Introduction to Modern Liquid Chromatography, Third Edition part 4 pdf

xxviii CONTENTS
17.3.5 Module Substitution, 820
17.3.6 Put It Back, 821
17.4 Common Symptoms of HPLC Problems, 821
17.4.1 Leaks, 822
17.4.1.1 Pre-pump Leaks, 822
17.4.1.2 Pump Leaks, 823
17.4.1.3 High-Pressure Leaks, 825
17.4.1.4 Autosampler Leaks, 825
17.4.1.5 Column Leaks, 828
17.4.1.6 Detector Leaks, 828
17.4.2 Abnormal Pressure, 830
17.4.2.1 Pressure Too High, 831
17.4.2.2 Pressure Too Low, 832
17.4.2.3 Pressure Too Variable, 833
17.4.3 Variation in Retention Time, 833
17.4.3.1 Flow-Rate Problems, 834
17.4.3.2 Column-Size Problems, 834
17.4.3.3 Mobile-Phase Problems, 834
17.4.3.4 Stationary-Phase Problems, 835
17.4.3.5 Temperature Problems, 836
17.4.3.6 Retention-Problem
Symptoms, 836
17.4.4 Peak Area, 838
17.4.4.1 Peak Area Too Large, 839
17.4.4.2 Peak Area Too Small, 840
17.4.4.3 Peak Area Too Variable, 840
17.4.5 Other Problems Associated with the
Chromatogram, 841
17.4.5.1 Baseline Drift Problems, 841
17.4.5.2 Baseline Noise Problems, 844

17.4.5.3 Peak Shape Problems, 847
17.4.6 Interpretation of System Performance
Tests, 856
17.4.6.1 Interpretation of Gradient
Performance Tests, 857
17.4.6.2 Interpretation of Additional
System Tests, 864
17.5 Troubleshooting Tables, 865
References, 876
APPENDIX I. PROPERTIES OF HPLC SOLVENTS 879
I.1 Solvent-Detector Compatibility, 879
I.1.1 UV Detection, 879
I.1.2 RI Detection, 881
CONTENTS xxix
I.1.3 MS Detection, 881
I.2 Solvent Polarity and Selectivity, 882
I.3 Solvent Safety, 885
References, 886
APPENDIX II. PREPARING BUFFERED MOBILE PHASES 887
II.1 Sequence of Operations, 887
II.2 Recipes for Some Commonly Used Buffers, 888
Reference, 890
Index 891

PREFACE
H
igh-performance liquid chromatography (HPLC) is today the premier technique
for chemical analysis and related applications, with an ability to separate,
analyze, and/or purify virtually any sample. The second edition of this book appeared
in 1979, and for tens of thousands of readers it eventually became their choice of

an HPLC reference book. The remarkable staying power of the second edition (with
significant sales into the first decade of the present century) can be attributed to
certain features which continue to be true for the present book. First, all three
editions have been closely tied to short courses presented by the three authors over
the past four decades, to an audience of more than 10,000 industrial, governmental,
and academic chromatographers. Teaching allows different approaches to a subject
to be tried and evaluated, and a pragmatic emphasis is essential when dealing with
practicing chromatographers as students. Second, all three editions have tried to
combine practical suggestions (‘‘how to?’’) with a theoretical background (‘‘why?’’).
Both theory and practice continue to be emphasized so that the reader can better
understand and evaluate the various recommendations presented here. Finally, each
of the three authors has been an active participant in HPLC research, development,
and/or routine application throughout most of their careers.
Since the preparation of the second edition in 1979, there have been major
improvements in columns and equipment, as well as numerous advances in
(1) our understanding of HPLC separation, (2) our ability to solve problems
that were troublesome in the past, and (3) the application of HPLC for new kinds of
samples. Whereas six different HPLC procedures received comparable attention in
the second edition, today reversed-phase chromatography (RPC) accounts for about
80% of all HPLC applications—and therefore receives major (but not exclusive)
attention in the present edition. Over the past three decades the use of HPLC for
biological samples, enantiomeric (chiral) separations, and sample purification has
expanded enormously, accompanied by a much better understanding of these and
other HPLC applications.
Commercial HPLC columns continue to be improved, and many new kinds
of columns have been introduced for specific applications, as well as for faster,
trouble-free operation. Prior to 1990, HPLC method development was an uncertain
process—often requiring several months for the acceptable separation of a sample.
xxxi
xxxii PREFACE

