Supercritical Fluid
Chromatography
This page intentionally left blank
1BO4UBOGPSE4FSJFTPO3FOFXBCMF&OFSHZ7PMVNF
Supercritical Fluid
Chromatography
Advances and Applications in Pharmaceutical Analysis
editors
Preben Maegaard
Anna Krenz
Wolfgang Palz
edited by
Webster
The Rise of Gregory
ModernK. Wind
Energy
Wind Power
for the World
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
© 2014 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works
Version Date: 20131218
International Standard Book Number-13: 978-981-4463-01-0 (eBook - PDF)
This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher
cannot assume responsibility for the validity of all materials or the consequences of their use. The
authors and publishers have attempted to trace the copyright holders of all material reproduced in
this publication and apologize to copyright holders if permission to publish in this form has not
been obtained. If any copyright material has not been acknowledged please write and let us know so
we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced,
transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or
hereafter invented, including photocopying, microfilming, and recording, or in any information
storage or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.
copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc.
(CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been
granted a photocopy license by the CCC, a separate system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and
are used only for identification and explanation without intent to infringe.
Visit the Taylor & Francis Web site at
http://www.taylorandfrancis.com
and the CRC Press Web site at
http://www.crcpress.com
Contents
Preface
1.
The SFC Market: “Yesterday, Today, and Tomorrow”
Gregory K. Webster
1.1
Introduction
1.2
Development of SFC
1.2.1 Capillary SFC
1.2.2 Packed-Column SFC
1.2.3 Preparative SFC
1.3
The SFC Market
1.4
SFC in the Pharmaceutical Industry
1.4.1 The Use of SFC in Discovery
1.4.2 IQ/OQ/PQ for SFC Instrumentation
1.4.3 Method Development for Achiral SFC
1.4.4 Achiral Preparative SFC
1.4.5 SFC for Chiral Method Development
Screening and Analysis
1.4.6 Chiral Preparative SFC
1.4.7 SFC in Process Analytical Chemistry
1.4.8 Analytical SFC for Impurities
1.4.9 SFC-MS
1.4.10 SFC of Natural Products
1.4.11 Polarimetry Detection in SFC
1.4.12 New Frontiers in SFC–USFC
1.4.13 Pilot-Scale SFC
2. The Use of SFC in Discovery Sciences
Kanaka Hettiarachchi, Andersen Yun, May Kong,
John R. Jacobsen, and Qifeng Xue
2.1
Introduction
2.2
High-Throughput Screening and Purification
2.2.1 Chromatographic Technologies
2.2.2 Laboratory Workflow
2.3
Implementation of SFC
2.3.1 SFC Fundamentals
xiv
1
1
4
4
5
5
6
6
7
7
7
7
8
8
9
9
10
11
12
12
13
15
15
16
16
17
19
19
vi
Contents
2.4
2.5
3.
2.6
2.7
2.3.2 Benefits of SFC
Instrumentation
2.4.1 Analytical Instrumentation
2.4.2 Preparative Instrumentation
Enantiomeric Separation in SFC
2.5.1 Chirality and Chromatographic
Separation
2.5.1.1 RPLC and SFC Separation
of Two Diastereomers
2.5.1.2 SFC Separation of a
Lipophilic Prodrug
Achiral Separation in SFC
2.6.1 Screening Samples
2.6.1.1 Separation of Routine
Compounds
2.6.1.2 Challenging Separations
2.6.2 Mass-Directed Purification with SFC
2.6.3 Achiral Purification Comparison
of RPLC-MS and SFC-MS
Remarks of SFC in Drug Discovery
Qualification of SFC Hardware and Validation of Systems
Ludwig Huber
3.1
Introduction
3.2
Analytical Instrument Qualification According
to USP <1058>
3.3
Qualification Planning
3.4
Design Qualification
3.4.1 The Importance of Requirement
Specifications
3.4.2 Vendor Assessment
3.5
Installation Qualification
3.6
Operational Qualification
3.7
Tests for Operational Qualification
3.8
Performance Qualification
3.9
Specific Considerations for Software and
Computer Systems
3.10 (Preventive) Maintenance and Repair
3.11 Change Control
3.12 Validation Reports
20
23
23
24
25
25
25
28
28
29
30
31
32
33
37
43
43
44
46
48
49
51
53
54
56
56
60
61
63
64
Contents
4. Method Development for Achiral SFC
Jeffrey W. Caldwell, Walton B. Caldwell,
Gregory K. Webster, and Zhenyu Wang
4.1
Introduction
4.2
Overview of Achiral SFC Separations
4.2.1 Achiral SFC vs. Achiral HPLC
4.2.2 Commercially Available Achiral
Stationary Phases for SFC
4.2.3 Novel Stationary Phases for SFC
4.2.3.1 “Amino” stationary phases
4.2.3.2 Hydroxylated stationary
phases
4.2.3.3 Stationary phase pore size
4.2.4 Column Formats for SFC
4.3
Achiral Method Development
4.3.1 Role of Modifier and Additive
4.3.2 Primary Screening on Mobile Phase
and Stationary Phase
4.3.3 Fine-Tuning on SFC Separation
4.4
Develop SFC Method for Mometasone Furoate
Impurity Analysis
4.5
Summary
5.
