isotonic, and Hypertonic Sodium Chloride Solution on Erythrocytes 128 Questions ........................................................................................................................................129 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water .....................................130 Introduction.....................................................................................................................................130 Background......................................................................................................................................131 Experiment Protocol ........................................................................................................................137 Part A. Preparing the Solutions 137 Part B. Phase Inversion 142 Questions ........................................................................................................................................144 Lab 13: Optimization of Powder Flow and Particle Size Determination................................................146 Introduction.....................................................................................................................................146 Background......................................................................................................................................147 Experiment Protocol ........................................................................................................................152 Part A. Compounding Powder Blends 152 Part B. Determining Tapped Density 153 Part C. Determining the Angle of Repose 154 Part D. Determining Powder Flowability 154 Part E. Sieve Analysis 157 Questions ........................................................................................................................................158 Lab 14: Pharmaceutical Granulations...................................................................................................159 Introduction.....................................................................................................................................159 Background......................................................................................................................................159 Experiment Protocol ........................................................................................................................161 Part A. Preparing a Standard Curve for Acetaminophen 161 Part B. Preparing the Powder Blends and Granulating162 Part C. Milling and Sizing 163 Questions ........................................................................................................................................163 Lab 15: Tableting, Capsuling, and Dissolution Testing ..........................................................................165 Introduction.....................................................................................................................................165 Background......................................................................................................................................166 Tableting Methods 168 Tablet Properties 168 Experiment Protocol ........................................................................................................................175 Lab Period 1:....................................................................................................................................176 Part A. Tableting 176 Part B. Stability (Shelf Life)177 Demonstration: Tablet Coating 178 Lab Period 2:....................................................................................................................................180 Part C. Tablet Dissolution 180 Part D. Formulating Capsules 180 Part E. Content Uniformity: Tablets and Capsules 181
Preface 5 Lab Period 3:....................................................................................................................................181 Part F. Capsule Dissolution ...........................................................................................................181 Part G. Detection of Degradation Products / Decomposition: Thin Layer Chromatography ............182 Summary of Formulation Testing..................................................................................................184 Questions ........................................................................................................................................184 Lab 17: Synthesis and Examination of Colloids.....................................................................................186 Introduction.....................................................................................................................................186 Background......................................................................................................................................186 Experiment Protocol ........................................................................................................................187 Part A. Yttrium Citrate Colloid.......................................................................................................188 Part B. Rhenium Heptasulphide Colloid, Method 1........................................................................189 Part C. Rhenium Heptasulphide Colloid, Method 2 (Performed as a Demonstration only)..............189 Part D. Analysis of Colloids ...........................................................................................................190 Questions ........................................................................................................................................191 Lab 18: Formulating Using Molds.........................................................................................................192 Introduction.....................................................................................................................................192 Background......................................................................................................................................192 Experiment Protocol ........................................................................................................................199 Part A. Formulating 325 mg Acetaminophen Suppositories (Calibrated Batch Volume Method) ....200 Part B. Formulating 20 mg Benzocaine Lollipops (Mass of Drug Negligible) ...................................202 Part C. Formulating 20 mg Hydrocortisone Troches (Displacement Factor Method) ......................204 Part D. Double Casting Method: 70 mg Hydrocortisone/150 mg Lidocaine Lip Balm ......................206 Questions ........................................................................................................................................208 APPENDIX ............................................................................................................................................209 U.S. Standard Sieve Sizes and Lab Sieve Inventory ............................................................................209 Quadro Comil Meshes......................................................................................................................210 Methocel and Avicel Grades.............................................................................................................211 Avicel Grade Usage Chart.................................................................................................................213 Capsule Properties...........................................................................................................................214 Working Ranges of Typical Granulating Fluids...................................................................................215 Viscosities of Typical Fluids...............................................................................................................215 Powder Flowability Indices...............................................................................................................215 Average HLB Values of Some Surface Active Agents..........................................................................216 General Physical Properties of Spans and Tweens.............................................................................218 HLB Requirement for Some Common Oil Components......................................................................220 Buffer Solution Preparation: Polyprotonic Acids and Bases...............................................................221 Dissociation Constants of Acids in Aqueous Solutions at 25°C...........................................................222 Dissociation Constants of Bases in Aqueous Solutions at 25°C...........................................................222 Sorensen Phosphate Buffers ............................................................................................................223 Fundamental Lab Calculations..........................................................................................................223 Preparing a Known Molar Concentration......................................................................................223 Weight-Volume Percent (%w/v) ...................................................................................................224 Dilution Equation .........................................................................................................................224 How to Use a Syringe Filter...........................................................................................................226 Capsule Filling: Quality Control.....................................................................................................227 YOUR NOTES....................................................................................................................................229
Preface Preface There are a lot of rules and guidelines that accompany working in a laboratory, as there is a lot of potential for you harming equipment, or far worse, the equipment harming you. Rising above the details, there are three basic tenets that will permeate through each laboratory: Be aware of the specific hazards and protect yourself accordingly; Think about the exercises as you are doing them, and learn the techniques and principles behind them; Have fun! A lab is a refreshing change from the classroom, where you get to try things out, rather than just being told how they work. Concepts in these labs are used in pharmaceutical industry, in pharmacies, and in research, particularly with respect to drug formulation, manufacture, and compounding. The protocols outlined in the labs provide suggestions on how to observe the phenomena of interest. However, there is more than one way to accomplish something, and there is certainly more than one way to measure something. In many cases, common sense will play an important part of your lab work. For instance, is it more accurate to measure out 5 mL of de-ionized water in a 10 mL graduated cylinder, or a 100 mL graduated cylinder? Subtle methods in the labs may be changed by your instructor, TA, or even by you, depending on the equipment and supplies available to you on your lab day. There is room for creativity. If you find a specific section, step, or explanation in this manual vague or difficult to follow, ask your TA or instructor for help. Please let us know, so we can improve the manual for future editions. The following icons are used in the margins throughout this manual: Useful tip on an experimental method. Read carefully. Important discussion point that is particularly useful in understanding the exercise. Important safety tip. Time-critical experimental step.
Introduction 7 Introduction General Information Check-in for the laboratory will be on September 15, 2017, during the first laboratory session. During the check-in, you will be given your locker key and should make sure all the equipment in your locker is complete and clean. To prepare for a lab, read the part of the lab manual pertaining to that lab exercise, understand the rationale of the exercise, watch any associated videos on the laboratory website, perform any calculations that may be necessary to prepare for the lab, be aware of any potential hazards, review the questions, and go to bed early the night before. The laboratories start on time. There will be various “surprise” pre-lab quizzes during the pre-lab tutorials. They will consist of five or six short questions related to the experiments being performed during that session. Students who are late are not eligible to write the quiz. Recommended Textbooks There are no required textbooks for the PHC340 laboratory. This manual will serve as the primary reference to the laboratory. The following textbooks are recommended to clarify concepts or to serve as useful general references: Sinko, Patrick J. Martin’s Physical Pharmacy and Pharmaceutical Sciences. Lippincott Williams & Wilkins; 6 edition (Feb 21, 2010) Troy, David B. Remington – The Science and Practice of Pharmacy. Lippincott Williams & Wilkins; 21 edition (May 19, 2005) Aulton, Michael E. Aulton’s Pharmaceutics: The Design and Manufacture of Medicines. A Churchill Livingstone Title; 3 edition (Nov 1, 2007) Allen, Loyd V. Jr. et al. Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Lippincott Williams & Wilkins; 9 edition (Jan 7, 2010) Rowe, Raymond C et al. Handbook of Pharmaceutical Excipients. Pharmaceutical Press; 6th edition (2009). Available online, U of T Library permalink: http://simplelink.library.utoronto.ca/url.cfm/141954 (UtorID login required) Teaching Staff The following people will be teaching, helping, and evaluating your work in the lab: PHM340Y Laboratory Coordinator E-mail Address David Dubins d.dubins@utoronto.ca PHM340Y Teaching Assistants E-mail Address Noor Al-Saden n.alsaden@mail.utoronto.ca Giovanna Schver giovanna.medeiros@mail.utoronto.ca Intro
Introduction Role of Teaching Assistants One Teaching Assistant (TA) will be assigned to each laboratory period. The following are the roles and duties of the TA: Before the lab: Ensure the availability of chemicals and supplies, and inform the Instructor if orders are required in advance Work with the instructor to ensure equipment in their assigned section is set up, functional and serviced Prepare buffers, reagents, and indicators in advance  Arrive before the lab in order to warm up any relevant equipment and appropriately set up the lab During the lab: Take attendance, checking student TCards Ensure laboratory safety Notify the Instructor of any injuries or hazards in the lab Handling of the disposal of hazardous chemicals Provide pre-laboratory lectures in an interactive format Supervising students regarding procedure, process, technique and safety elements of laboratory session Coordinate equitable access to equipment Collect student attendance via sign-in sheets Supervise the progress of student work - by asking appropriate questions, not only by providing answers Provide directions and clarify instructions Ensuring the cleanliness of the lab, and coordinating laboratory clean-up  Ensure equipment is clean and shut down at the end of the lab (especially spectrophotometers) After the lab: Assisting in lab check-in and check-out Evaluate submitted laboratory reports, quizzes, products and work plans Recording and entering marks on the Blackboard system Attending and supervising student tours Attendance Attendance in labs is mandatory. Attendance in each lab, and the lab tour, will be recorded. If you miss a lab or lab tour due to medical, personal, family, or other unavoidable reasons, you must provide supportive documentation (e.g. a doctor’s note) to the course Instructor for consideration of accommodation. The U of T Verification of Illness or Injury form is available online at: http://www.illnessverification.utoronto.ca/ If accommodation is granted, you will be asked to complete a make-up assignment on the same topic of the missed course material. Otherwise, if you miss a single laboratory session, you will obtain a zero for that laboratory. If you miss one laboratory session of a laboratory that spans PHC 340Y Lab Manual 2017-2018
Introduction 9 more than one session, your final mark will be multiplied by the ratio of the number of sessions you attended, provided that you participate in writing the final report. Lateness Policy You may submit lab reports via email, or by hard copy. Labs are generally due one week from performing the lab, by the beginning of class. For late submissions, there will be an academic penalty imposed of 10% per day, in accordance with departmental policies. Submissions will not be accepted beyond 1 week from the original due date. Each lab will also be subject to a cleanliness and timeliness penalty. Cleanliness penalties will be issued if a lab area or lab scale is left untidy. Timeliness penalties will be issued if you remain inside the lab more than 10 minutes past the end of the scheduled lab. Budget your time in the lab to allow for sufficient clean-up once you are finished. Lab worksheets, when made available, will have the following box beside the final score to indicate if a penalty has been issued: Raw Score _____ -1% Cleanliness -1% Timeliness Pre-Lab Penalty (max 5%) _____ Late Penalty (10%/day) _____ Intro PHC 340Y Lab Manual 2017-2018
10 Introduction Laboratory and Lecture Schedule All pre-labs and labs will take place in PB860. Labs Lectures Fall Term 2017 (PB860) (PB255) Thursday Monday 9am-1pm 3pm-4pm Lecture 1 – Acid/Base Equilibria (Keith Pardee) 8am-9am, 07-Sep-17 Lab 1* – Safety Lecture, Locker Check-in, Examination of UV 07-Sep-17 Spectroscopy and Preparation of a Standard Curve Lecture 2 – Phase Partitioning (Keith Pardee) 11-Sep-17 Lab 2 – Preparation of pH Buffers 14-Sep-17 Lecture 3 – Mixing (Keith Pardee) 18-Sep-17 Lab 3 ‡ – Effect of pH on the Partition Coefficient of a Slightly Soluble 21-Sep-17 Weak Acid Lecture 4 – Polymorph and Salt (Ping Lee) 25-Sep-17 Lab 4* - Characterization of Drug Candidates (I) – Measuring Solubility 28-Sep-17 and pKa Lab 5* – Characterization of Drug Candidates (II) – Co-solvency, Salt 05-Oct-17 Selection and Polymorph Identification Lab 6* – Thermodynamics of Mixing – Enthalpy and Volume 12-Oct-17 Workshop: Writing Formal Lab Reports for PHC 340 (Heather Sanguins) Lecture 5 – Rheology (Keith Pardee) 16-Oct-17 Lab 7* – Examination of Viscosity and Suspending Agents 19-Oct-17 Lecture 6 – Chemical Kinetics & Stability (Ping Lee) 23-Oct-17 Lab 8 ‡ – Kinetics of Acetylsalicylic Acid Hydrolysis 26-Oct-17 Lecture 7 – Diffusion and Membrane Transport 1 (Ping Lee) 30-Oct-17 Lab 9 – Diffusion and Membrane Transport 1: Permeation Measurement 02-Nov-17 [Exercise 2: Quassignment for Lab 11] Lecture 9 – Colligative Properties (Keith Pardee) 13-Nov-17 (intentionally out of order) Lab 11* – Tonicity and Pharmaceutics 16-Nov-17 (intentionally out of order) Lecture 8 – Diffusion and Membrane Transport 2 (Ping Lee) 20-Nov-17 Lab 10 ‡ – Diffusion and Membrane Transport 2: Drug Release from 23-Nov-17 Ointment Bases Lecture 10 – Molecules at Interfaces (Keith Pardee) 27-Nov-17 Lab 12* – Estimation of Critical Micelle Concentration (CMC) of a 30-Nov-17 Surfactant in Water Lecture 11 – Particle Size and Powder Flow (Ping Lee) 04-Dec-17 Labs Lectures Winter Term 2018 Thursday Wednesday 1pm-5pm 11a-12p Lab 13* – Optimization of Powder Flow and Particle Size Determination 04-Jan-18 Lecture 12 – Pharmaceutical Granulation (Ping Lee) 10-Jan-18 Lab 14 – Pharmaceutical Granulations, Part 1 11-Jan-18 PHC340 Midterm 17-Jan-18 Lab 14 ‡ – Pharmaceutical Granulations, Part 2 18-Jan-18 Lecture 13 – Tabeting and Dissolution Testing (Ping Lee) 24-Jan-18 Lab 15 – Tableting and Dissolution Testing, Part 1 25-Jan-18 Lecture 14 – Measurement, Part 1 (Keith Pardee) 31-Jan-18 Lab 15 – Tableting and Dissolution Testing, Part 2 01-Feb-18 PHC 340Y Lab Manual 2017-2018
