Pharmaceutical Microscopy

Robert Allen Carlton

Pharmaceutical Microscopy Robert Allen Carlton Senior Scientific Investigator GlaxoSmithKline Pharmaceuticals Physical Properties 709 Swedeland Road King of Prussia, PA 19406 USA [email protected]

ISBN 978-1-4419-8830-0 e-ISBN 978-1-4419-8831-7 DOI 10.1007/978-1-4419-8831-7 Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011925544

© Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

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Springer is part of Springer Science+Business Media (www.springer.com) Preface

A simple glance at the table of contents demonstrates the breadth of microscopy techniques applied to pharmaceutical microscopy. We range from simple stereo- microscopy to polarized light microscopy, and from electron microscopy to IR and Raman spectroscopy. This can be an intimidating set of techniques and instruments to attempt to learn and to use in just a cursory fashion, much less to become expert in use and interpretation. I am fortunate to have been given the opportunity to learn to use all of these techniques over my career. I began microscopy by measuring glass fibers on a projection microscope in a closet and have moved from there to all of the techniques listed in this book. I did the majority of my education part time after I started in industry and have consciously directed my academic studies toward industrial microscopy. My doctorate is in chemistry (physical) and my dis- sertation covered quantitative analysis using energy dispersive X-ray spectrometry in the environmental scanning electron microscope. I am fortunate to have had sup- portive managers as well as supportive academic advisors. I learned optical microscopy from McCrone Research Institute, from listening to talks by industrial microscopists, from reading microscopy works, and by long hours with eyes applied to eyepieces. Walter McCrone’s courses and conferences are what helped me decide that my goal was to become a chemical microscopist. Skip Palenik and John Delly were the two microscopists I most admired, but I must say that there were so many fine industrial microscopists working in the 1980s. I use the past tense because industrial specialization in science is not as common today as it was then. I think we have lost something important but such opinions may be tilting at windmills. Still, I believe that the satisfaction of becoming a mas- ter craftsman (woman as well) is worth the effort and pays dividends to business. Oh, did I mention how much I learned from chats at the bar after conferences bend- ing elbows and ears? I cannot forget that source of “learning.” I have worked in the pharmaceutical industry since 1992. In that time, I have worked on hundreds of compounds and had maybe a dozen approved. My work has extended from very early-stage discovery before first time in human studies to post launch. I have transferred microscope size methods to manufacturing facilities. Most pharmaceutical scientists work in a very narrow range of development – a development that can take a decade from start to finish. Solid-state scientists, on the other hand, often work from the very early stages looking at polymorphism and

v vi Preface particle size all the way to marketing approval and manufacture. It is a fine way to learn the industry and the particular challenges to drug development. I use personal pronouns such as ‘I’, ‘you’, and ‘we’ much more than is common in scientific books. I hope you do not find the practice too jarring or distracting. I chose to present the information in this fashion because so much of the contents involves personal preference and experience. Also, I am hoping to achieve the sens that you and I are sitting in front of a microscope and I am sharing with you what I have learned from more than 30 experience in industrial and pharmaceutical microscopy. I am an enthusiastic proponent of microscopy in solid-state pharmaceutical development. I have gained a great deal of personal satisfaction from using micros- copy to solve industrial problems and I hope this book helps you have the same experience.