Achiral Preparative Supercritical Fluid Chromatography
Vivi Lazarescu, Mark J. Mulvihill, and Lifu Ma
5.1
Introduction
5.2
Evolution of Achiral SFC Instrumentation
5.3
Stationary Phases for Achiral SFC
5.4
Method Development: Experimental
5.4.1 The Value of Pre-Purification
5.4.2 Column Selection
5.4.3 Mobile Phase Modifier and Additives
5.4.4 Flow Rate
5.4.5 Gradient Ramp Rate
5.4.6 Sample Solvents
5.4.7 Triage between SFC and HPLC
5.5
Singleton Achiral Purification of Difficult
Samples for Discovery Research Support
5.6
Approaches for SFC Purification of Compound
Libraries
65
65
66
66
67
70
70
74
76
80
80
80
82
84
86
93
97
97
100
108
112
112
113
114
115
115
116
116
116
119
vii
viii
Contents
5.7
5.8
5.6.1 UV-Triggered or Fixed Window
Fraction Collection
5.6.2 Mass-Triggered Fraction Collection
Multicolumn (Dual-Column) Approach for
Achiral SFC Purifications
Summary
6. Supercritical Fluid Chromatography for Chiral Method
Development Screening and Analysis
Gregory K. Webster and Ted J. Szczerba
6.1
Introduction
6.2
Overview of Chiral SFC Separations
6.2.1 Stereoselectivity
6.2.2 Chiral SFC vs. Chiral HPLC
6.2.3 Commercially Available Chiral
Stationary Phases for SFC
6.2.4 Mobile Phases for Chiral SFC
6.2.5 Co-Solvents in Chiral SFC
6.2.5 Co-Solvent Modifiers in Chiral SFC
6.3
Chiral Method Development
6.3.1 Synthetic Approach
6.3.2 Method Development Screens
6.3.3 Application
6.3.3.1 Screening success rate
6.4
Summary
7.
Chiral Preparative Supercritical Fluid Chromatography
Manuel C. Ventura
7.1
Introduction
7.2
Toward Useful Chiral Stationary Phases
7.3
SFC Application to Preparative Separation
7.3.1 Basic Background
7.3.2 Mobile Phases for Prep SFC
7.3.3 Instrumentation for Prep SFC
7.4
Strategy for Chiral Preparative SFC Separation
of New Drug-Like Molecules
7.4.1 Analytical Method Development
7.4.2 Preparative Purification
7.5
Applications for Chiral Prep SFC: Successes
and Challenges
121
125
130
135
145
145
147
147
148
149
157
157
159
161
161
163
165
166
168
171
171
172
173
173
174
175
177
177
179
181
Contents
8.
9.