Introduction 11 Lecture 15 – Measurement, Part 2 (Keith Pardee) 07-Feb-18 Lab 15 ‡ – Tableting and Dissolution Testing, Part 3 08-Feb-18 Lecture 16 – Synthetic Biology and Human Health, Part 1 (Keith Pardee) 14-Feb-18 Lab 16 – Mystery Laboratory 15-Feb-18 Lecture 17 – Synthetic Biology and Human Health, Part 2 (Keith Pardee) 28-Feb-18 Reading Week Feb 20-23 Lab 17 ‡ – Synthesis and Examination of Colloids 01-Mar-18 Lecture 18 – Mold Calculations (David Dubins) 07-Mar-18 Lab 18* – Formulating Using Molds 08-Mar-18 Lab 19 – Advanced Formulations Project, Part 1 15-Mar-18 Lecture 19 – Ethics & Academic Integrity (Alison Thompson) 21-Mar-18 Industrial Tour (to be confirmed) 22-Mar-18 Lecture 20– Ethics & Academic Integrity (Alison Thompson) 28-Mar-18 Lab 19 ‡ – Advanced Formulations Project, Part 2. Lab Check-Out. 29-Mar-18 PHC340 Final Exam During final exam period ‡ Lab Report to be completed for evaluation * Lab Worksheet to be completed for evaluation Intro PHC 340Y Lab Manual 2017-2018
Introduction Locker Check-In / Check-Out Check-In: September 15 th , 2017 Your Locker #: ____________ You will be assigned your own locker. The contents of your locker have been arranged by students of previous years. It is your privilege to use the locker and your responsibility to maintain the locker. Today, make sure you have all the glassware according to the “Content of your Locker” list. You may also want to clean the glassware. Replacement of damaged equipment can be obtained from the back shelves or from your Teaching Assistants (TAs). Take time to review the laboratory safety section of this manual and locate the following safety equipment in the laboratory. Indicate the location in the space provided below: Safety Equipment Location Fire Extinguishers Fire Alarm Eye Wash Fountains Safety Shower First Aid Box When your group has completed the locker check-in, notify your teaching assistant and he/she will ask you a few safety questions. Locker key issued _________________________ (student signature) Locker Check-Out: Marth 30 th , 2018 A fee of $10.00 will be charged for locker key replacement. You are encouraged to attach the key to a secure key ring or case. Lab Check-Out Procedure Clean your lab bench and any dirty glassware; Throw out any remaining formulations or garbage; Empty your locker water bottle; Verify that your locker contents are complete; Return any extra glassware to the laboratory back shelves; Get a TA or Instructor to verify the above, and sign your check-out list (next page); Be assigned a special area in the lab to clean; Lock your locker, and return your lab key when the above is completed. Keep this and the following page in your laboratory manual. PHC 340Y Lab Manual 2017-2018
Introduction 13 Volumetric Flask Graduated Cylinder Erlenmeyer Flask Bulb Grad. Pipette Pipette Locker Contents 2017-2018 Student Name Locker # Date Name of Apparatus/Item Qty In Out 50 mL Volumetric Flask 2 100 mL Volumetric Flask 2 200 mL or 250 mL Volumetric Flask 3 10 mL Graduated Cylinder 1 25 mL Graduated Cylinder 1 100 mL Graduated Cylinder 2 50 mL Erlenmeyer Flask 2 125 mL Erlenmeyer Flask 2 250 mL Erlenmeyer Flask 2 500 mL Erlenmeyer Flask 2 5 cm Glass Funnel 1 7.5 cm Glass Funnel 1 10 cm Glass Funnel 1 Test Tube Rack 1 1 mL Bulb Pipette 1 5 mL Bulb Pipette 1 10 mL Bulb Pipette 1 20 mL or 25 mL Bulb Pipette 1 1 mL Graduated Pipette 1 10 mL Graduated Pipette 1 Thermometer (°C) 1 Watch Glass small 1 Watch Glass large 1 8” Glass Stirring Rod 1 50 mL Beaker 2 150 mL Beaker 2 250 mL Beaker 2 400 mL Beaker 2 600 mL Beaker 2 Glass Slab 1 3” Ceramic Evaporating Dish 1 6” Ceramic Evaporating Dish 1 Plastic Wash Bottle 1 Ceramic Mortar & Pestle Set (Glass set additional in some cases) 1 Funnel Clamp and Holders 1 Sharpie Lab Marker 1 TA Signature - Lab Check-In TA Signature - Lab Check-Out Watch Glass Beaker Evaporating Dish Mortar & Pestle Funnel Clamp Intro PHC 340Y Lab Manual 2017-2018
Introduction Recording Data, Analysis, and Results In this laboratory, we are attempting to introduce laboratory practices that are employed in research and development labs in the pharmaceutical industry. Practices such as daily initialing of laboratory results and the use of bound books are used to increase security and in some cases to document intellectual property. Hard cover bound laboratory notebooks will be used to record your data. At the beginning of selected laboratories, the TA will give you a laboratory worksheet which you will use to record your data, present your results and interpretation for grading. The worksheets will also contain specific questions to answer about the labs. Other times you will record data in your lab book. During the laboratory, you may work in groups of two, and sometimes in larger groups, for the collection of data. Any data that is collected during the lab period must be recorded in each member of the group’s data booklet. The analysis of the data, presentation and calculations are to be done individually, and recorded in the appropriate section in the data booklet. All lab books are to be initialed by both the student and an instructor at the end of each laboratory session. It is your responsibility to make sure that your book is initialed. Books will be initialed after satisfactory laboratory clean-up has been completed. A lab has a lot of potential for entropy (read: mess). Please keep your lab area tidy. The lab reports will be graded according to the Report Format which is outlined in this introduction. In some cases, grades may also be assigned to the quality of the product, and product label that you made during the lab. Lab reports will be not be returned to you until all students in the class have completed the same exercise. In addition to the lab reports and quizzes, there will be a written exam in December as part of the PHM340 Mid-term exam. Questions related to the work in the Winter Term will be included in the April final exam. Try to work cooperatively with others in your group. If there are unresolved conflicts, approach your TA or the lab coordinator to seek a solution. Plagiarism and Falsification At some point in your laboratory, you might look at your results and think, “OH NO! This can’t be right!” You will be nervous. You will wonder what happened. What went wrong? Worse off, you might be tempted to misreport the results for that ONE point that should have fallen on the line. However, you are reminded to always report what you observed, rather than what you would have liked to observe. Provided you made the correct calculations and performed your exercises meticulously and carefully, you will not lose marks for less than perfect looking observations. Real data rarely look perfect. Things don’t always work. If they did, there would be no need for formulation scientists. If you encounter suspicious looking data, identify your concerns in your analysis, and explain where you think things may have gone wrong (sources of error). If your entire data set is concerning you, seek the assistance of your T.A. or instructor. There could be a malfunction in the equipment, a problem with the method, or a systematic error in your calculations. If you have time, you can repeat the outlying measurements to refute or confirm their validity. PHC 340Y Lab Manual 2017-2018
Introduction 15 DO NOT PLAGIARISE OR FALSIFY YOUR DATA. Doing so is an offence under the University of Toronto Governing Council’s Code of Behaviour on Academic Matters. Clean-up Check-List Your experiment is done. Are you all ready to go? Here are some helpful tips on leaving the lab clean for the next group of students: I cleaned all lab equipment (especially balances!), so other students can use them. I rinsed out my pipettes and burettes with water, so crystallization won’t gum up the tips. I washed and shook out all my glassware, and put it back in my locker so it’s clean for my next lab. I wiped my work area, lab bench, and bench top (including the balances I used). I properly labeled and handed in my preparation (if there is one to hand in). I properly disposed of all chemicals: solid and semi-solid inert waste in the garbage,  liquid inert waste down the sinks hazardous chemicals in appropriately labeled waste bottles in the fume hoods I double-checked the fume hood. It’s clean, and I didn’t leave anything in it. Assignment of Grades Laboratory Reports*, Exercises 65 % Quizzes and Problem Sets (weighted equally) 5 % Mid-term and Final exam (weighted equally) 30 % Total 100 % *Lab reports are weighted in proportion to the number of lab periods. Guidelines for Writing Pre-Labs, Worksheets and Individual Laboratory Reports Pre-Labs Prior to the lab, regardless of whether a worksheet or formal lab report is assigned, you will be expected to prepare a pre-lab in your lab notebook, which should include the following sections: Purpose: Why are you doing this lab? What scientific questions will be addressed? Procedures: In flow-chart form, organize your activities in the lab. This will help you prepare for complicated procedures, and allow you to be more efficient in the lab. Pre-labs will be checked at the beginning of the lab, and will be worth 5% of the lab report or worksheet mark. Preparing a proper pre-lab will help you succeed in surprise quizzes. Individual Lab Worksheets For selected labs, worksheets will be handed out in the beginning and will be made available for download from Blackboard. For these labs, filling out the worksheets and answering the worksheet questions is all that is required for the lab. For these labs, the mark breakdown will be indicated on the worksheets. Intro PHC 340Y Lab Manual 2017-2018
Introduction NOTE: Where applicable, your submitted, properly labeled product will constitute a proportion of the “Presentation, neatness” component. Other laboratories will involve creating a formal lab report. The following is a guide on what is expected for these reports. As each lab is individual, the marking scheme may vary slightly for each lab. Rationale of Laboratory Reports The purpose of writing a scientific report is to communicate your findings with the outside world. Enough detail should be conveyed so that someone who did not do the experiment could repeat it, and be able to fairly compare their results with yours. Writing laboratory reports (and technical writing in general) is an extremely useful and valuable skill to develop. Avoid providing one word answers and bullet points. Use sentence form, and summarize where appropriate. The ability to condense the purpose, observations, and results into an abstract will help the reader connect with the material, and will put your results in perspective for the reader. This process will help prepare you for writing scientific publications. Be consistent with grammar. For events that happened in the lab, use the past tense for reports, and the passive voice. e.g.: “1 mg of the free acid of sulfathiazole was incubated at 25 C in 10 mL of phosphate buffer for 1 hour, with agitation every 15 minutes.” For scientific principles, use the present tense. e.g.: “Ethanol is a co-solvent, and disrupts the hydrogen bonding between water molecules and the surface of the drug molecule.” Details on Writing a Formal Laboratory Report Pre-Lab (5%) Your pre-lab mark will be evaluated at the beginning of each laboratory. Title Page (1%) Please include lab number and title, student name(s), date submitted, and course code. Abstract (10%) No more than 200 words, an abstract is a mini-version of the entire lab report. It provides a brief introduction, purpose, a summary of results (not the raw data itself but parameters estimated), conclusions, and the relevance of the conclusions to the field of study. It is usually the last section that you will write, although it comes first in the report. Introduction (5%) This section should be 1-2 paragraphs long, and include the purpose of the experiment and a brief overview. What is the main purpose of the lab? Which scientific principles are being investigated? What is the value of the results to the field of study? A good introduction will spark the interest of the reader and explain the purpose of the work.  Experimental (10%) This section should be no more than 2 pages long, but depending on the PHC 340Y Lab Manual 2017-2018
Introduction 17 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).  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.”  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 r 2 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 Intro PHC 340Y Lab Manual 2017-2018
Introduction Deviations from the lab protocol Errors in the lab protocol Limitations of the method used to evaluate the phenomena of study Equipment malfunction or improper use of the equipment If the error was a result of experimental design, suggest how the design could be improved. If relevant, put your results in the context of literature values. Were they in agreement? e.g.: The pKa of sulfathiazole was estimated to be 5.98. This is not in good agreement with a published value of 7.14 (reference 1). You may also discuss other related theories.  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. 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.: Fioritto AF et al., Int J Pharmaceutics (2007);330:105-113. 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 2017-2018
Introduction 19 forgetting which beaker you poured it in. It will save you aggravation to get in the habit early of clearly labeling your preparations as you go along. Chemical Disposal There are large green buckets available for broken glassware in the lab. Please use them instead of the garbage, to respect the safety of the cleaning staff.  There will be designated waste jars for hazardous waste and organic solvents in the fume hoods for each lab. When appropriate, there will also be a designated container for sharps (e.g. needles).  Solid and semi-solid chemically inert waste (e.g. petrolatum) will gum up the drains, and are properly disposed of in the garbage. If you are unsure how to properly dispose something, ask your TA or instructor. ACIDS CORRODE PIPES, AND SHOULD BE DISPOSED OF IN WASTE BOTTLES ONLY. Dress Code For your protection, you are required to wear the following protective gear, at all times during the lab: A lab coat Safety Goggles Closed-Toed Shoes (no sandals or open-toed shoes) Clothing that covers your legs The following special protective equipment is available for specific tasks, or on your request: Latex (and non-allergenic neoprene) gloves N95 Masks Protective hair covers Dress Code Rationale If you have ever taken a laboratory course, you have likely already heard much of the following safety advice at some point. Common sense plays a large part in lab safety. However, it is useful to outline a few principles that pertain to the labs in this manual, so they are fresh in your mind. Laboratory coats offer first line protection to your clothes and body against chemical burns. They work best when they are done up – an open lab coat will not properly protect you from a spill. Closed-toed shoes protect your feet from chemical spills.  Safety glasses will help to shield your eyes from any chemical splashes, including boiling solutions.  Latex (and nitrile) gloves are available for use in the laboratory. In particular, hydrochloric acid (HCl), potassium hydroxide (KOH), and sodium hydroxide (NaOH) are extremely corrosive. Gloves should be worn if you are going to be handling these solutions. Gloves also offer protection if you have a known specific allergy or sensitivity to a certain chemical. Working with Hazardous Chemicals  When in doubt, treat all chemicals as hazardous, until you are familiarized with their Intro PHC 340Y Lab Manual 2017-2018
Introduction properties. Consult the Material Safety Data Sheets (MSDS) or your TA for relevant information. Whenever possible, or necessary, handling chemicals in a fume hood will protect you as well as those around you from toxic and flammable fumes. Handle all volatile and flammable solvents in a fume hood. Do not put a sealed container over any heat source, as it may explode. If you are not sure how to use something, ask your TA.  Notify your TA if there is any broken glassware, so they can safely clean and dispose of any chemical or sharps hazards.  Notify your TA immediately if there is a mercury spill. They will have access to a mercury spill kit.  Be cautious when testing for odours. Never inhale a chemical directly. Fan the vapours towards your nose. Many vapours can cause irreparable damage. Never ingest any excipients or products in the teaching laboratory. Other safety references: Merck Index Material Safety Data Sheets (MSDS), a part of the WHMIS (Workplace Hazardous Material Information System) right-to-know system Fisher Scientific Catalog o Sigma-Aldrich MSDS Follow these guidelines to decrease the risks of working with chemicals: Work with a minimum amount of chemicals necessary. Read the warning labels and/or consult the MSDS before using a chemical.  When storing, using or disposing of chemicals, avoid accidental mixing of incompatible chemicals such as acids and bases, flammables and toxics, flammables and oxidizers, oxidizers and reducers.  Highly toxic and flammable chemicals must be stored in ventilated areas in unbreakable, chemically resistant containers. Emergency Response The University Emergency phone number is 416-978-2222. In Case of Personal Injury Inform the Teaching Assistant, or the Laboratory Coordinator of any injury acquired during a lab, no matter how slight it may appear.  An open or even partially healed cut is dangerous, since it allows easier penetration of chemicals. Cover any exposed areas with a bandage when working in the laboratory. Protective latex gloves are available from your TA.  In case of chemical eye injury, hold the eye open in the eye-wash, even if painful, and wash the eye for 15-20 minutes.  In case of chemical body burns, use cold water to wash chemicals from the skin immediately, and thoroughly. Hot water may increase the absorbency of the chemical. PHC 340Y Lab Manual 2017-2018