April 2011 Robert A Carlton Collegeville PA Acknowledgments

I want to thank my wife, Brenda, for her help, support and understanding in this endeavor. I know, you are probably thinking this is the trivial, obligatory “thank the marriage mate” sentence. It is not. All it requires is one unruly sentence, a para- graph that wrests control and refuses to yield, one misbehaving figure to turn an otherwise chummy, congenial writer into a raving, mad lunatic. Brenda has been patient and understanding when I have needed it the most. I do appreciate the care and the sacrifices. Brenda also did a thorough edit of the book which was particu- larly helpful since she is an author in her own right (Drug Money, Accident on 13th and Jefferson). Thank you from the bottom of my heart. My daughter Danielle Broadaway helped with a number of figures and with my Web site. Thank you. My parents, Joan and Bob Carlton are a continuing source of inspiration and encouragement. I had the opportunity to work with two young interns from Singapore. Hwee- Jing Ong and Yuniatine who did much of the actual lab work related to caffeine and carbamazepine. It was a distinct pleasure to work with both of them. They are both bright, capable scientists and I expect good things from them in the future. My editor at Springer, David Packer, has been a source of encouragement and good advice throughout the writing process. Dr. Jeffrey Brum, Dr. Ronald Mueller, and Dr. Greg Webber of GlaxoSmithKline have all supported my work and graciously allowed me to use instrumentation in our labs. Jeff has been particularly understanding and supportive. I have benefited from talks with Dr. Frederick Vogt and Dr. Mark Strohmeier of GSK about microspectroscopy. I have received help and advice on the same subject from Dr John Refner of the John Jay School of Criminal Justice and from Pauline Leary of Smiths Detection. Fran Adar of Horiba Jobin-Yvon has contributed good advice on Raman microspectroscopy. I have had the benefit of many good microscopy teachers including Skip Palenik, John Delly, Walter McCrone, and Charles Lyman among many others. I also want to thank a generation of industrial microscopists who freely gave of their knowl- edge in technical meetings and in conversations afterwards. They are an inspiring example.

vii viii Acknowledgments

Finally, in order to finish this book in a reasonable time frame, I had to have help with our extensive gardens at home. Tina, Kalee, Sutton, Jasmine, George, Aubree, Gavin, and Dave provided that help willingly and with good humor. Without their help the task would have been much harder and taken much longer. Contents

1 Introduction...... 1 1.1 Microscopy and Industrial Microscopy...... 1 1.2 Pharmaceutical Microscopy...... 1 1.3 Purpose of the Book...... 2 1.4 Plan of the Book...... 4 1.5 How Best to Use the Book...... 5 References...... 6

2 Polarized Light Microscopy...... 7 2.1 Introduction...... 7 2.2 Properties of Light...... 8 2.3 Basic Optics...... 11 2.4 Crystallography...... 15 2.5 Optical Crystallography...... 19 2.5.1 No Polars, Brightfield...... 22 2.5.2 One Polar...... 28 2.5.3 Crossed Polars...... 28 2.5.4 Summary of Optical Crystallography...... 37 2.6 Measurement of Refractive Indices...... 37 2.6.1 Introduction...... 37 2.6.2 Cubic Crystals and Amorphous Materials: Basic Immersion Technique...... 38 2.6.3 Uniaxial Crystals...... 41 2.6.4 Biaxial Crystal Search Method...... 42 2.6.5 Biaxial Crystals with Aid of Interference Figures...... 45 2.6.6 Summary of Refractive Index Measurements...... 46 2.7 Instruments and Köhler Illumination...... 47 2.7.1 Objectives...... 48 2.7.2 Oculars (Eyepiece)...... 49 2.7.3 Light Source...... 49 2.7.4 Substage Condenser...... 50 2.7.5 Microscope Stand and Stage...... 50 2.7.6 Polarizer and Analyzer...... 51

ix x Contents

2.7.7 Compensators...... 51 2.7.8 Köhler Illumination...... 51 2.8 Stereomicroscopy...... 54 2.9 Photomicrography...... 55 2.10 Summary...... 57 2.11 Exercises...... 57 A. Sample Preparation and Preparation of Crystals...... 57 B. Microscope Setup: Köhler Illumination...... 58 C. Polarized Light...... 59 D. Interference colors...... 60 E. Refractive Index: Cubic, Uniaxial, Biaxial, High low...... 61 F. Optical Crystallography...... 62 References...... 63 Internet References...... 64

3 Thermal Microscopy...... 65 3.1 Introduction...... 65 3.2 Instruments and Methods...... 66 3.3 Calibration...... 69 3.4 Test Strategy...... 71 3.5 Observations...... 72 3.5.1 Melting Point...... 72 3.5.2 Solid–Solid Transformations...... 75 3.5.3 Solid–Liquid–Solid Transformations...... 75 3.5.4 Sublimation...... 76 3.5.5 Cooling...... 78 3.5.6 Dehydration and Desolvation...... 78 3.6 Fusion Techniques and Other Applications...... 79 3.7 Summary...... 81 3.8 Exercises...... 81 A. Sample Preparation and Experimental Setup...... 81 B. Calibration and Performance Checks...... 82 C. Effects of Heating Rate...... 82 D. Phase Transformations...... 83 E. Fusion...... 83 References...... 83 Internet References...... 84