7.6
7.5.1 Toward Pharmaceutical Application
of Chiral Preparative SFC
7.5.2 Preparative Application of Chiral
Stationary Phases
7.5.3 Solubility and Mobile Phase Issues
in Prep SFC
7.5.4 Recycling Chiral SFC Separation
Processes
Conclusion
SFC in Process Analytical Chemistry
Yanqun Zhao
8.1
Introduction
8.2
Chiral Purity Analysis and Method
Development
8.2.1 Introduction
8.2.2 Chiral Purity Analysis by SFC in
Process Analytical Chemistry
8.2.3 Method Development Using SFC
Screening
8.2.4 Column Selection
8.2.5 Modifier and Modifier Composition
8.2.6 Effect of Additive
8.2.7 Method Transfer between SFC and
HPLC
8.3
SFC Instrument Qualification and Method
Validation
8.4
Impurity Isolation and Material Purification
8.4.1 Impurity Isolation
8.4.2 Material Purification
8.5
SFC with Mass Detection
8.5.1 Using a Mass Detector
8.5.2 Applications
8.6
Achiral Separations
8.7
Summary and Conclusion
Analytical SFC for Impurities
Yun Huang
9.1
Introduction
9.2
Qualification of Analytical SFC System
181
181
184
187
188
195
195
197
197
197
199
202
203
204
205
207
209
209
212
212
213
214
216
218
225
226
231
ix
x
Contents
9.3
9.4
9.2.1 Analytical Instrument Qualification
Overview
9.2.2 Qualification of Analytical SFC System
Analytical SFC as Primary Chiral Purity Tool
for Clinical Release and Stability Testing
9.3.1 Method Validation Parameters
9.3.2 Feasibility Study on Chiral SFC Used
for Clinical Release and Stability Testing
9.3.2.1 Method validation results and
discussions for PF-00981823
9.3.2.2 SFC method development and
validation for PD-0348292
9.3.3 Method Transferability
9.3.3.1 Study design
9.3.3.2 Results and discussions
Conclusions
10. Supercritical Fluid Chromatography–Mass Spectrometry
Laila Kott
10.1 Introduction
10.2 Sources
10.2.1 Vacuum Sources
10.2.1.1 Direct introduction
10.2.1.2 Thermospray interface
10.2.1.3 Particle beam interface
10.2.2 Atmospheric Sources
10.2.2.1 Atmospheric pressure
chemical ionization
10.2.2.2 Electrospray ionization
10.2.2.3 Atmospheric pressure
photoionization
10.3 Source and Mass Analyzer Interfaces
10.3.1 Flow Splitting Prior to the Back
Pressure Regulator
10.3.2 Total Flow Using a Pressure
Regulating Fluid Interface
10.3.3 Total Flow Using a BPR
10.3.4 Total Flow Using a Passive BPR
10.4 Mass Analyzers
10.5 Types of Analyses
231
233
239
240
241
243
246
255
255
256
260
265
265
266
266
267
267
268
268
269
272
273
276
276
277
279
280
280
282
Contents
10.6
10.5.1 Chiral SFC-MS
10.5.2 Achiral SFC-MS
10.5.3 Prep SFC-MS
10.5.4 Structure Elucidation
Traditional Problems
11. Supercritical Fluid Chromatography of Natural
Products
Ying Wang
11.1 Introduction
11.2 Analytical Supercritical Fluid Chromatography
of Plant Metabolites
11.2.1 Sesquiterpenes
11.2.2 Diterpenes
11.2.3 Triterpenes
11.2.4 Alkaloids
11.2.5 Flavonoids
11.2.6 Kava Lactones
11.3 Analytical Supercritical Fluid Chromatography
of Microbial Metabolites
11.3.1 Macrolides
11.3.2 Cyclic Peptides
11.3.3 Polyethers
11.3.4 Trichothecenes
11.3.5 Chloramphenicol
11.4 Preparative Supercritical Fluid
Chromatography of Natural Products
11.4.1 Preparative Supercritical Fluid
Chromatography of Plant Metabolites
11.4.2 Preparative Supercritical Fluid
Chromatography of Microbial
Metabolites
11.5 Conclusions and Prospects
12. Polarimetric Detection in Supercritical Fluid
Chromatography
Gary W. Yanik
12.1 Introduction
12.2 Theory of Operation
12.3 Comparison of ALP, UV, and CD
285
286
287
288
288
297
297
299
299
304
309
311
313
314
316
316
318
320
320
323
324
324
326
326
333
333
334
337
xi
xii
Contents
12.4
12.5
12.6
Analytes
12.4.1 Small Molecule Pharmaceutical
Candidates
12.4.2 Antibiotics and Sugars: Compounds
without Chromophores
12.4.3 Amino Acids
12.4.4 Natural Products
12.4.5 Foods, Flavors, and Fragrances
12.4.6 Fertilizers and Pesticides
Applications
12.5.1 SFC Method Development
12.5.2 SFC Preparatory Purification: Peak
Collection
Summary
13. Supercritical Fluid Chromatography with
Ultra-Performance Particles
Ziqiang Wang
13.1 Introduction
13.1.1 Supercritical Fluid Chromatography
13.2 Current Status of SFC Performance
13.3 Characteristics of Ultra-Performance
Particles for SFC Considerations
13.4 Published studies on SFC with
Ultra-Performance Particles
13.5 Summary
13.6 Future Directions of SFC Development
14. Pilot and Production-Scale Supercritical Fluid
Chromatography
Geoffrey B. Cox
14.1 Introduction
14.2 Large-Scale SFC: The Potential
14.3 Scale-Up Issues
14.3.1 Column Size
14.3.2 Particle Size
14.3.3 Speed
14.3.4 Sample Introduction
14.4 Large-Scale Equipment
14.4.1 Batch Systems
338
338
338
340
340
341
341
341
341
343
346
347
347
347
349
352
355
366
366
371
371
372
375
376
377
381
381
382
382
Contents
14.5
14.6
Index
14.4.2 Alternate Pumping Recycle in SFC
14.4.3 SMB-SFC
Applications at Pilot and Industrial Scale
14.5.1 Fish Oils
14.5.2 Cyclosporin
14.5.3 Palm Oil Components
Future
383
385
388
389
390
390
390
395
xiii
This page intentionally left blank
Preface
After a Pittsburgh Conference symposium, Stanford Chong of Pan
Sanford approached me to ask if I was interested in writing a book
on supercritical fluid chromatography (SFC). I quickly replied,
“No!” Thinking about it further, I saw a need to update the many
applications of SFC in the pharmaceutical industry. I told Stanford
that I would look to industry experts to help guide me on this
endeavor. Thank you, Stanford, and my good friend, Laila, for your
support and encouragement on this endeavor.