Introduction 21 In Case of Spills Chemicals spilled in the laboratory must be cleaned up immediately to reduce and eliminate hazards. The Chemical Spill Cart is located in the laboratory outside the entrance of Room 865. In the event of a localized, minor spill, use the following procedure: Responding to a Minor Spill Report all spills to the TA. Notify other students who are working in the area. Confine the spill to a small area. Do not allow the spill to spread. If the material involved is flammable, turn off any ignition sources/electrical equipment present. Ventilation should be established to dispel vapour, if necessary, and if safe to do so.  Absorb and neutralize the spilled liquid chemical. For example, strong acids should first be neutralized with sodium bicarbonate, then washed with water. It is always advisable to add acid into water when mixing, since water has a much larger heat capacity and will therefore be able to absorb any resulting heat much better. You can always remember the catch phrase: “Do as you aughta, add acid to watah”.  The TA or Instructor should handle a mercury spill. Spilled mercury is collected with a mercury collector. Sprinkle the affected area with sulfur powder. The sulfur-mercury powder is then swept up and discarded in the appropriate labeled container. When cleaning up a spill, wear the proper protective equipment, such as gloves and goggles.  After the spilled chemicals have been removed, wash the area with warm, soapy water to remove any residue left behind. In the event of a major spill that exceeds the clean-up capabilities of the laboratory, the following procedure is to be followed: Responding to a Major Spill Notify everyone to evacuate the area immediately.  Contact the University of Toronto Emergency Number 416-978-2222 and state the location of spill, extent of the spill, and the chemical involved. Or, call 911. Wait in a safe area until the response team arrives. In Case of Fire If the fire is contained in beakers or flasks, smother the fire simply by covering the vessels so that no oxygen can enter. If electrical equipment is on fire, unplug it quickly or cut the power if possible.  If your clothing is on fire, do not run. Stop, drop, and roll. If the clothing of someone next to you is on fire, help him to the floor and use your lab coat or fire blanket, or whatever is available to smother the fire. Once the fire is extinguished, help the person away from the general fire area.  If the fire is small and contained, a qualified person should attempt to use a fire extinguisher to eliminate the fire. Many fire extinguishers handle multiple types of fires. There are 4 major classes: Intro PHC 340Y Lab Manual 2017-2018
22 Introduction Fire Extinguisher Appropriate for: Examples: Class Class A Ordinary combustibles (paper, wood, cardboard) Class B Flammable/Combustible (gasoline, organic Liquids and Gasses solvents) Class C Electrical Equipment Computers, monitors, melting point apparatus Class D Combustible metals Magnesium, titanium, potassium, sodium Class K Grease fires Cooking Oils, fats The fire extinguishers in Room 860 are rated for Classes A, B, and C. They are located by each exit, and outline the following procedure: (PASS) Pull the pin out  Aim at the base of the fire Squeeze the handle Sweep the nozzle back and forth  If the fire is too large to be contained with a fire extinguisher, pull the fire alarm, and evacuate the building. Once out of harm’s way, call the University of Toronto Emergency 978-2222 or call 911. Specify the site and extent of the fire.  Wait outside the building, away from the main entrance so that you do not block the entrance when the fire personnel arrive. If the Fire Alarm Sounds Evacuate the building quickly, using the stairwells. The elevators will automatically go out of service. Do not try to use them.  Wait in the designated emergency area (the area between the Medical Sciences Building and the Leslie L. Dan Pharmacy Building), Keep clear of the building. Do not re-enter the building until authorized by a Fire Officer. PHC 340Y Lab Manual 2017-2018
Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve 23 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve Read the introduction and lab protocol completely 1 Watch the following related lab videos on the laboratory website: La b Preparing for the Lab  UV/Vis Spectrophotometry - Determining Absorbance (http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv) Calculate the volume of stock required for each standard solution in the calibration curve. Group Allocation You will be working in groups of 2 students Part A: Prepare a calibration curve for hydrochlorothiazide What You’ll Be Doing Part B: Plot your calibration curve Demonstration: Using a spectrophotometer Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/calibration.xls What You’re Handing In Lab 1 Worksheet (due at the beginning of the next lab) Introduction One of the fundamental tools to be used in any pharmaceutics laboratory is the analysis of the drug that is the subject of the experiment. In this introductory session a standard solution will be prepared and some of the principles related to the Beer-Lambert Law will be examined. The standard curve will be able to be used in a later session. Background Lambert’s Law Lambert showed that each unit length of material through which light passes absorbs the same fraction of the incident or entering light and compares the relation between the incident light (Io) and the transmitted light (IT) for various thicknesses t. Io loge IT  t Where: Ȁ⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀ᨀĀĀĀ⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀ is the intensity of light is the thickness of the substance  the absorption coefficient Conversion to log 10 results in the equation: log10 I 0   t Kt 2.3026IT Where K is the extinction coefficient generally defined as the reciprocal of the thickness (t in cm) required in order to reduce the intensity of the incident light to its original intensity. PHC 340Y Lab Manual 2017-2018
Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve Beer’s Law Despite what it sounds like, Beer’s Law does not describe the relationship between number of beers consumed and physical attraction. Beer examined the relationship between absorption and the concentration of coloured solutions. The equation is similar: log 10 I0  k 1 c IT If this is performed in a cell with a uniform thickness then a measure of the length l may be added: (2) log I 0  k 1 cl or log I 0  A 10 I T 10 I T The value of k 1 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:  = A (1 %w/v, 1 cm) × l is equal to the slope of the calibration curve (absorbance vs. concentration):  A = × l × c Where: = extinction coefficient ((concentration units) -1 cm -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. Extinction 0 .5 0 .4 0 .3 0 .2 0 .1 0 0 1 2 3 4 5 6 7 8 9 ml Standard Fe (0. 02 mg Fe/ ml) Figure 1. A Standard Curve for an Iron Solution PHC 340Y Lab Manual 2017-2018
Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve 25 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 8 th 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. 1 2 1 0 ) 8 ,1cm 6 E(1% 4 2 0 1 90 2 00 2 10 2 20 2 30 2 40 2 50 2 60 2 70 2 80 2 90 Wa v elength Figure 2. Graph Showing the Change in E (1%, 1cm) at Several Wavelengths Areas of the curve where the change [E (1%, 1cm)] is large should never be used in drug analysis. On the curve in Figure 2, wavelengths of 205, 230, and 265 nm are sub-optimal. Experiment Protocol Chemicals Supplies Special Equipment Hydrochlorothiazide (5 mg/mL) in Plastic transfer pipettes Helios UV/Vis Sodium Hydroxide Solution (0.1 N) UV Cuvettes (Plastic) Spectrophotometer Volumetric Sodium Hydroxide (40.0 g/mol) Parafilm flasks The following solutions are prepared or provided by the TA: 250 mL of Hydrochlorothiazide stock solution (5 mg/mL in 0.1 N NaOH) The Helios spectrophotometers will be turned on prior to the laboratory. Each person will do their own measurements. Part A. Preparing a Calibration Curve Prepare 1 L of 0.1 N Sodium Hydroxide solution. Note: do not leave sodium hydroxide pellets exposed to air. Close the cap of the bottle when not weighing pellets. Wear gloves when weighing and handling sodium hydroxide. Lab1 PHC 340Y Lab Manual 2017-2018
Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve Prepare dilutions of the hydrochlorothiazide stock solution in 0.1 N NaOH as follows, in triplicate: 1:10, 1:50, 1:100, 1:200, 1:250 To clarify, “in triplicate” means that you create each solution three times, rather than measure the absorbance of the same solution three times, to get an estimate of the error associated with creating the standard solutions. Measuring the standards in triplicate will allow you to report the average, standard deviation, and %RSD at each standard concentration. An efficient way to accomplish this is to have three different people run the same curve in parallel. The same spectrophotometer must be used. 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. 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. Repeat the above steps with each of the five dilutions of the sample. Measure the absorbance of the stock solution (remember 3 determinations). Calculate the average and standard deviation for each concentration. SPECTROSCOPY NOTES Fill the cuvette to the etched line (approx ¾ full) Make sure the cuvette is facing the correct way (the light path should go through the clear windows through the longest path length, not the ridged sides) To avoid fingerprints, only handle the cuvettes by the ridged sides, not the clear windows. Fill the cuvette slowly, and gently tap to release bubbles clinging to the sides of the cuvette Gently wipe the clear windows with a Kimwipe prior to measuring Make sure the sample door is closed before measuring absorbance Make sure you use the same UV spectrophotometer for calibration and sample measurements. *NOTE: Plastic UV cuvettes are tapered towards the bottom, to accommodate a smaller sample volume. The fill line is just above the clear part of the cuvette window. The “V” shaped arrow on the Plastic UV cuvette indicates the side of the cuvette that the UV beam will travel through the entire 1 cm path length (not widthwise, which is only 0.5 cm): Fill line (fill to at least here) Spectrophotometer beam travels this way PHC 340Y Lab Manual 2017-2018
Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve 27 Lab1 Beam direction Beam direction Helios Spectrophotometer (PB 860) Varian Spectrophotometer (PB 819) Part B. Plotting Your Calibration Curve You will be preparing two calibration curves using calibration.xls (available in the Downloads section of the laboratory website): One curve with all of your collected data;  One curve with the linear portion of the curve (excluding the higher concentrations). Questions Describe the shape of the curve that results from your data. Does the best-fit curve go through zero? Is this necessary for Beer’s law to be valid? Which of the linear fits in the two curves in Part B would you use to convert OD to concentration? Why? What is the accuracy of your measurements? What is the precision? Hydrochlorothiazide is a very weak acid. Why is 0.1 N NaOH used to help dissolve hydrochlorothiazide? PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers Lab 2: Preparation of pH Buffers Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:  Measuring pH Preparing for the Lab (http://phm.utoronto.ca/~ddubins/DL/pH.wmv)  UV/Vis Spectrophotometry - Determining Absorbance (http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv) Calculate the volume of stock required for each standard solution in the calibration curve. Group Allocation You will be working in groups of 2 students Part A: Preparing Phosphate (Sorensen’s) Buffer @ pH 7.4 Part B: Preparing McIlvane’s Buffer at 2 assigned pH values What You’ll Be Doing Demonstration: Using a pH meter Your buffers will be stored in the cold room on the 9 th floor, for use in Lab 3. Spreadsheets You Will Need Not applicable. What You’re Handing In Not Applicable. Retain and store buffers prepared in this lab for use in Lab 3 (McIlvaine’s) and Lab 4 (Sorensen’s). Introduction Buffers are fundamental to wet chemistry, although the basic idea of buffers extends far beyond solutions. The primary idea behind a buffer is to dampen or minimize the effects of changes to or within the system so that the impact on the system is not as bad. There are buffers in electrical systems, irrigation systems, computers, and mechanics. Shock absorbers, for instance, prevent you from feeling bumps in the road when you are driving. Similarly, in wet chemistry, buffers can help reduce or minimize external stresses (changes in temperature or pressure), or chemical reactions from changing the overall pH of a solution. It is critical to select a buffer that is well suited to the system you are studying. Will the temperature of the system be changing? Will the pressure be changing? How does a change in either affect the pKa of the buffer? What is the pH value you would like to maintain? A buffer is most effective when the pH of the solution is in the vicinity of its pKa value (±1 pH unit). In this laboratory, you will learn how to calibrate and use a pH meter. You will be preparing two buffer systems: Sorensen’s Buffer and McIlvaine’s Buffer. You will be using these buffers in the following two labs. References Glasstone, Samuel. An Introduction to Electrochemistry. New York, NY USA (1942). p372. http://www.chembuddy.com/?left=pH-calculation&right=pH-buffer-capacity http://biotech.about.com/od/buffersandmedia/ht/phosphatebuffer.htm http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer http://www.sigmaaldrich.com/life-science/core-bioreagents/biological- buffers/learning-center/buffer-reference-center.html 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 PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers 29 theoretical (or even published) values of how much of each buffer component to add are just that – theoretical. The actual recipe required could be different, depending on the purity of components, errors in weighing and measurement, and even the quality of water used. There is a lot of theory involved, but at the end of the day, the calculated theoretical values serve only as a guide. Making a buffer is relatively quick and straightforward once you’ve tried it a few times. In order to understand buffers and how they work, a “crash course” in pH and pKa is offered in this section. Definition of pH and pKa An acid will dissociate in water to a conjugate base and proton. Consequently, acids are typically thought of as proton donors: Ka (1) [HA]  [H + ] + [A - ] Acid Proton Conjugate Base Ka is the equilibrium constant that determines the extent that the acid will dissociate in water: (2) Ka  [H ][A ] [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: (5) 10-pKa 10 pH [A  ] [HA] (6) [HA] 10pKa [A  ] 10 pH [HA] 10 pKapH [A  ] According to the Henderson-Hasselbalch buffer relationship, pH, pKa, and the buffer component concentrations for a weak acid are related as follows: [base] [acid]  10 p K a p H Here, the ‘acid’ is the proton donor (HA), and the ‘base’ is the conjugate base (A - ) in Equation (1). This is a very convenient form of the equation, because it allows us to see the following: Key Concepts if the pKa is greater than the pH, there will be more of the acid form in the solution. If the pKa is equal to the pH, there will be an equal amount of acid and base in the Lab2 PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers solution. If the pKa is less than the pH, there will be more of the base form in the solution. A strong acid is defined as one that will dissociate completely. Consequently, the lower the pKa of the acid, the stronger the acid. The same scheme can be re-written to describe the reaction of a base with water, to form its conjugate acid: 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 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; pKb = -log[Kb]; pKa = 14 – pKb Try it out for yourself. This saves us having to remember two sets of Hendersson-Hasselbalch equations. If the pKa of a base is greater than the pH, there will be more conjugate acid. It need only be remembered that [B-H + ] is the concentration of conjugate acid and [B] is the concentration of base. The higher the pKa of a base, the stronger the base. By using the appropriate experimental conditions, the pKa of a drug may be measured directly with a pH meter. Pairing a Weak Acid with its Salt: Sorensen’s Buffer Many buffer systems are weak acids paired with their respective salts. One example is citric acid paired with sodium citrate. The reason that two solutions are made at the beginning – a solution of the acid of the buffering agent, and another solution of its salt – is so that we may titrate one with the other to attain the exact pH we are looking for. It is assumed that when the salt of a buffer is dissolved in water, it will dissociate completely and go into solution in the ionic form. It is important to note however that a buffering system can be as simple as a weak acid added to de-ionized water, with the pH of solution adjusted close to the pKa using either NaOH or HCl. Buffering systems are not limited to weak acids, they can also be weak bases (e.g. ammonia + ammonium chloride). Weak bases may be used for solutions where the pH desired is above 7. We will focus our discussion on weak acid buffers paired with salts of their conjugate OH bases. Since it was already stated a buffer is most effective within 1 O P OH pKa2 = 6.86 pH unit of its pKa, you would think that a given buffer would only be useful in the vicinity of one pH value. However, many pKa1 = 2.15OHbuffers have more than one acidic group attached, which vary in affinity to their respective protons. Sorensen’s Buffer Phosphoric Acid (phosphate buffer) has three acidic groups, each with different pKa values (see right panel). PHC 340Y Lab Manual 2017-2018 pKa3 = 12.32