4 Scanning Electron Microscopy and Energy-Dispersive X-Ray Spectrometry...... 85 4.1 Introduction...... 85 4.2 Scanning Electron Microscopy...... 86 4.2.1 Basic Operation of SEM...... 86 4.2.2 Electron Beam–Specimen Interactions...... 89 4.2.3 Backscatter Electron Emission and Detection...... 91 Contents xi

4.2.4 Secondary Electron Emission and Detection...... 94 4.2.5 SEM Instruments...... 98 4.3 Sample Preparation and Imaging Strategy for Pharmaceuticals...... 102 4.3.1 Drug Substance...... 102 4.3.2 Drug Product...... 107 4.4 Energy Dispersive X-Ray Spectrometry...... 108 4.4.1 Theory...... 108 4.4.2 Qualitative Analysis...... 114 4.4.3 Quantitative Analysis...... 117 4.4.4 Elemental Mapping...... 120 4.4.5 EDS with Low-Vacuum SEM...... 123 4.5 Summary...... 125 4.6 Exercises...... 126 A. Sample Preparation...... 126 B. Secondary and Backscattered Electron Imaging...... 127 C. Low-Vacuum SEM...... 127 D. EDS Qualitative Analysis...... 128 E. Elemental Mapping...... 128 References...... 129 Internet References...... 130

5 Infrared and Raman Microscopy...... 131 5.1 Introduction...... 131 5.2 Infrared Microscopy...... 132 5.2.1 Theory...... 132 5.2.2 Instruments...... 134 5.2.3 Sample Preparation and Testing...... 136 5.2.4 Calibration and Performance Checks...... 140 5.3 Raman Microscopy...... 141 5.3.1 Theory...... 141 5.3.2 Instruments...... 142 5.3.3 Sample Preparation and Testing...... 144 5.3.4 Calibration and Performance Checks...... 146 5.4 Applications...... 147 5.4.1 Solid-State Analysis...... 147 5.4.2 Contaminant Analysis...... 151 5.4.3 Chemical Imaging...... 152 5.5 Summary...... 153 5.6 Exercises...... 154 A. Basic Operation...... 154 B. Operating Parameters...... 154 C. Spectral Interpretation...... 155 D. Mapping...... 155 References...... 155 Internet Resources...... 156 xii Contents

6 Specialized Microscopy Techniques...... 157 6.1 Introduction...... 157 6.2 Confocal Microscopy...... 157 6.3 Scanning Probe Microscopy...... 159 6.4 Transmission Electron Microscopy...... 160 6.5 Differential Interference Contrast and Hoffmann Modulation Contrast...... 162 6.6 Phase Contrast...... 163 6.7 Dispersion Staining...... 165 6.8 Dark Field and Rheinberg Illumination...... 167 6.9 Flourescence Microscopy...... 168 6.10 Freeze-Dry Microscopy...... 170 6.11 Thermal Microscopy in the SEM...... 170 6.12 Summary...... 171 References...... 171 Internet Reference...... 172

7 Image Analysis...... 173 7.1 Introduction...... 173 7.2 General Principles...... 174 7.3 Sample Preparation...... 175 7.3.1 Introduction...... 175 7.3.2 Optical Microscopy Sample Preparation...... 176 7.3.3 SEM Sample Preparation...... 179 7.4 Image Production...... 181 7.4.1 General Considerations...... 181 7.4.2 Image Production with the Optical Microscope...... 182 7.4.3 Image Production with the SEM...... 182 7.4.4 Choice of Microscopy: Resolution and Magnification...... 183 7.5 Image Collection...... 183 7.6 Image Processing...... 185 7.7 Segmentation...... 187 7.8 Binary Operations...... 188 7.8.1 Feature Improvements...... 188 7.8.2 Removal of Unwanted Features and Noise...... 191 7.9 Measurements...... 193 7.9.1 General Considerations...... 193 7.9.2 Field Measurements...... 194 7.9.3 Object Measurements...... 194 7.10 Calibration, Performance Checks, and Size Standards...... 202 7.11 Data Presentation and Statistics...... 203 7.12 Exercises...... 207 A. Sample Preparation...... 208 B. Image Collection...... 208 C. Image Processing...... 208 Contents xiii