When I look at supercritical fluid chromatography (SFC), the first
concept that comes to mind is that of an analytical toolbox. A past coworker, or “Pope Van Deempter” as we sometimes called him, often
liked to label people by their specialty. He was a “chromatographer.”
Because my graduate work was mostly in atomic spectrometry, he
would call me a “spectroscopist.” This would challenge me because
(1) my academic days have been in the rear view mirror for a quite
a while and (2) such labeling is opposite of what I was taught in
regard to analytical chemistry. If I had to be labeled as a scientist, I
would much prefer the title “analytical chemist” for a true analytical
chemist tries not to marry oneself to a single technique, but uses
the “right” technique for the task at hand. We value the generalist
who can maintain a broad view as much as the specialist in a single
technique. In truth, both are needed in the pharmaceutical industry.
The success of SFC is along these same lines. For much of its
existence, other techniques could perform in a similar manner, but
SFC has several niches where it is the “right tool” for the task at hand.
Conversely, whereas SFC can be investigated as a tool for several
applications, it should not be used in areas where other techniques
have technical, efficiency, or cost advantages.
SFC developed its place in the pharmaceutical industry because
it has simply outperformed other techniques in preparative
chromatography. From there, it developed other applications at the
analytical, preparative, and production scale as scientists looked to
broaden their use of this analytical tool. In some areas, such as in
chiral analysis, SFC has become the primary technique. In others,
xvi
Preface
such as in achiral analytical chromatography, the advances have
been less apparent to date and researchers are still looking for
improvements. This book is a mix. We have included areas where
SFC is the dominant technique as well as areas where its application
is still emerging in the pharmaceutical industry.
When I graduated from college, the pharmaceutical industry
accounted for approximately seventy percent of the jobs for
young chemists and accounted for most of the sales for analytical
instruments. While we all know this number seems to be changing,
pharma today still plays a dominant role in the chemical industry.
The goal of this book was not to rewrite what others have written
and pioneered in SFC but rather to add to this existing body of work
as to how SFC is being used in the pharmaceutical industry today. In
this last regard, I invited current pharmaceutical industry scientists
who are currently using SFC in a specific role to write about their
applications. I would like to thank the authors for their contributions
resulting from their experiences, dedication, and work. In addition,
I would like to thank Phillip Searle, Erin Jordan, Paul David, Cindy
Pommerening, Ken Miller, and Christine Havrilla of AbbVie, as well
as the authors, for their help in reviewing the content.
I would like to thank my dog, Murphy, who continually reminds
me that life is more about the joy of chasing a tennis ball than
the stress of meeting deadlines. Finally, I would like to thank my
wife, Tammy, who shares my life and reviews my grammar on our
wonderful journey together.
Gregory K. Webster
December 2013
Chapter 1
The SFC Market: “Yesterday, Today,
and Tomorrow”
Gregory K. Webster
AbbVie, Global Research and Development, 1 N. Waukegan Rd.,
North Chicago, IL 60064 USA
[email protected]
1.1 Introduction
Chemistry majors in the 1980s were aware of the excitement of a new
and “revolutionary” technique that was coming. The technique was
called supercritical fluid chromatography (SFC). Capillary SFC was
thought to be the next great innovation in column chromatography.