Lab 2: Preparation of pH Buffers 31 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 O - + H + O P OH P 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 OH Na + - + + H2OO P O Na O P OH + HO - O - Na + O - Na + Sodium Phosphate Dibasic Sodium Phosphate Monobasic (salt of conjugate base) (weak acid) Lab2 PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers However, to a first approximation, we treat the system as if the salt completely dissociates and stays in the ionic form. A buffer is usually prepared in concentrations ranging from 0.1 – 10 M. The way that a buffer works, is that provided there are both forms (acid and conjugate base) of the buffer present (i.e. the pH is around the pKa), then if another acid dissociates to add a proton to solution, the proton will be absorbed by the buffer’s conjugate base instead of lowering the pH. If a base is added to the solution, it will result in a hydroxide ion, which will in turn react with the buffer’s weak acid instead of raising the pH. In this way, the balance of hydrogen ions is protected, and changes in pH are much smaller than they would have been in the absence of buffer. Calculating the Amount of Acid and Salt of Conjugate Base Required There are essentially four decisions to make when selecting a buffer: What is the desired pH of the solution? What type of buffer system will you choose? Which pKa will you be making use of? 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]  10p Ka p H [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: [base] [acid]  [HA] [A]  10 6.867.4  10 0.54  0.2884 Simplifying (15): (16) [HA] = 0.2884 [A - ] Since we want the buffer to be 0.1 M, we also have a mass balance to think about: (17) [HA] + [A - ] = 0.1 M Substituting equation (16) into (17), we can solve for [A-], the concentration of conjugate base we will be adding in the salt form: 0.2884[A - ] + [A - ] = 0.1 M 1.2884[A - ] = 0.1 M [A - ] = 0.0776 M PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers 33 Substituting equation (20) into (17), we can solve for [HA], the concentration of acid that will be added in the un-ionized acid form: [HA] + 0.0776 = 0.1 M [HA] = 0.0224 M So now we have to do the important part: we have to translate a theoretical calculation into reality. We need to create two solutions and mix them together, such that the final concentration of sodium phosphate monobasic is 0.0224 M, and the concentration of dibasic is 0.0776 M. If we would like to start with two stock concentrations, 250 mL each, at a concentration of 0.2 M: Sodium Phosphate Monobasic (NaH2PO4*H20) m  C  MW  V m  0.2 mol L 137.99 mol g  0.250 L m  6.900 g Sodium Phosphate Dibasic (Na2HPO4*7H20) m  C  MW  V mol g m  0.2 L 268.07mol0.250 L Lab2 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 C1V1  C 2 V2 C1V1  C 2 V2 V C 2 V2 V C 2 V2 1 C1 1 C1 0.0224 mol  250 mL 0.0776 mol  250 mL L L V1  V1  0.2mol 0.2mol 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 PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers burette. The pH electrode is inserted into the beaker, and solution is titrated until the desired pH is attained. Once it is attained, for this particular protocol, an equal volume of water (volume in beaker + volume of solution titrated) is added to bring the concentration of buffer from 0.2 M to 0.1 M. Question: Why can’t you just prepare 0.1 M of each solution, and add them together? Wouldn’t that give you a 0.1 M Sorensen’s buffer? Answer: You can. The reason we start with 0.2 M stock solutions in this case is out of convenience, because the burette can only hold 50.0 mL. A 250 mL solution of 0.1 M sodium phosphate monobasic would require 56 mL for the final mix, which wouldn’t all fit in one burette. What makes these calculations lengthy is compensated by the simplicity of published tables in the literature of volumes of each solution to attain the desired pH. In practice, you need not perform the calculations routinely, you can use the tables as a starting point and titrate to your desired pH. However, studying the theory behind these calculations will make all the difference in your understanding of how a buffer works. Buffer Capacity One question that might arise is how well will the buffer protect the pH from changing? This will depend on the pKa of the buffer, pH of the solution (the closer to the pKa the pH is, the more effective the buffer will be), and on the concentration of buffering agent used. One definition of buffer capacity is the amount of (external) acid or base required to change the pH of 1 L of the solution by 1 pH unit. The higher the buffer capacity, the larger this number will be. A formula to calculate buffer capacity is presented here: dn K w  C buf K [H  ]  (23) β   2.303  [H  ]  a    2 dpH  [H ] (Ka [H ])    Where, : buffer capacity (mol/(L*pH unit)). number of moles of acid or base added (assumed to be monoprotic) Kw: The equilibrium constant of water (1.00×10 -14 at 25 °C) Cbuff: the concentration of buffering agent The summation sign in Equation (23) means that you can enter more than one Ka of your buffering agent if it has multiple acidic groups, or the equation can be used for buffers with multiple components. It is beyond the scope of this discussion to derive Equation (22). However, we may use it to calculate the buffer capacity of our Sorensen’s buffer. At a pH 7.4: PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers 35 [H  ]  10 pH 107.4  3.9811 10 8 K a 10 K a 106.86  1.3804 10 7 Kw   C buf K a [H  ] β  2.303  [H ]     2 [H ] (K a  [H ])     110 -14 0.1 M 1.3804 10 7  3.9811 10 8   β  2.303  3.9811 10 8   8 7 8 2 3.9811 10 (1.3804 10  3.9811 10 )     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 BufferCapacity (mmol/(L* pH) 100 80 60 40 20 0 2 3 4 5 6 7 8 pH We can see a peak (left panel, above) at the second pKa of phosphoric acid (6.88), and the buffer capacity decreases as the pH falls farther away from the pKa. At 1 pH unit away (pH 6 or pH 8) the buffer capacity is reduced to less than half of what it was at the pKa. Some buffering systems make use of more than one buffering agent. For instance, McIlvaine’s buffer contains both phosphoric acid (pKa values: 2.15, 6.86, and 12.32), and citric acid (pKa values: 3.13, 4.76, and 6.40). Within the range pH 2 – 8, you are never farther than 1 pH unit away from a pKa. The buffer capacity graph for McIlvaine’s buffer is more complicated (right panel, above). We can use buffer capacity to back-calculate what concentration of buffer we require (Cbuf), depending on what changes in pH we would like the system to tolerate. This will affect Equation (11), the mass balance of buffering agent. In practice, a concentration of 0.1 M is usually used. Did you know? The mathematics of acid/base chemistry are exactly the same as that for any other equilibrium reaction, e.g. drug/receptor interactions. So by learning the mathematics of buffers, you have just learned how to calculate binding constants of drugs with their targets. Lab2 PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers Remember that the Ka values for weak acids and bases are dependent on the temperature and pressure of the solution. Many experiments make use of this, and measure the equilibrium constants at different temperatures or pressures to examine other interesting properties of the systems studied. In practice, Sorensen’s buffer is simply referred to as “phosphate buffer”. Key Concepts: Many buffering systems are weak acids paired with the salt of their conjugate bases, or weak acids titrated to the viscinity of their pKa with NaOH or HCl. A buffering agent is useful within 1 pH unit of its pKa.  Understanding the mathematics of buffering systems is important, but ultimately, your pH meter decides how much of each agent to add. Experiment Protocol Chemicals Supplies Special Equipment Sodium Phosphate Monobasic N/A pH Meter (verify MW on the bottle used) Mixing plate and magnetic stir Sodium Phosphate Dibasic (verify bar MW on the bottle used) 100 mL graduated cylinder Citric Acid (MW 210.14 g/mol) 250 mL volumetric flask pH Standardizing Buffers 140 mL beaker 50 mL burette, burette clamp, retort stand The following solutions are prepared or provided by the TA: pH Meter Standardizing Buffers (pH 4, 7) Part A. Preparing Sorensen’s Buffer Prepare 250 mL of a 0.2 M solution of sodium phosphate monobasic. Prepare 250 mL of a 0.2 M solution of sodium phosphate dibasic. Load a burette with 50 mL of the 0.2 M sodium phosphate monobasic solution. Set up the burette on a retort stand with a burette clamp.  Measure out 97 mL of sodium phosphate dibasic using a 100 mL graduated cylinder. Pour into a 140 mL beaker. Calibrate your pH meter with the standard solutions provided.  Set the 140 mL beaker on top of a mixing plate under the burette, and insert the pH electrode.  Make sure the tip of the pH electrode is submerged, and does not touch the bottom of the beaker.  Place a magnetic stir bar in the 140 mL beaker, and set mixing to a low speed. Do not allow the stir bar to hit the pH meter, or a vortex to appear.  Slowly titrate the sodium phosphate dibasic solution with the sodium phosphate monobasic solution, until you attain a pH of 7.40. PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers 37 Record the volume of sodium phosphate monobasic added.  Transfer the titrated mixture to a 250 mL beaker. Add an equal volume of water to create a 0.1 M Sorensen’s Buffer. Seal your buffer with parafilm, label it appropriately, and store it for Lab 4. Part B. Preparing McIlvaines’s Buffer McIlvaine’s Buffer is a mixture of phosphoric and citric acids. It is useful in the range pH 2.2 – pH The two solutions to be prepared are sodium phosphate dibasic, and citric acid. In this exercise, you will be assigned two of the following pH values: 2.5, 3, 3.5, 4, and 4.5. You may be assigned different pH buffers by group. The following is a published chart on volume (in mL) of each solution to combine for the expected pH, through the useful range of McIlvaine’s Buffer. It makes 20 mL of buffer: Lab2 Source: http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer In a 250 mL flask, prepare 0.2 M sodium phosphate dibasic solution. NOTE: You may have enough stock left over from making the Sorensen’s Buffer. In a 250 mL volumetric flask, prepare 0.1 M citric acid solution.  Load a burette with 50 mL of the 0.2 M sodium phosphate dibasic solution. Set up the burette on a retort stand with a burette clamp. Measure out 100 mL of citric acid using a 100 mL graduated cylinder. Pour into a 250 mL PHC 340Y Lab Manual 2017-2018
Lab 2: Preparation of pH Buffers beaker. Set the 250 mL beaker on top of a mixing plate under the burette, and insert the pH electrode.  Place a magnetic stir bar in the 250 mL beaker, and set mixing to a low speed. Do not allow the stir bar to hit the pH meter, or a vortex to appear.  Slowly titrate the citric acid solution with the sodium phosphate dibasic solution, until you attain the desired pH. NOTE: Depending on the pH selected, you may have to re-fill the burette with the sodium phosphate dibasic solution. Record the total volume of sodium phosphate dibasic added. Seal your buffer with parafilm, label it appropriately.  The TA will store your buffers in the cold room on the 9 th floor, for Lab 3. In general, many aqueous buffers will grow bacteria at room temperature, and are best stored at cold temperatures (e.g. at 5 °C in media bottles, or -20 °C in small aliquots, depending on the buffer). Questions Explain using the Henderson-Hasselbalch equation why the pH of the Sorensen’s buffer shouldn’t change when you add an equal volume of water for the final step. The pKa of salicylic acid is 2.97. What form (ionized or un-ionized) will it predominantly be: In the stomach, at pH 2? In the gut, at pH 7? If the ionized form (salicylate) cannot pass through cell membranes, where would you expect the drug to be absorbed? 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 BOH +H2O HO H OHHO Boric Acid Borate What is the useful pH range of borate buffer? Create a protocol for preparing 500 mL of 0.1 M Borate buffer, at pH 8, assuming you already have a solution of 0.2 M NaOH. How will you prepare your stock solutions? How much of each will you require? Calculate the buffer capacity of 0.1 M Borate buffer: At pH 8. At pH 5. Does your answer for (a) double at pH 8 for a Cbuff of 0.2 M? PHC 340Y Lab Manual 2017-2018
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
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2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
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2003 pharmaceutical chemistry laboratory manual 1
2003 pharmaceutical chemistry laboratory manual 1
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2003 pharmaceutical chemistry laboratory manual 1

  • 1. Student Name: _________________________________ Pharmaceutical Chemistry (PMY 331 & PMY 332) Laboratory Manual Prepared by: Lungwani T.M. Muungo, PhD 2003
  • 2. Pharmaceutical Chemistry Laboratory Manual Pharmaceutics Principles and Evaluation Pharmaceutical Chemistry (PMY 331 & PMY 332) Department of Pharmacy School of Medicine University of Zambia Programme Coordinator: Dr. Lungwani T. Muungo Lungwani.muungo@unza.zm
  • 3. This manual was prepared for use by students enrolled in the undergraduate Pharmacy Program at the Department of Pharmacy, University of Zambia. Permission is granted to copy the manual provided no charge is made beyond reasonable reimbursement for duplication and handling costs, and provided that this notice is retained in all such copies. This manual was compiled the HOD for official use in 2003. Special thanks to staff members at the Department of Chemistry that actively participated in the laboratory work when pharmacy students were allocated. Specific contributions included optimizing the experimental procedure to better detect phase inversion, ensuring a robust incoming voltage by introducing and modifying the 9V DC adaptors, developing and testing the metal electrodes, and adopting the pharmaceutical lab protocol. Lungwani T.M. Muungo, PhD
  • 4. Preface 1 Table of Contents Preface.....................................................................................................................................................6 Introduction.............................................................................................................................................7 General Information.............................................................................................................................7 Recommended Textbooks................................................................................................................7 Teaching Staff ......................................................................................................................................7 Role of Teaching Assistants ..............................................................................................................8 Attendance......................................................................................................................................8 Lateness Policy.....................................................................................................................................9 Laboratory and Lecture Schedule ....................................................................................................... 10 Locker Check-In / Check-Out .............................................................................................................. 12 Recording Data, Analysis, and Results................................................................................................. 14 Plagiarism and Falsification ............................................................................................................ 14 Clean-up Check-List............................................................................................................................ 15 Assignment of Grades ........................................................................................................................ 15 Guidelines for Writing Pre-Labs, Worksheets and Individual Laboratory Reports................................. 15 Laboratory Safety............................................................................................................................... 18 Chemical Inventory ........................................................................................................................ 18 Labeling of Preparations................................................................................................................. 18 Chemical Disposal .......................................................................................................................... 19 Dress Code..................................................................................................................................... 19 Dress Code Rationale ..................................................................................................................... 19 Working with Hazardous Chemicals................................................................................................ 19 Emergency Response ..................................................................................................................... 20 In Case of Personal Injury ............................................................................................................... 20 In Case of Spills .............................................................................................................................. 21 In Case of Fire ................................................................................................................................ 21 If the Fire Alarm Sounds................................................................................................................. 22 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve.......................................23 Introduction....................................................................................................................................... 23 Background........................................................................................................................................ 23 Experiment Protocol .......................................................................................................................... 25 Part A. Preparing a Calibration Curve.............................................................................................. 25 Part B. Plotting Your Calibration Curve............................................................................................ 27 Questions .......................................................................................................................................... 27 Lab 2: Preparation of pH Buffers............................................................................................................28 Introduction....................................................................................................................................... 28 Background........................................................................................................................................ 28 Definition of pH and pKa................................................................................................................. 29 Buffer Capacity............................................................................................................................... 34 Experiment Protocol .......................................................................................................................... 36 Part A. Preparing Sorensen’s Buffer................................................................................................ 36 Part B. Preparing McIlvaines’s Buffer.............................................................................................. 37