D. Image Segmentation...... 209 E. Binary Operations...... 209 F. Measurements...... 209 G. Statistics and Data Presentation...... 210 References...... 210 Internet References...... 211

8 Polymorphism...... 213 8.1 Introduction...... 213 8.2 Theory...... 214 8.2.1 Solid-State Forms...... 214 8.2.2 Form Relationships...... 218 8.3 Solid-State Form Discovery and Selection...... 222 8.3.1 General Considerations...... 222 8.3.2 Form Discovery...... 223 8.3.3 Form Selection...... 226 8.4 Analytical Characterization of Solid-State Forms...... 227 8.5 Caffeine Polymorphism: A Practical Example...... 228 8.5.1 Introduction...... 228 8.5.2 Caffeine Properties...... 229 8.5.3 Caffeine Form Discovery – Initial Tests...... 230 8.5.4 Form Discovery – Polymorph Screening...... 234 8.5.5 Form Stability and Form Relationships...... 238 8.5.6 Thermodynamic Relationship of Anhydrous Polymorphs...... 240 8.5.7 Caffeine Hydrate...... 241 8.5.8 Caffeine Example Summary...... 242 8.6 Summary...... 243 References...... 244 Caffeine Polymorphism...... 245 Regulatory Guidance...... 246 Additional General References...... 246

9 Size and Shape Analysis...... 247 9.1 Introduction...... 247 9.2 Qualitative Shape Analysis...... 248 9.3 Image Analysis Examples...... 250 9.3.1 Image Analysis of Hydrocortisone Cream with Optical Microscopy...... 250 9.3.2 Image Analysis of Carbon Black with SEM...... 254 9.3.3 Image Analysis of Generic Naproxen Sodium Tablet: Backscatter and EDS...... 258 9.3.4 Summary of Exercises...... 264 9.4 Method Development and Error Analysis...... 264 9.4.1 General Considerations...... 264 xiv Contents

9.4.2 Method Development...... 265 9.4.3 Error Analysis and Validation...... 266 9.5 Specifications...... 273 9.6 Automation...... 274 9.7 Summary...... 276 References...... 276 Internet Reference...... 276

10 Contaminant Analysis...... 277 10.1 Introduction...... 277 10.2 Robert’s Rules for Contaminant Identification...... 279 10.2.1 Rule #1: Think Before You Act...... 279 10.2.2 Rule #2: Get All the Information Before You Start...... 279 10.2.3 Rule #3: Clearly Establish the Goal of the Investigation...... 280 10.2.4 Rule #4: Every Experiment Should Test a Hypothesis..... 281 10.2.5 Rule #5: Start Simple and Progress to Complex...... 281 10.2.6 Rule #6: Maintain Clear Distinction Between Fact and Hypothesis...... 283 10.2.7 Rule #7: Verify Conclusions...... 283 10.2.8 Rule #8: Document as You Work...... 284 10.2.9 Rule #9: Have a Clear Exit Strategy...... 285 10.3 Practical Examples...... 286 10.3.1 Introduction and Method of Analysis...... 286 10.3.2 Dust...... 286 10.3.3 Activated Charcoal in Powdered Sugar...... 289 10.3.4 Polymer Tubing in Corn Starch...... 294 10.3.5 Silicone Oil in Water...... 299 10.4 Laboratory Requirements for Contaminant Identification...... 301 10.5 Summary...... 302 Reference...... 303 General References...... 303 Internet References...... 303

11 Conclusion...... 305 11.1 General Summary...... 305 11.2 Training and Education...... 307 11.3 Experts vs. Generalists...... 308 11.4 Conclusion...... 309

Index...... 311