As undergraduate students, we didn’t quite know much about this
technique; but the news at the time was that this technique was
projected to take over gas and liquid chromatography (LC) and bring
analytical separation science into a new dynamic in chromatographic
analysis. The use of a supercritical fluid mobile phase had a potential
advantage in not only chromatographic efficiencies but also cost and
ease of use. However, as with capillary electrophoresis, the advanced
marketing and hype of early capillary SFC was never achieved.
Supercritical Fluid Chromatography: Advances and Applications in Pharmaceutical Analysis
Edited by Gregory K. Webster
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4463-00-3 (Hardcover), 978-981-4463-01-0 (eBook)
www.panstanford.com
2
The SFC Market
While SFC found a niche in the petrochemical industry where
nonpolar aromatics are of interest, the relatively polar nature of
pharmaceuticals limited their ability to be analyzed by SFC. The earlier
excitement of SFC fizzled. Instead, as a capillary chromatography
technique, SFC was challenged by its inability to solvate enough
polar molecules to maintain the attention of the chemical industry
as a whole, and the pharmaceutical industry in particular. Primarily
through the efforts of Berger [1], SFC evolved into a packed column
technique that found a niche in preparative and chiral analysis.
Before SFC could make strides in achiral analysis, SFC was essentially
sidetracked as a technique for effective impurity discrimination
by the Ultra High Performance Liquid Chromatography (UHPLC)
revolution.
Today’s SFC has had a rocky road to get here. As we will see in
this text, traversing this path has been worth it. Although still today
SFC has yet to match the advanced levels projected in the 1980s, it
has established itself as a valuable chromatographic separation tool
in the pharmaceutical analytical chemist’s toolbox. The introduction
of traditional LC column format for packed column SFC eliminated
the difficulties many laboratories had in running capillary SFC. The
advent of chiral column chromatography created the demand for
efficient normal phase separations.
SFC is now the stalwart technique in preparative-scale chiral
chromatography and is rapidly becoming the technique of choice for
routine analytical applications of chiral chromatography as well. Since
nearly 40% of drugs in use are known to be chiral and approximately
a quarter of these are administered as pure enantiomers, SFC is
involved in a substantial analytical and preparative market.
Today’s SFC instrumentation enables the analytical chemist
to develop highly efficient chromatographic methods and fast reequilibration. The dynamics of a supercritical fluid mobile phase
enables chromatographic coupling and ease in interfacing with
mass spectrometric (MS) detection. Preparative SFC has proven to
significantly reduce development costs, minimize waste handling,
and replace alkane solvents in many laboratories. Since SFC adds
no additional carbon dioxide to the atmosphere, it is designated
as a “green” analytical technique. It has several proven advantages
over traditional HPLC (Table 1.1). The benefits of modern SFC have
lead to the availability of a chromatographic tool that enables fast
speeds and high resolution with low operating costs. SFC provides
Introduction
performance now only beginning to be achieved through the use of
UHPLC.
Table 1.1
Advantages of SFC over HPLC
Advantages
Opportunities
The higher diffusivity/lower viscosity
using supercritical CO2 mobile phases
leads to faster methods with higher
efficiency than traditional liquid
chromatography. (3-10×)
Analyte solubility in mobile
phases
Method development screening systems
are faster.
Preparative SFC fractions are collected in
small volumes of volatile organic solvent.
Equipment
SFC mobile phases are more compatible
with mass spectrometer systems.
Higher efficiency in SFC allows
preparative injection stacking for rapid
collection of fractions.
CO2 mobile phases improve operational
costs thru reduced solvent consumption
and solvent disposal.
CO2 mobile phases with alcoholic
modifiers that are much greener than
other solvents are generally used in NPLC
and RPLC.
With the advances of SFC, chromatography is returning to its true
capability in orthogonal analysis. In recent years, differences in C18
stationary phases were deemed “orthogonal” due to the differences
in selectivity seen with various phases [2, 3]. Traditionally,
chromatographers challenged the purity of their chromatograps by
analyzing their sample in both reversed phase and its “orthogonal”
compliment of normal phase chromatography. Normal phase
chromatography has become less popular for analytical applications
over the last few decades, and mass spectrometry has become more
routine for chromatographic detection. Thus, challenging method
selectivity by changing chromatographic modes became less
commonplace, allowing this new definition of orthogonality to creep
3
.PDF 
412 
216 
98 