  • 5. 2 Preface Questions .......................................................................................................................................... 38 Lab 3: Effect of pH on the Partition Coefficient of a Slightly Soluble Weak Acid.....................................39 Introduction....................................................................................................................................... 39 Background........................................................................................................................................ 39 Experiment Protocol .......................................................................................................................... 43 Part A. UV Absorbance Standard Curve of Sodium Salicylate 44 Part B. Determination of the Partition Coefficient 45 Part C. Direct Measurement of the Partition Coefficient 46 Questions .......................................................................................................................................... 47 Lab 4: Characterization of Drug Candidates (I) – Measuring Solubility and pKa ......................................48 Introduction....................................................................................................................................... 48 Background........................................................................................................................................ 49 pKa and Intrinsic Solubility 49 Calculation of pHp 51 Polymorphism 55 Experiment Protocol .......................................................................................................................... 56 Part A. Intrinsic Solubility Determination 56 Part B. Preparing Different Salts of Sulfathiazole 57 Part C. Preparing Different Polymorphs of Sulfathiazole 57 Part D. pKa Determination 58 Part E. Melting Point Determination 59 Part F. Macroscopic Evaluation 60 Questions .......................................................................................................................................... 60 Lab 5: Characterization of Drug Candidates (II) – Co-solvency, Salt Selection, and Polymorph Identification .........................................................................................................................................61 Introduction....................................................................................................................................... 61 Background........................................................................................................................................ 62 Experiment Protocol .......................................................................................................................... 63 Part A. Co-solvency 63 Part B. Salt Selection 64 Part C. Polymorph Identification 64 Questions .......................................................................................................................................... 64 Lab 6: Thermodynamics of Mixing – Enthalpy and Volume....................................................................65 Introduction....................................................................................................................................... 65 Background........................................................................................................................................ 65 Specific Heat Capacity 65 Partial Molar Quantities 68 Experiment Protocol .......................................................................................................................... 69 Part A. Calibration of the Calorimeter 70 Part B. Specific Heat Capacity of Copper Metal 71 Part C. Heat of Reaction and Heat of Hydration 71 Part D. Measurement of Molar Enthalpy of Reaction 71 Part E. Illustration of Partial Molar Volume 71 Lab 7: Examination of Viscosity and Suspending Agents ........................................................................73 PHC 340Y Lab Manual 2017-2018
  • 6. Preface 3 Introduction....................................................................................................................................... 73 Background........................................................................................................................................ 74 Experiment Protocol .......................................................................................................................... 79 Part A. Characteristics of a Polymeric Solution: Intrinsic Viscosity ................................................... 79 Part B. Characteristics of a Polymeric Solution: Fluid Type .............................................................. 80 Part C. Measurement of the Sedimentation Rate of an Ion Exchange Resin (Glass Beads)................ 80 Questions .......................................................................................................................................... 81 Lab 8: Kinetics of Acetylsalicylic Acid Hydrolysis ....................................................................................82 Introduction....................................................................................................................................... 82 Background........................................................................................................................................ 83 Half-Life and Shelf-Life ................................................................................................................... 85 Temperature dependency of Kinetics: The Arrhenius Equation ....................................................... 85 Kinetics of ASA Hydrolysis............................................................................................................... 86 Calculating the Amount of ASA as a Function of Time ..................................................................... 87 Experiment Protocol .......................................................................................................................... 88 Part A. Acetylsalicylic Acid Hydrolysis – Effect of Temperature........................................................ 89 Part B. Acetylsalicylic Acid Hydrolysis – Effect of Concentration ...................................................... 90 Part C. Acetylsalicylic Acid Hydrolysis – Effect of a Suspension ........................................................ 90 Lab 9: Diffusion and Membrane Transport (I) – Permeation Measurement ...........................................93 Introduction....................................................................................................................................... 93 Background........................................................................................................................................ 93 Experiment Protocol .......................................................................................................................... 99 Part A. Standard Curve: Salicylate................................................................................................... 99 Part B. The Diffusion Experiment..................................................................................................100 Questions ........................................................................................................................................101 Lab 10: Diffusion and Membrane Transport (II) – Drug Release from Ointment Bases.........................103 Introduction.....................................................................................................................................103 Background......................................................................................................................................104 Experiment Protocol ........................................................................................................................108 Part A. UV Absorbance Standard Curve of Salicylic Acid ................................................................108 Part B. Ointment Base Preparation...............................................................................................110 1. Hydrocarbon Base........................................................................................................................111 2. Absorption Base...........................................................................................................................112 3. Emulsion Bases W/O Type............................................................................................................113 4a. Emulsion Bases O/W Type ..........................................................................................................114 4b. Emulsion Bases O/W Type..........................................................................................................115 5. Hydrophillic/Water Soluble Bases.................................................................................................115 6. Poloxamer Gel/Cream ..................................................................................................................116 Part C. Salicylic Acid Base Compounding and Drug Release ...........................................................117 Part D. Using an Ointment Mill .....................................................................................................119 Results & Questions.........................................................................................................................119 Lab 11: Tonicity and Pharmaceutics.....................................................................................................121 Introduction.....................................................................................................................................121 Background......................................................................................................................................122 Experiment Protocol ........................................................................................................................126 Part A. Determination of the Tonicity of Sodium Chloride Solutions..............................................126 PHC 340Y Lab Manual 2017-2018
  • 7. 4 Preface Part B. Determination of the Tonicity of Atropine Sulfate Solutions 127 Part C. Calculation and Preparation of an Isotonic Solution of Atropine Sulfate 128 Part D. Preparation of an Isotonic Phosphate Buffer 128 Part E. Demonstration of the Action of a Hypotonic, Isotonic, and Hypertonic Sodium Chloride Solution on Erythrocytes 128 Questions ........................................................................................................................................129 Lab 12: Estimation of Critical Micelle Concentration of a Surfactant in Water .....................................130 Introduction.....................................................................................................................................130 Background......................................................................................................................................131 Experiment Protocol ........................................................................................................................137 Part A. Preparing the Solutions 137 Part B. Phase Inversion 142 Questions ........................................................................................................................................144 Lab 13: Optimization of Powder Flow and Particle Size Determination................................................146 Introduction.....................................................................................................................................146 Background......................................................................................................................................147 Experiment Protocol ........................................................................................................................152 Part A. Compounding Powder Blends 152 Part B. Determining Tapped Density 153 Part C. Determining the Angle of Repose 154 Part D. Determining Powder Flowability 154 Part E. Sieve Analysis 157 Questions ........................................................................................................................................158 Lab 14: Pharmaceutical Granulations...................................................................................................159 Introduction.....................................................................................................................................159 Background......................................................................................................................................159 Experiment Protocol ........................................................................................................................161 Part A. Preparing a Standard Curve for Acetaminophen 161 Part B. Preparing the Powder Blends and Granulating162 Part C. Milling and Sizing 163 Questions ........................................................................................................................................163 Lab 15: Tableting, Capsuling, and Dissolution Testing ..........................................................................165 Introduction.....................................................................................................................................165 Background......................................................................................................................................166 Tableting Methods 168 Tablet Properties 168 Experiment Protocol ........................................................................................................................175 Lab Period 1:....................................................................................................................................176 Part A. Tableting 176 Part B. Stability (Shelf Life)177 Demonstration: Tablet Coating 178 Lab Period 2:....................................................................................................................................180 Part C. Tablet Dissolution 180 Part D. Formulating Capsules 180 Part E. Content Uniformity: Tablets and Capsules 181
  • 8. Preface 5 Lab Period 3:....................................................................................................................................181 Part F. Capsule Dissolution ...........................................................................................................181 Part G. Detection of Degradation Products / Decomposition: Thin Layer Chromatography ............182 Summary of Formulation Testing..................................................................................................184 Questions ........................................................................................................................................184 Lab 17: Synthesis and Examination of Colloids.....................................................................................186 Introduction.....................................................................................................................................186 Background......................................................................................................................................186 Experiment Protocol ........................................................................................................................187 Part A. Yttrium Citrate Colloid.......................................................................................................188 Part B. Rhenium Heptasulphide Colloid, Method 1........................................................................189 Part C. Rhenium Heptasulphide Colloid, Method 2 (Performed as a Demonstration only)..............189 Part D. Analysis of Colloids ...........................................................................................................190 Questions ........................................................................................................................................191 Lab 18: Formulating Using Molds.........................................................................................................192 Introduction.....................................................................................................................................192 Background......................................................................................................................................192 Experiment Protocol ........................................................................................................................199 Part A. Formulating 325 mg Acetaminophen Suppositories (Calibrated Batch Volume Method) ....200 Part B. Formulating 20 mg Benzocaine Lollipops (Mass of Drug Negligible) ...................................202 Part C. Formulating 20 mg Hydrocortisone Troches (Displacement Factor Method) ......................204 Part D. Double Casting Method: 70 mg Hydrocortisone/150 mg Lidocaine Lip Balm ......................206 Questions ........................................................................................................................................208 APPENDIX ............................................................................................................................................209 U.S. Standard Sieve Sizes and Lab Sieve Inventory ............................................................................209 Quadro Comil Meshes......................................................................................................................210 Methocel and Avicel Grades.............................................................................................................211 Avicel Grade Usage Chart.................................................................................................................213 Capsule Properties...........................................................................................................................214 Working Ranges of Typical Granulating Fluids...................................................................................215 Viscosities of Typical Fluids...............................................................................................................215 Powder Flowability Indices...............................................................................................................215 Average HLB Values of Some Surface Active Agents..........................................................................216 General Physical Properties of Spans and Tweens.............................................................................218 HLB Requirement for Some Common Oil Components......................................................................220 Buffer Solution Preparation: Polyprotonic Acids and Bases...............................................................221 Dissociation Constants of Acids in Aqueous Solutions at 25°C...........................................................222 Dissociation Constants of Bases in Aqueous Solutions at 25°C...........................................................222 Sorensen Phosphate Buffers ............................................................................................................223 Fundamental Lab Calculations..........................................................................................................223 Preparing a Known Molar Concentration......................................................................................223 Weight-Volume Percent (%w/v) ...................................................................................................224 Dilution Equation .........................................................................................................................224 How to Use a Syringe Filter...........................................................................................................226 Capsule Filling: Quality Control.....................................................................................................227 YOUR NOTES....................................................................................................................................229
  • 9. Preface Preface There are a lot of rules and guidelines that accompany working in a laboratory, as there is a lot of potential for you harming equipment, or far worse, the equipment harming you. Rising above the details, there are three basic tenets that will permeate through each laboratory: Be aware of the specific hazards and protect yourself accordingly; Think about the exercises as you are doing them, and learn the techniques and principles behind them; Have fun! A lab is a refreshing change from the classroom, where you get to try things out, rather than just being told how they work. Concepts in these labs are used in pharmaceutical industry, in pharmacies, and in research, particularly with respect to drug formulation, manufacture, and compounding. The protocols outlined in the labs provide suggestions on how to observe the phenomena of interest. However, there is more than one way to accomplish something, and there is certainly more than one way to measure something. In many cases, common sense will play an important part of your lab work. For instance, is it more accurate to measure out 5 mL of de-ionized water in a 10 mL graduated cylinder, or a 100 mL graduated cylinder? Subtle methods in the labs may be changed by your instructor, TA, or even by you, depending on the equipment and supplies available to you on your lab day. There is room for creativity. If you find a specific section, step, or explanation in this manual vague or difficult to follow, ask your TA or instructor for help. Please let us know, so we can improve the manual for future editions. The following icons are used in the margins throughout this manual: Useful tip on an experimental method. Read carefully. Important discussion point that is particularly useful in understanding the exercise. Important safety tip. Time-critical experimental step.
  • 10. Introduction 7 Introduction General Information Check-in for the laboratory will be on September 15, 2017, during the first laboratory session. During the check-in, you will be given your locker key and should make sure all the equipment in your locker is complete and clean. To prepare for a lab, read the part of the lab manual pertaining to that lab exercise, understand the rationale of the exercise, watch any associated videos on the laboratory website, perform any calculations that may be necessary to prepare for the lab, be aware of any potential hazards, review the questions, and go to bed early the night before. The laboratories start on time. There will be various “surprise” pre-lab quizzes during the pre-lab tutorials. They will consist of five or six short questions related to the experiments being performed during that session. Students who are late are not eligible to write the quiz. Recommended Textbooks There are no required textbooks for the PHC340 laboratory. This manual will serve as the primary reference to the laboratory. The following textbooks are recommended to clarify concepts or to serve as useful general references: Sinko, Patrick J. Martin’s Physical Pharmacy and Pharmaceutical Sciences. Lippincott Williams & Wilkins; 6 edition (Feb 21, 2010) Troy, David B. Remington – The Science and Practice of Pharmacy. Lippincott Williams & Wilkins; 21 edition (May 19, 2005) Aulton, Michael E. Aulton’s Pharmaceutics: The Design and Manufacture of Medicines. A Churchill Livingstone Title; 3 edition (Nov 1, 2007) Allen, Loyd V. Jr. et al. Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Lippincott Williams & Wilkins; 9 edition (Jan 7, 2010) Rowe, Raymond C et al. Handbook of Pharmaceutical Excipients. Pharmaceutical Press; 6th edition (2009). Available online, U of T Library permalink: http://simplelink.library.utoronto.ca/url.cfm/141954 (UtorID login required) Teaching Staff The following people will be teaching, helping, and evaluating your work in the lab: PHM340Y Laboratory Coordinator E-mail Address David Dubins d.dubins@utoronto.ca PHM340Y Teaching Assistants E-mail Address Noor Al-Saden n.alsaden@mail.utoronto.ca Giovanna Schver giovanna.medeiros@mail.utoronto.ca Intro
  • 11. Introduction Role of Teaching Assistants One Teaching Assistant (TA) will be assigned to each laboratory period. The following are the roles and duties of the TA: Before the lab: Ensure the availability of chemicals and supplies, and inform the Instructor if orders are required in advance Work with the instructor to ensure equipment in their assigned section is set up, functional and serviced Prepare buffers, reagents, and indicators in advance  Arrive before the lab in order to warm up any relevant equipment and appropriately set up the lab During the lab: Take attendance, checking student TCards Ensure laboratory safety Notify the Instructor of any injuries or hazards in the lab Handling of the disposal of hazardous chemicals Provide pre-laboratory lectures in an interactive format Supervising students regarding procedure, process, technique and safety elements of laboratory session Coordinate equitable access to equipment Collect student attendance via sign-in sheets Supervise the progress of student work - by asking appropriate questions, not only by providing answers Provide directions and clarify instructions Ensuring the cleanliness of the lab, and coordinating laboratory clean-up  Ensure equipment is clean and shut down at the end of the lab (especially spectrophotometers) After the lab: Assisting in lab check-in and check-out Evaluate submitted laboratory reports, quizzes, products and work plans Recording and entering marks on the Blackboard system Attending and supervising student tours Attendance Attendance in labs is mandatory. Attendance in each lab, and the lab tour, will be recorded. If you miss a lab or lab tour due to medical, personal, family, or other unavoidable reasons, you must provide supportive documentation (e.g. a doctor’s note) to the course Instructor for consideration of accommodation. The U of T Verification of Illness or Injury form is available online at: http://www.illnessverification.utoronto.ca/ If accommodation is granted, you will be asked to complete a make-up assignment on the same topic of the missed course material. Otherwise, if you miss a single laboratory session, you will obtain a zero for that laboratory. If you miss one laboratory session of a laboratory that spans PHC 340Y Lab Manual 2017-2018
  • 12. Introduction 9 more than one session, your final mark will be multiplied by the ratio of the number of sessions you attended, provided that you participate in writing the final report. Lateness Policy You may submit lab reports via email, or by hard copy. Labs are generally due one week from performing the lab, by the beginning of class. For late submissions, there will be an academic penalty imposed of 10% per day, in accordance with departmental policies. Submissions will not be accepted beyond 1 week from the original due date. Each lab will also be subject to a cleanliness and timeliness penalty. Cleanliness penalties will be issued if a lab area or lab scale is left untidy. Timeliness penalties will be issued if you remain inside the lab more than 10 minutes past the end of the scheduled lab. Budget your time in the lab to allow for sufficient clean-up once you are finished. Lab worksheets, when made available, will have the following box beside the final score to indicate if a penalty has been issued: Raw Score _____ -1% Cleanliness -1% Timeliness Pre-Lab Penalty (max 5%) _____ Late Penalty (10%/day) _____ Intro PHC 340Y Lab Manual 2017-2018
  • 13. 10 Introduction Laboratory and Lecture Schedule All pre-labs and labs will take place in PB860. Labs Lectures Fall Term 2017 (PB860) (PB255) Thursday Monday 9am-1pm 3pm-4pm Lecture 1 – Acid/Base Equilibria (Keith Pardee) 8am-9am, 07-Sep-17 Lab 1* – Safety Lecture, Locker Check-in, Examination of UV 07-Sep-17 Spectroscopy and Preparation of a Standard Curve Lecture 2 – Phase Partitioning (Keith Pardee) 11-Sep-17 Lab 2 – Preparation of pH Buffers 14-Sep-17 Lecture 3 – Mixing (Keith Pardee) 18-Sep-17 Lab 3 ‡ – Effect of pH on the Partition Coefficient of a Slightly Soluble 21-Sep-17 Weak Acid Lecture 4 – Polymorph and Salt (Ping Lee) 25-Sep-17 Lab 4* - Characterization of Drug Candidates (I) – Measuring Solubility 28-Sep-17 and pKa Lab 5* – Characterization of Drug Candidates (II) – Co-solvency, Salt 05-Oct-17 Selection and Polymorph Identification Lab 6* – Thermodynamics of Mixing – Enthalpy and Volume 12-Oct-17 Workshop: Writing Formal Lab Reports for PHC 340 (Heather Sanguins) Lecture 5 – Rheology (Keith Pardee) 16-Oct-17 Lab 7* – Examination of Viscosity and Suspending Agents 19-Oct-17 Lecture 6 – Chemical Kinetics & Stability (Ping Lee) 23-Oct-17 Lab 8 ‡ – Kinetics of Acetylsalicylic Acid Hydrolysis 26-Oct-17 Lecture 7 – Diffusion and Membrane Transport 1 (Ping Lee) 30-Oct-17 Lab 9 – Diffusion and Membrane Transport 1: Permeation Measurement 02-Nov-17 [Exercise 2: Quassignment for Lab 11] Lecture 9 – Colligative Properties (Keith Pardee) 13-Nov-17 (intentionally out of order) Lab 11* – Tonicity and Pharmaceutics 16-Nov-17 (intentionally out of order) Lecture 8 – Diffusion and Membrane Transport 2 (Ping Lee) 20-Nov-17 Lab 10 ‡ – Diffusion and Membrane Transport 2: Drug Release from 23-Nov-17 Ointment Bases Lecture 10 – Molecules at Interfaces (Keith Pardee) 27-Nov-17 Lab 12* – Estimation of Critical Micelle Concentration (CMC) of a 30-Nov-17 Surfactant in Water Lecture 11 – Particle Size and Powder Flow (Ping Lee) 04-Dec-17 Labs Lectures Winter Term 2018 Thursday Wednesday 1pm-5pm 11a-12p Lab 13* – Optimization of Powder Flow and Particle Size Determination 04-Jan-18 Lecture 12 – Pharmaceutical Granulation (Ping Lee) 10-Jan-18 Lab 14 – Pharmaceutical Granulations, Part 1 11-Jan-18 PHC340 Midterm 17-Jan-18 Lab 14 ‡ – Pharmaceutical Granulations, Part 2 18-Jan-18 Lecture 13 – Tabeting and Dissolution Testing (Ping Lee) 24-Jan-18 Lab 15 – Tableting and Dissolution Testing, Part 1 25-Jan-18 Lecture 14 – Measurement, Part 1 (Keith Pardee) 31-Jan-18 Lab 15 – Tableting and Dissolution Testing, Part 2 01-Feb-18 PHC 340Y Lab Manual 2017-2018
  • 14. Introduction 11 Lecture 15 – Measurement, Part 2 (Keith Pardee) 07-Feb-18 Lab 15 ‡ – Tableting and Dissolution Testing, Part 3 08-Feb-18 Lecture 16 – Synthetic Biology and Human Health, Part 1 (Keith Pardee) 14-Feb-18 Lab 16 – Mystery Laboratory 15-Feb-18 Lecture 17 – Synthetic Biology and Human Health, Part 2 (Keith Pardee) 28-Feb-18 Reading Week Feb 20-23 Lab 17 ‡ – Synthesis and Examination of Colloids 01-Mar-18 Lecture 18 – Mold Calculations (David Dubins) 07-Mar-18 Lab 18* – Formulating Using Molds 08-Mar-18 Lab 19 – Advanced Formulations Project, Part 1 15-Mar-18 Lecture 19 – Ethics & Academic Integrity (Alison Thompson) 21-Mar-18 Industrial Tour (to be confirmed) 22-Mar-18 Lecture 20– Ethics & Academic Integrity (Alison Thompson) 28-Mar-18 Lab 19 ‡ – Advanced Formulations Project, Part 2. Lab Check-Out. 29-Mar-18 PHC340 Final Exam During final exam period ‡ Lab Report to be completed for evaluation * Lab Worksheet to be completed for evaluation Intro PHC 340Y Lab Manual 2017-2018
  • 15. Introduction Locker Check-In / Check-Out Check-In: September 15 th , 2017 Your Locker #: ____________ You will be assigned your own locker. The contents of your locker have been arranged by students of previous years. It is your privilege to use the locker and your responsibility to maintain the locker. Today, make sure you have all the glassware according to the “Content of your Locker” list. You may also want to clean the glassware. Replacement of damaged equipment can be obtained from the back shelves or from your Teaching Assistants (TAs). Take time to review the laboratory safety section of this manual and locate the following safety equipment in the laboratory. Indicate the location in the space provided below: Safety Equipment Location Fire Extinguishers Fire Alarm Eye Wash Fountains Safety Shower First Aid Box When your group has completed the locker check-in, notify your teaching assistant and he/she will ask you a few safety questions. Locker key issued _________________________ (student signature) Locker Check-Out: Marth 30 th , 2018 A fee of $10.00 will be charged for locker key replacement. You are encouraged to attach the key to a secure key ring or case. Lab Check-Out Procedure Clean your lab bench and any dirty glassware; Throw out any remaining formulations or garbage; Empty your locker water bottle; Verify that your locker contents are complete; Return any extra glassware to the laboratory back shelves; Get a TA or Instructor to verify the above, and sign your check-out list (next page); Be assigned a special area in the lab to clean; Lock your locker, and return your lab key when the above is completed. Keep this and the following page in your laboratory manual. PHC 340Y Lab Manual 2017-2018
  • 16. Introduction 13 Volumetric Flask Graduated Cylinder Erlenmeyer Flask Bulb Grad. Pipette Pipette Locker Contents 2017-2018 Student Name Locker # Date Name of Apparatus/Item Qty In Out 50 mL Volumetric Flask 2 100 mL Volumetric Flask 2 200 mL or 250 mL Volumetric Flask 3 10 mL Graduated Cylinder 1 25 mL Graduated Cylinder 1 100 mL Graduated Cylinder 2 50 mL Erlenmeyer Flask 2 125 mL Erlenmeyer Flask 2 250 mL Erlenmeyer Flask 2 500 mL Erlenmeyer Flask 2 5 cm Glass Funnel 1 7.5 cm Glass Funnel 1 10 cm Glass Funnel 1 Test Tube Rack 1 1 mL Bulb Pipette 1 5 mL Bulb Pipette 1 10 mL Bulb Pipette 1 20 mL or 25 mL Bulb Pipette 1 1 mL Graduated Pipette 1 10 mL Graduated Pipette 1 Thermometer (°C) 1 Watch Glass small 1 Watch Glass large 1 8” Glass Stirring Rod 1 50 mL Beaker 2 150 mL Beaker 2 250 mL Beaker 2 400 mL Beaker 2 600 mL Beaker 2 Glass Slab 1 3” Ceramic Evaporating Dish 1 6” Ceramic Evaporating Dish 1 Plastic Wash Bottle 1 Ceramic Mortar & Pestle Set (Glass set additional in some cases) 1 Funnel Clamp and Holders 1 Sharpie Lab Marker 1 TA Signature - Lab Check-In TA Signature - Lab Check-Out Watch Glass Beaker Evaporating Dish Mortar & Pestle Funnel Clamp Intro PHC 340Y Lab Manual 2017-2018
  • 17. Introduction Recording Data, Analysis, and Results In this laboratory, we are attempting to introduce laboratory practices that are employed in research and development labs in the pharmaceutical industry. Practices such as daily initialing of laboratory results and the use of bound books are used to increase security and in some cases to document intellectual property. Hard cover bound laboratory notebooks will be used to record your data. At the beginning of selected laboratories, the TA will give you a laboratory worksheet which you will use to record your data, present your results and interpretation for grading. The worksheets will also contain specific questions to answer about the labs. Other times you will record data in your lab book. During the laboratory, you may work in groups of two, and sometimes in larger groups, for the collection of data. Any data that is collected during the lab period must be recorded in each member of the group’s data booklet. The analysis of the data, presentation and calculations are to be done individually, and recorded in the appropriate section in the data booklet. All lab books are to be initialed by both the student and an instructor at the end of each laboratory session. It is your responsibility to make sure that your book is initialed. Books will be initialed after satisfactory laboratory clean-up has been completed. A lab has a lot of potential for entropy (read: mess). Please keep your lab area tidy. The lab reports will be graded according to the Report Format which is outlined in this introduction. In some cases, grades may also be assigned to the quality of the product, and product label that you made during the lab. Lab reports will be not be returned to you until all students in the class have completed the same exercise. In addition to the lab reports and quizzes, there will be a written exam in December as part of the PHM340 Mid-term exam. Questions related to the work in the Winter Term will be included in the April final exam. Try to work cooperatively with others in your group. If there are unresolved conflicts, approach your TA or the lab coordinator to seek a solution. Plagiarism and Falsification At some point in your laboratory, you might look at your results and think, “OH NO! This can’t be right!” You will be nervous. You will wonder what happened. What went wrong? Worse off, you might be tempted to misreport the results for that ONE point that should have fallen on the line. However, you are reminded to always report what you observed, rather than what you would have liked to observe. Provided you made the correct calculations and performed your exercises meticulously and carefully, you will not lose marks for less than perfect looking observations. Real data rarely look perfect. Things don’t always work. If they did, there would be no need for formulation scientists. If you encounter suspicious looking data, identify your concerns in your analysis, and explain where you think things may have gone wrong (sources of error). If your entire data set is concerning you, seek the assistance of your T.A. or instructor. There could be a malfunction in the equipment, a problem with the method, or a systematic error in your calculations. If you have time, you can repeat the outlying measurements to refute or confirm their validity. PHC 340Y Lab Manual 2017-2018
  • 18. Introduction 15 DO NOT PLAGIARISE OR FALSIFY YOUR DATA. Doing so is an offence under the University of Toronto Governing Council’s Code of Behaviour on Academic Matters. Clean-up Check-List Your experiment is done. Are you all ready to go? Here are some helpful tips on leaving the lab clean for the next group of students: I cleaned all lab equipment (especially balances!), so other students can use them. I rinsed out my pipettes and burettes with water, so crystallization won’t gum up the tips. I washed and shook out all my glassware, and put it back in my locker so it’s clean for my next lab. I wiped my work area, lab bench, and bench top (including the balances I used). I properly labeled and handed in my preparation (if there is one to hand in). I properly disposed of all chemicals: solid and semi-solid inert waste in the garbage,  liquid inert waste down the sinks hazardous chemicals in appropriately labeled waste bottles in the fume hoods I double-checked the fume hood. It’s clean, and I didn’t leave anything in it. Assignment of Grades Laboratory Reports*, Exercises 65 % Quizzes and Problem Sets (weighted equally) 5 % Mid-term and Final exam (weighted equally) 30 % Total 100 % *Lab reports are weighted in proportion to the number of lab periods. Guidelines for Writing Pre-Labs, Worksheets and Individual Laboratory Reports Pre-Labs Prior to the lab, regardless of whether a worksheet or formal lab report is assigned, you will be expected to prepare a pre-lab in your lab notebook, which should include the following sections: Purpose: Why are you doing this lab? What scientific questions will be addressed? Procedures: In flow-chart form, organize your activities in the lab. This will help you prepare for complicated procedures, and allow you to be more efficient in the lab. Pre-labs will be checked at the beginning of the lab, and will be worth 5% of the lab report or worksheet mark. Preparing a proper pre-lab will help you succeed in surprise quizzes. Individual Lab Worksheets For selected labs, worksheets will be handed out in the beginning and will be made available for download from Blackboard. For these labs, filling out the worksheets and answering the worksheet questions is all that is required for the lab. For these labs, the mark breakdown will be indicated on the worksheets. Intro PHC 340Y Lab Manual 2017-2018
  • 19. Introduction NOTE: Where applicable, your submitted, properly labeled product will constitute a proportion of the “Presentation, neatness” component. Other laboratories will involve creating a formal lab report. The following is a guide on what is expected for these reports. As each lab is individual, the marking scheme may vary slightly for each lab. Rationale of Laboratory Reports The purpose of writing a scientific report is to communicate your findings with the outside world. Enough detail should be conveyed so that someone who did not do the experiment could repeat it, and be able to fairly compare their results with yours. Writing laboratory reports (and technical writing in general) is an extremely useful and valuable skill to develop. Avoid providing one word answers and bullet points. Use sentence form, and summarize where appropriate. The ability to condense the purpose, observations, and results into an abstract will help the reader connect with the material, and will put your results in perspective for the reader. This process will help prepare you for writing scientific publications. Be consistent with grammar. For events that happened in the lab, use the past tense for reports, and the passive voice. e.g.: “1 mg of the free acid of sulfathiazole was incubated at 25 C in 10 mL of phosphate buffer for 1 hour, with agitation every 15 minutes.” For scientific principles, use the present tense. e.g.: “Ethanol is a co-solvent, and disrupts the hydrogen bonding between water molecules and the surface of the drug molecule.” Details on Writing a Formal Laboratory Report Pre-Lab (5%) Your pre-lab mark will be evaluated at the beginning of each laboratory. Title Page (1%) Please include lab number and title, student name(s), date submitted, and course code. Abstract (10%) No more than 200 words, an abstract is a mini-version of the entire lab report. It provides a brief introduction, purpose, a summary of results (not the raw data itself but parameters estimated), conclusions, and the relevance of the conclusions to the field of study. It is usually the last section that you will write, although it comes first in the report. Introduction (5%) This section should be 1-2 paragraphs long, and include the purpose of the experiment and a brief overview. What is the main purpose of the lab? Which scientific principles are being investigated? What is the value of the results to the field of study? A good introduction will spark the interest of the reader and explain the purpose of the work.  Experimental (10%) This section should be no more than 2 pages long, but depending on the PHC 340Y Lab Manual 2017-2018
  • 20. Introduction 17 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).  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.”  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 r 2 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 Intro PHC 340Y Lab Manual 2017-2018
  • 21. Introduction Deviations from the lab protocol Errors in the lab protocol Limitations of the method used to evaluate the phenomena of study Equipment malfunction or improper use of the equipment If the error was a result of experimental design, suggest how the design could be improved. If relevant, put your results in the context of literature values. Were they in agreement? e.g.: The pKa of sulfathiazole was estimated to be 5.98. This is not in good agreement with a published value of 7.14 (reference 1). You may also discuss other related theories.  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. 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.: Fioritto AF et al., Int J Pharmaceutics (2007);330:105-113. 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 2017-2018
  • 22. Introduction 19 forgetting which beaker you poured it in. It will save you aggravation to get in the habit early of clearly labeling your preparations as you go along. Chemical Disposal There are large green buckets available for broken glassware in the lab. Please use them instead of the garbage, to respect the safety of the cleaning staff.  There will be designated waste jars for hazardous waste and organic solvents in the fume hoods for each lab. When appropriate, there will also be a designated container for sharps (e.g. needles).  Solid and semi-solid chemically inert waste (e.g. petrolatum) will gum up the drains, and are properly disposed of in the garbage. If you are unsure how to properly dispose something, ask your TA or instructor. ACIDS CORRODE PIPES, AND SHOULD BE DISPOSED OF IN WASTE BOTTLES ONLY. Dress Code For your protection, you are required to wear the following protective gear, at all times during the lab: A lab coat Safety Goggles Closed-Toed Shoes (no sandals or open-toed shoes) Clothing that covers your legs The following special protective equipment is available for specific tasks, or on your request: Latex (and non-allergenic neoprene) gloves N95 Masks Protective hair covers Dress Code Rationale If you have ever taken a laboratory course, you have likely already heard much of the following safety advice at some point. Common sense plays a large part in lab safety. However, it is useful to outline a few principles that pertain to the labs in this manual, so they are fresh in your mind. Laboratory coats offer first line protection to your clothes and body against chemical burns. They work best when they are done up – an open lab coat will not properly protect you from a spill. Closed-toed shoes protect your feet from chemical spills.  Safety glasses will help to shield your eyes from any chemical splashes, including boiling solutions.  Latex (and nitrile) gloves are available for use in the laboratory. In particular, hydrochloric acid (HCl), potassium hydroxide (KOH), and sodium hydroxide (NaOH) are extremely corrosive. Gloves should be worn if you are going to be handling these solutions. Gloves also offer protection if you have a known specific allergy or sensitivity to a certain chemical. Working with Hazardous Chemicals  When in doubt, treat all chemicals as hazardous, until you are familiarized with their Intro PHC 340Y Lab Manual 2017-2018
  • 23. Introduction properties. Consult the Material Safety Data Sheets (MSDS) or your TA for relevant information. Whenever possible, or necessary, handling chemicals in a fume hood will protect you as well as those around you from toxic and flammable fumes. Handle all volatile and flammable solvents in a fume hood. Do not put a sealed container over any heat source, as it may explode. If you are not sure how to use something, ask your TA.  Notify your TA if there is any broken glassware, so they can safely clean and dispose of any chemical or sharps hazards.  Notify your TA immediately if there is a mercury spill. They will have access to a mercury spill kit.  Be cautious when testing for odours. Never inhale a chemical directly. Fan the vapours towards your nose. Many vapours can cause irreparable damage. Never ingest any excipients or products in the teaching laboratory. Other safety references: Merck Index Material Safety Data Sheets (MSDS), a part of the WHMIS (Workplace Hazardous Material Information System) right-to-know system Fisher Scientific Catalog o Sigma-Aldrich MSDS Follow these guidelines to decrease the risks of working with chemicals: Work with a minimum amount of chemicals necessary. Read the warning labels and/or consult the MSDS before using a chemical.  When storing, using or disposing of chemicals, avoid accidental mixing of incompatible chemicals such as acids and bases, flammables and toxics, flammables and oxidizers, oxidizers and reducers.  Highly toxic and flammable chemicals must be stored in ventilated areas in unbreakable, chemically resistant containers. Emergency Response The University Emergency phone number is 416-978-2222. In Case of Personal Injury Inform the Teaching Assistant, or the Laboratory Coordinator of any injury acquired during a lab, no matter how slight it may appear.  An open or even partially healed cut is dangerous, since it allows easier penetration of chemicals. Cover any exposed areas with a bandage when working in the laboratory. Protective latex gloves are available from your TA.  In case of chemical eye injury, hold the eye open in the eye-wash, even if painful, and wash the eye for 15-20 minutes.  In case of chemical body burns, use cold water to wash chemicals from the skin immediately, and thoroughly. Hot water may increase the absorbency of the chemical. PHC 340Y Lab Manual 2017-2018
  • 24. Introduction 21 In Case of Spills Chemicals spilled in the laboratory must be cleaned up immediately to reduce and eliminate hazards. The Chemical Spill Cart is located in the laboratory outside the entrance of Room 865. In the event of a localized, minor spill, use the following procedure: Responding to a Minor Spill Report all spills to the TA. Notify other students who are working in the area. Confine the spill to a small area. Do not allow the spill to spread. If the material involved is flammable, turn off any ignition sources/electrical equipment present. Ventilation should be established to dispel vapour, if necessary, and if safe to do so.  Absorb and neutralize the spilled liquid chemical. For example, strong acids should first be neutralized with sodium bicarbonate, then washed with water. It is always advisable to add acid into water when mixing, since water has a much larger heat capacity and will therefore be able to absorb any resulting heat much better. You can always remember the catch phrase: “Do as you aughta, add acid to watah”.  The TA or Instructor should handle a mercury spill. Spilled mercury is collected with a mercury collector. Sprinkle the affected area with sulfur powder. The sulfur-mercury powder is then swept up and discarded in the appropriate labeled container. When cleaning up a spill, wear the proper protective equipment, such as gloves and goggles.  After the spilled chemicals have been removed, wash the area with warm, soapy water to remove any residue left behind. In the event of a major spill that exceeds the clean-up capabilities of the laboratory, the following procedure is to be followed: Responding to a Major Spill Notify everyone to evacuate the area immediately.  Contact the University of Toronto Emergency Number 416-978-2222 and state the location of spill, extent of the spill, and the chemical involved. Or, call 911. Wait in a safe area until the response team arrives. In Case of Fire If the fire is contained in beakers or flasks, smother the fire simply by covering the vessels so that no oxygen can enter. If electrical equipment is on fire, unplug it quickly or cut the power if possible.  If your clothing is on fire, do not run. Stop, drop, and roll. If the clothing of someone next to you is on fire, help him to the floor and use your lab coat or fire blanket, or whatever is available to smother the fire. Once the fire is extinguished, help the person away from the general fire area.  If the fire is small and contained, a qualified person should attempt to use a fire extinguisher to eliminate the fire. Many fire extinguishers handle multiple types of fires. There are 4 major classes: Intro PHC 340Y Lab Manual 2017-2018
  • 25. 22 Introduction Fire Extinguisher Appropriate for: Examples: Class Class A Ordinary combustibles (paper, wood, cardboard) Class B Flammable/Combustible (gasoline, organic Liquids and Gasses solvents) Class C Electrical Equipment Computers, monitors, melting point apparatus Class D Combustible metals Magnesium, titanium, potassium, sodium Class K Grease fires Cooking Oils, fats The fire extinguishers in Room 860 are rated for Classes A, B, and C. They are located by each exit, and outline the following procedure: (PASS) Pull the pin out  Aim at the base of the fire Squeeze the handle Sweep the nozzle back and forth  If the fire is too large to be contained with a fire extinguisher, pull the fire alarm, and evacuate the building. Once out of harm’s way, call the University of Toronto Emergency 978-2222 or call 911. Specify the site and extent of the fire.  Wait outside the building, away from the main entrance so that you do not block the entrance when the fire personnel arrive. If the Fire Alarm Sounds Evacuate the building quickly, using the stairwells. The elevators will automatically go out of service. Do not try to use them.  Wait in the designated emergency area (the area between the Medical Sciences Building and the Leslie L. Dan Pharmacy Building), Keep clear of the building. Do not re-enter the building until authorized by a Fire Officer. PHC 340Y Lab Manual 2017-2018
  • 26. Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve 23 Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve Read the introduction and lab protocol completely 1 Watch the following related lab videos on the laboratory website: La b Preparing for the Lab  UV/Vis Spectrophotometry - Determining Absorbance (http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv) Calculate the volume of stock required for each standard solution in the calibration curve. Group Allocation You will be working in groups of 2 students Part A: Prepare a calibration curve for hydrochlorothiazide What You’ll Be Doing Part B: Plot your calibration curve Demonstration: Using a spectrophotometer Spreadsheets You Will Need http://phm.utoronto.ca/~ddubins/DL/calibration.xls What You’re Handing In Lab 1 Worksheet (due at the beginning of the next lab) Introduction One of the fundamental tools to be used in any pharmaceutics laboratory is the analysis of the drug that is the subject of the experiment. In this introductory session a standard solution will be prepared and some of the principles related to the Beer-Lambert Law will be examined. The standard curve will be able to be used in a later session. Background Lambert’s Law Lambert showed that each unit length of material through which light passes absorbs the same fraction of the incident or entering light and compares the relation between the incident light (Io) and the transmitted light (IT) for various thicknesses t. Io loge IT  t Where: Ȁ⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀ᨀĀĀĀ⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀Ā⸀ is the intensity of light is the thickness of the substance  the absorption coefficient Conversion to log 10 results in the equation: log10 I 0   t Kt 2.3026IT Where K is the extinction coefficient generally defined as the reciprocal of the thickness (t in cm) required in order to reduce the intensity of the incident light to its original intensity. PHC 340Y Lab Manual 2017-2018
  • 27. Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve Beer’s Law Despite what it sounds like, Beer’s Law does not describe the relationship between number of beers consumed and physical attraction. Beer examined the relationship between absorption and the concentration of coloured solutions. The equation is similar: log 10 I0  k 1 c IT If this is performed in a cell with a uniform thickness then a measure of the length l may be added: (2) log I 0  k 1 cl or log I 0  A 10 I T 10 I T The value of k 1 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:  = A (1 %w/v, 1 cm) × l is equal to the slope of the calibration curve (absorbance vs. concentration):  A = × l × c Where: = extinction coefficient ((concentration units) -1 cm -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. Extinction 0 .5 0 .4 0 .3 0 .2 0 .1 0 0 1 2 3 4 5 6 7 8 9 ml Standard Fe (0. 02 mg Fe/ ml) Figure 1. A Standard Curve for an Iron Solution PHC 340Y Lab Manual 2017-2018
  • 28. Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve 25 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 8 th 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. 1 2 1 0 ) 8 ,1cm 6 E(1% 4 2 0 1 90 2 00 2 10 2 20 2 30 2 40 2 50 2 60 2 70 2 80 2 90 Wa v elength Figure 2. Graph Showing the Change in E (1%, 1cm) at Several Wavelengths Areas of the curve where the change [E (1%, 1cm)] is large should never be used in drug analysis. On the curve in Figure 2, wavelengths of 205, 230, and 265 nm are sub-optimal. Experiment Protocol Chemicals Supplies Special Equipment Hydrochlorothiazide (5 mg/mL) in Plastic transfer pipettes Helios UV/Vis Sodium Hydroxide Solution (0.1 N) UV Cuvettes (Plastic) Spectrophotometer Volumetric Sodium Hydroxide (40.0 g/mol) Parafilm flasks The following solutions are prepared or provided by the TA: 250 mL of Hydrochlorothiazide stock solution (5 mg/mL in 0.1 N NaOH) The Helios spectrophotometers will be turned on prior to the laboratory. Each person will do their own measurements. Part A. Preparing a Calibration Curve Prepare 1 L of 0.1 N Sodium Hydroxide solution. Note: do not leave sodium hydroxide pellets exposed to air. Close the cap of the bottle when not weighing pellets. Wear gloves when weighing and handling sodium hydroxide. Lab1 PHC 340Y Lab Manual 2017-2018
  • 29. Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve Prepare dilutions of the hydrochlorothiazide stock solution in 0.1 N NaOH as follows, in triplicate: 1:10, 1:50, 1:100, 1:200, 1:250 To clarify, “in triplicate” means that you create each solution three times, rather than measure the absorbance of the same solution three times, to get an estimate of the error associated with creating the standard solutions. Measuring the standards in triplicate will allow you to report the average, standard deviation, and %RSD at each standard concentration. An efficient way to accomplish this is to have three different people run the same curve in parallel. The same spectrophotometer must be used. 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. 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. Repeat the above steps with each of the five dilutions of the sample. Measure the absorbance of the stock solution (remember 3 determinations). Calculate the average and standard deviation for each concentration. SPECTROSCOPY NOTES Fill the cuvette to the etched line (approx ¾ full) Make sure the cuvette is facing the correct way (the light path should go through the clear windows through the longest path length, not the ridged sides) To avoid fingerprints, only handle the cuvettes by the ridged sides, not the clear windows. Fill the cuvette slowly, and gently tap to release bubbles clinging to the sides of the cuvette Gently wipe the clear windows with a Kimwipe prior to measuring Make sure the sample door is closed before measuring absorbance Make sure you use the same UV spectrophotometer for calibration and sample measurements. *NOTE: Plastic UV cuvettes are tapered towards the bottom, to accommodate a smaller sample volume. The fill line is just above the clear part of the cuvette window. The “V” shaped arrow on the Plastic UV cuvette indicates the side of the cuvette that the UV beam will travel through the entire 1 cm path length (not widthwise, which is only 0.5 cm): Fill line (fill to at least here) Spectrophotometer beam travels this way PHC 340Y Lab Manual 2017-2018
  • 30. Lab 1: Examination of UV Spectroscopy and Preparation of a Standard Curve 27 Lab1 Beam direction Beam direction Helios Spectrophotometer (PB 860) Varian Spectrophotometer (PB 819) Part B. Plotting Your Calibration Curve You will be preparing two calibration curves using calibration.xls (available in the Downloads section of the laboratory website): One curve with all of your collected data;  One curve with the linear portion of the curve (excluding the higher concentrations). Questions Describe the shape of the curve that results from your data. Does the best-fit curve go through zero? Is this necessary for Beer’s law to be valid? Which of the linear fits in the two curves in Part B would you use to convert OD to concentration? Why? What is the accuracy of your measurements? What is the precision? Hydrochlorothiazide is a very weak acid. Why is 0.1 N NaOH used to help dissolve hydrochlorothiazide? PHC 340Y Lab Manual 2017-2018
  • 31. Lab 2: Preparation of pH Buffers Lab 2: Preparation of pH Buffers Read the introduction and lab protocol completely Watch the following related lab videos on the laboratory website:  Measuring pH Preparing for the Lab (http://phm.utoronto.ca/~ddubins/DL/pH.wmv)  UV/Vis Spectrophotometry - Determining Absorbance (http://phm.utoronto.ca/~ddubins/DL/Spectrophotometry.wmv) Calculate the volume of stock required for each standard solution in the calibration curve. Group Allocation You will be working in groups of 2 students Part A: Preparing Phosphate (Sorensen’s) Buffer @ pH 7.4 Part B: Preparing McIlvane’s Buffer at 2 assigned pH values What You’ll Be Doing Demonstration: Using a pH meter Your buffers will be stored in the cold room on the 9 th floor, for use in Lab 3. Spreadsheets You Will Need Not applicable. What You’re Handing In Not Applicable. Retain and store buffers prepared in this lab for use in Lab 3 (McIlvaine’s) and Lab 4 (Sorensen’s). Introduction Buffers are fundamental to wet chemistry, although the basic idea of buffers extends far beyond solutions. The primary idea behind a buffer is to dampen or minimize the effects of changes to or within the system so that the impact on the system is not as bad. There are buffers in electrical systems, irrigation systems, computers, and mechanics. Shock absorbers, for instance, prevent you from feeling bumps in the road when you are driving. Similarly, in wet chemistry, buffers can help reduce or minimize external stresses (changes in temperature or pressure), or chemical reactions from changing the overall pH of a solution. It is critical to select a buffer that is well suited to the system you are studying. Will the temperature of the system be changing? Will the pressure be changing? How does a change in either affect the pKa of the buffer? What is the pH value you would like to maintain? A buffer is most effective when the pH of the solution is in the vicinity of its pKa value (±1 pH unit). In this laboratory, you will learn how to calibrate and use a pH meter. You will be preparing two buffer systems: Sorensen’s Buffer and McIlvaine’s Buffer. You will be using these buffers in the following two labs. References Glasstone, Samuel. An Introduction to Electrochemistry. New York, NY USA (1942). p372. http://www.chembuddy.com/?left=pH-calculation&right=pH-buffer-capacity http://biotech.about.com/od/buffersandmedia/ht/phosphatebuffer.htm http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer http://www.sigmaaldrich.com/life-science/core-bioreagents/biological- buffers/learning-center/buffer-reference-center.html 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 PHC 340Y Lab Manual 2017-2018
  • 32. Lab 2: Preparation of pH Buffers 29 theoretical (or even published) values of how much of each buffer component to add are just that – theoretical. The actual recipe required could be different, depending on the purity of components, errors in weighing and measurement, and even the quality of water used. There is a lot of theory involved, but at the end of the day, the calculated theoretical values serve only as a guide. Making a buffer is relatively quick and straightforward once you’ve tried it a few times. In order to understand buffers and how they work, a “crash course” in pH and pKa is offered in this section. Definition of pH and pKa An acid will dissociate in water to a conjugate base and proton. Consequently, acids are typically thought of as proton donors: Ka (1) [HA]  [H + ] + [A - ] Acid Proton Conjugate Base Ka is the equilibrium constant that determines the extent that the acid will dissociate in water: (2) Ka  [H ][A ] [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: (5) 10-pKa 10 pH [A  ] [HA] (6) [HA] 10pKa [A  ] 10 pH [HA] 10 pKapH [A  ] According to the Henderson-Hasselbalch buffer relationship, pH, pKa, and the buffer component concentrations for a weak acid are related as follows: [base] [acid]  10 p K a p H Here, the ‘acid’ is the proton donor (HA), and the ‘base’ is the conjugate base (A - ) in Equation (1). This is a very convenient form of the equation, because it allows us to see the following: Key Concepts if the pKa is greater than the pH, there will be more of the acid form in the solution. If the pKa is equal to the pH, there will be an equal amount of acid and base in the Lab2 PHC 340Y Lab Manual 2017-2018
  • 33. Lab 2: Preparation of pH Buffers solution. If the pKa is less than the pH, there will be more of the base form in the solution. A strong acid is defined as one that will dissociate completely. Consequently, the lower the pKa of the acid, the stronger the acid. The same scheme can be re-written to describe the reaction of a base with water, to form its conjugate acid: 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 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; pKb = -log[Kb]; pKa = 14 – pKb Try it out for yourself. This saves us having to remember two sets of Hendersson-Hasselbalch equations. If the pKa of a base is greater than the pH, there will be more conjugate acid. It need only be remembered that [B-H + ] is the concentration of conjugate acid and [B] is the concentration of base. The higher the pKa of a base, the stronger the base. By using the appropriate experimental conditions, the pKa of a drug may be measured directly with a pH meter. Pairing a Weak Acid with its Salt: Sorensen’s Buffer Many buffer systems are weak acids paired with their respective salts. One example is citric acid paired with sodium citrate. The reason that two solutions are made at the beginning – a solution of the acid of the buffering agent, and another solution of its salt – is so that we may titrate one with the other to attain the exact pH we are looking for. It is assumed that when the salt of a buffer is dissolved in water, it will dissociate completely and go into solution in the ionic form. It is important to note however that a buffering system can be as simple as a weak acid added to de-ionized water, with the pH of solution adjusted close to the pKa using either NaOH or HCl. Buffering systems are not limited to weak acids, they can also be weak bases (e.g. ammonia + ammonium chloride). Weak bases may be used for solutions where the pH desired is above 7. We will focus our discussion on weak acid buffers paired with salts of their conjugate OH bases. Since it was already stated a buffer is most effective within 1 O P OH pKa2 = 6.86 pH unit of its pKa, you would think that a given buffer would only be useful in the vicinity of one pH value. However, many pKa1 = 2.15OHbuffers have more than one acidic group attached, which vary in affinity to their respective protons. Sorensen’s Buffer Phosphoric Acid (phosphate buffer) has three acidic groups, each with different pKa values (see right panel). PHC 340Y Lab Manual 2017-2018 pKa3 = 12.32
  • 34. Lab 2: Preparation of pH Buffers 31 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 O - + H + O P OH P 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 OH Na + - + + H2OO P O Na O P OH + HO - O - Na + O - Na + Sodium Phosphate Dibasic Sodium Phosphate Monobasic (salt of conjugate base) (weak acid) Lab2 PHC 340Y Lab Manual 2017-2018
  • 35. Lab 2: Preparation of pH Buffers However, to a first approximation, we treat the system as if the salt completely dissociates and stays in the ionic form. A buffer is usually prepared in concentrations ranging from 0.1 – 10 M. The way that a buffer works, is that provided there are both forms (acid and conjugate base) of the buffer present (i.e. the pH is around the pKa), then if another acid dissociates to add a proton to solution, the proton will be absorbed by the buffer’s conjugate base instead of lowering the pH. If a base is added to the solution, it will result in a hydroxide ion, which will in turn react with the buffer’s weak acid instead of raising the pH. In this way, the balance of hydrogen ions is protected, and changes in pH are much smaller than they would have been in the absence of buffer. Calculating the Amount of Acid and Salt of Conjugate Base Required There are essentially four decisions to make when selecting a buffer: What is the desired pH of the solution? What type of buffer system will you choose? Which pKa will you be making use of? 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]  10p Ka p H [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: [base] [acid]  [HA] [A]  10 6.867.4  10 0.54  0.2884 Simplifying (15): (16) [HA] = 0.2884 [A - ] Since we want the buffer to be 0.1 M, we also have a mass balance to think about: (17) [HA] + [A - ] = 0.1 M Substituting equation (16) into (17), we can solve for [A-], the concentration of conjugate base we will be adding in the salt form: 0.2884[A - ] + [A - ] = 0.1 M 1.2884[A - ] = 0.1 M [A - ] = 0.0776 M PHC 340Y Lab Manual 2017-2018
  • 36. Lab 2: Preparation of pH Buffers 33 Substituting equation (20) into (17), we can solve for [HA], the concentration of acid that will be added in the un-ionized acid form: [HA] + 0.0776 = 0.1 M [HA] = 0.0224 M So now we have to do the important part: we have to translate a theoretical calculation into reality. We need to create two solutions and mix them together, such that the final concentration of sodium phosphate monobasic is 0.0224 M, and the concentration of dibasic is 0.0776 M. If we would like to start with two stock concentrations, 250 mL each, at a concentration of 0.2 M: Sodium Phosphate Monobasic (NaH2PO4*H20) m  C  MW  V m  0.2 mol L 137.99 mol g  0.250 L m  6.900 g Sodium Phosphate Dibasic (Na2HPO4*7H20) m  C  MW  V mol g m  0.2 L 268.07mol0.250 L Lab2 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 C1V1  C 2 V2 C1V1  C 2 V2 V C 2 V2 V C 2 V2 1 C1 1 C1 0.0224 mol  250 mL 0.0776 mol  250 mL L L V1  V1  0.2mol 0.2mol 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 PHC 340Y Lab Manual 2017-2018
  • 37. Lab 2: Preparation of pH Buffers burette. The pH electrode is inserted into the beaker, and solution is titrated until the desired pH is attained. Once it is attained, for this particular protocol, an equal volume of water (volume in beaker + volume of solution titrated) is added to bring the concentration of buffer from 0.2 M to 0.1 M. Question: Why can’t you just prepare 0.1 M of each solution, and add them together? Wouldn’t that give you a 0.1 M Sorensen’s buffer? Answer: You can. The reason we start with 0.2 M stock solutions in this case is out of convenience, because the burette can only hold 50.0 mL. A 250 mL solution of 0.1 M sodium phosphate monobasic would require 56 mL for the final mix, which wouldn’t all fit in one burette. What makes these calculations lengthy is compensated by the simplicity of published tables in the literature of volumes of each solution to attain the desired pH. In practice, you need not perform the calculations routinely, you can use the tables as a starting point and titrate to your desired pH. However, studying the theory behind these calculations will make all the difference in your understanding of how a buffer works. Buffer Capacity One question that might arise is how well will the buffer protect the pH from changing? This will depend on the pKa of the buffer, pH of the solution (the closer to the pKa the pH is, the more effective the buffer will be), and on the concentration of buffering agent used. One definition of buffer capacity is the amount of (external) acid or base required to change the pH of 1 L of the solution by 1 pH unit. The higher the buffer capacity, the larger this number will be. A formula to calculate buffer capacity is presented here: dn K w  C buf K [H  ]  (23) β   2.303  [H  ]  a    2 dpH  [H ] (Ka [H ])    Where, : buffer capacity (mol/(L*pH unit)). number of moles of acid or base added (assumed to be monoprotic) Kw: The equilibrium constant of water (1.00×10 -14 at 25 °C) Cbuff: the concentration of buffering agent The summation sign in Equation (23) means that you can enter more than one Ka of your buffering agent if it has multiple acidic groups, or the equation can be used for buffers with multiple components. It is beyond the scope of this discussion to derive Equation (22). However, we may use it to calculate the buffer capacity of our Sorensen’s buffer. At a pH 7.4: PHC 340Y Lab Manual 2017-2018
  • 38. Lab 2: Preparation of pH Buffers 35 [H  ]  10 pH 107.4  3.9811 10 8 K a 10 K a 106.86  1.3804 10 7 Kw   C buf K a [H  ] β  2.303  [H ]     2 [H ] (K a  [H ])     110 -14 0.1 M 1.3804 10 7  3.9811 10 8   β  2.303  3.9811 10 8   8 7 8 2 3.9811 10 (1.3804 10  3.9811 10 )     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 BufferCapacity (mmol/(L* pH) 100 80 60 40 20 0 2 3 4 5 6 7 8 pH We can see a peak (left panel, above) at the second pKa of phosphoric acid (6.88), and the buffer capacity decreases as the pH falls farther away from the pKa. At 1 pH unit away (pH 6 or pH 8) the buffer capacity is reduced to less than half of what it was at the pKa. Some buffering systems make use of more than one buffering agent. For instance, McIlvaine’s buffer contains both phosphoric acid (pKa values: 2.15, 6.86, and 12.32), and citric acid (pKa values: 3.13, 4.76, and 6.40). Within the range pH 2 – 8, you are never farther than 1 pH unit away from a pKa. The buffer capacity graph for McIlvaine’s buffer is more complicated (right panel, above). We can use buffer capacity to back-calculate what concentration of buffer we require (Cbuf), depending on what changes in pH we would like the system to tolerate. This will affect Equation (11), the mass balance of buffering agent. In practice, a concentration of 0.1 M is usually used. Did you know? The mathematics of acid/base chemistry are exactly the same as that for any other equilibrium reaction, e.g. drug/receptor interactions. So by learning the mathematics of buffers, you have just learned how to calculate binding constants of drugs with their targets. Lab2 PHC 340Y Lab Manual 2017-2018
  • 39. Lab 2: Preparation of pH Buffers Remember that the Ka values for weak acids and bases are dependent on the temperature and pressure of the solution. Many experiments make use of this, and measure the equilibrium constants at different temperatures or pressures to examine other interesting properties of the systems studied. In practice, Sorensen’s buffer is simply referred to as “phosphate buffer”. Key Concepts: Many buffering systems are weak acids paired with the salt of their conjugate bases, or weak acids titrated to the viscinity of their pKa with NaOH or HCl. A buffering agent is useful within 1 pH unit of its pKa.  Understanding the mathematics of buffering systems is important, but ultimately, your pH meter decides how much of each agent to add. Experiment Protocol Chemicals Supplies Special Equipment Sodium Phosphate Monobasic N/A pH Meter (verify MW on the bottle used) Mixing plate and magnetic stir Sodium Phosphate Dibasic (verify bar MW on the bottle used) 100 mL graduated cylinder Citric Acid (MW 210.14 g/mol) 250 mL volumetric flask pH Standardizing Buffers 140 mL beaker 50 mL burette, burette clamp, retort stand The following solutions are prepared or provided by the TA: pH Meter Standardizing Buffers (pH 4, 7) Part A. Preparing Sorensen’s Buffer Prepare 250 mL of a 0.2 M solution of sodium phosphate monobasic. Prepare 250 mL of a 0.2 M solution of sodium phosphate dibasic. Load a burette with 50 mL of the 0.2 M sodium phosphate monobasic solution. Set up the burette on a retort stand with a burette clamp.  Measure out 97 mL of sodium phosphate dibasic using a 100 mL graduated cylinder. Pour into a 140 mL beaker. Calibrate your pH meter with the standard solutions provided.  Set the 140 mL beaker on top of a mixing plate under the burette, and insert the pH electrode.  Make sure the tip of the pH electrode is submerged, and does not touch the bottom of the beaker.  Place a magnetic stir bar in the 140 mL beaker, and set mixing to a low speed. Do not allow the stir bar to hit the pH meter, or a vortex to appear.  Slowly titrate the sodium phosphate dibasic solution with the sodium phosphate monobasic solution, until you attain a pH of 7.40. PHC 340Y Lab Manual 2017-2018
  • 40. Lab 2: Preparation of pH Buffers 37 Record the volume of sodium phosphate monobasic added.  Transfer the titrated mixture to a 250 mL beaker. Add an equal volume of water to create a 0.1 M Sorensen’s Buffer. Seal your buffer with parafilm, label it appropriately, and store it for Lab 4. Part B. Preparing McIlvaines’s Buffer McIlvaine’s Buffer is a mixture of phosphoric and citric acids. It is useful in the range pH 2.2 – pH The two solutions to be prepared are sodium phosphate dibasic, and citric acid. In this exercise, you will be assigned two of the following pH values: 2.5, 3, 3.5, 4, and 4.5. You may be assigned different pH buffers by group. The following is a published chart on volume (in mL) of each solution to combine for the expected pH, through the useful range of McIlvaine’s Buffer. It makes 20 mL of buffer: Lab2 Source: http://stanxterm.aecom.yu.edu/wiki/index.php?page=McIlvaine_buffer In a 250 mL flask, prepare 0.2 M sodium phosphate dibasic solution. NOTE: You may have enough stock left over from making the Sorensen’s Buffer. In a 250 mL volumetric flask, prepare 0.1 M citric acid solution.  Load a burette with 50 mL of the 0.2 M sodium phosphate dibasic solution. Set up the burette on a retort stand with a burette clamp. Measure out 100 mL of citric acid using a 100 mL graduated cylinder. Pour into a 250 mL PHC 340Y Lab Manual 2017-2018
  • 41. Lab 2: Preparation of pH Buffers beaker. Set the 250 mL beaker on top of a mixing plate under the burette, and insert the pH electrode.  Place a magnetic stir bar in the 250 mL beaker, and set mixing to a low speed. Do not allow the stir bar to hit the pH meter, or a vortex to appear.  Slowly titrate the citric acid solution with the sodium phosphate dibasic solution, until you attain the desired pH. NOTE: Depending on the pH selected, you may have to re-fill the burette with the sodium phosphate dibasic solution. Record the total volume of sodium phosphate dibasic added. Seal your buffer with parafilm, label it appropriately.  The TA will store your buffers in the cold room on the 9 th floor, for Lab 3. In general, many aqueous buffers will grow bacteria at room temperature, and are best stored at cold temperatures (e.g. at 5 °C in media bottles, or -20 °C in small aliquots, depending on the buffer). Questions Explain using the Henderson-Hasselbalch equation why the pH of the Sorensen’s buffer shouldn’t change when you add an equal volume of water for the final step. The pKa of salicylic acid is 2.97. What form (ionized or un-ionized) will it predominantly be: In the stomach, at pH 2? In the gut, at pH 7? If the ionized form (salicylate) cannot pass through cell membranes, where would you expect the drug to be absorbed? 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 BOH +H2O HO H OHHO Boric Acid Borate What is the useful pH range of borate buffer? Create a protocol for preparing 500 mL of 0.1 M Borate buffer, at pH 8, assuming you already have a solution of 0.2 M NaOH. How will you prepare your stock solutions? How much of each will you require? Calculate the buffer capacity of 0.1 M Borate buffer: At pH 8. At pH 5. Does your answer for (a) double at pH 8 for a Cbuff of 0.2 M? PHC 340Y Lab Manual 2017-2018