Since then it has become possible to greatly accelerate method development, espe-
cially with the help of appropriate software. At the same time HPLC practice is
increasingly carried out in a regulatory environment that can slow the release of a
final method. These various advances and changes in the way HPLC is carried out
have mandated major changes in the present edition.
The organization of the present book, while similar to that of the second edition,
has been significantly modified in light of subsequent research and experience.
Chapter 1 provides a general background for HPLC, with a summary of how its
use compares with other modern separation techniques. Chapter 1 also reviews
some of the history of HPLC. Chapter 2 develops the basis of HPLC separation
and the general effects of different experimental conditions. Chapters 3 and 4 deal
with equipment and detection, respectively. In 1979 the detector was still the weak
link in the use of HPLC, but today the widespread use of diode-array UV and
mass-spectrometric detection—as well as the availability of several special-purpose
detectors—has largely addressed this problem. Chapter 5 deals with the column:
the ‘‘heart’’ of the HPLC system. In 1979, numerous problems were associated
with the column: peak tailing—especially for basic samples, column instability at
elevated temperatures or extremes in mobile-phase pH, and batch-to-batch column
variability; today these problems are much less common. We also now know a good
deal about how performance varies among different columns, allowing a better
choice of column for specific applications. Finally, improvements in the column are
largely responsible for our current ability to carry out ultra-fast separations (run
times of a few minutes or less) and to better separate mixtures that contain hundreds
or even thousands of components.
Chapter 6, which deals with the reversed-phase separation of non-ionic
samples, extends the discussion of Chapter 2 for these important HPLC appli-
cations. A similar treatment for normal-phase chromatography (NPC) is given in
Chapter 8, including special attention to hydrophilic interaction liquid chromatog-
raphy (HILIC). In Chapter 7 the separation of ionized or ionizable samples is
treated, whether by RPC, ion-pair chromatography, or ion-exchange chromatogra-

phy. Gradient elution is introduced in Chapter 9 for small-molecule samples, and
as an essential prerequisite for the separation of large biomolecules in Chapter 13;
two-dimensional separation—another technique of growing importance—is also
discussed. Chapter 10 covers the use of computer-facilitated method development
(computer simulation). Other important, general topics are covered in Chapters 11
(Qualitative and Quantitative Analysis) and 12 (Method Validation).
Chapter 13 introduces the separation of large molecules, including both
biological and synthetic polymers. HPLC procedures that are uniquely useful for
these separations are emphasized: reversed-phase, ion-exchange, and size-exclusion,
as well as related two-dimensional separations. Chapter 14 (Enantiomer Separations)
marks a decisive shift in approach, as the resolution of enantiomers requires columns
and conditions that are sample-specific—unlike most of the HPLC applications
described in earlier chapters.
Chapter 15 deals with preparative separations (‘‘prep-LC’’), where much
larger sample weights are introduced to the column. The big change since 1979
for prep-LC is that we now have a much better understanding of how such
separations vary with conditions, in turn making method development much more
systematic and efficient. Chapter 16 (Sample Preparation) provides a comprehensive
PREFACE xxxiii
coverage of this important supplement to HPLC separation. As in the case of
other HPLC-related topics, the past 30 years have seen numerous developments
that today make sample preparation a routine addition to many HPLC procedures.
Finally, Chapter 17 deals with HPLC troubleshooting. Despite all our advances in
equipment, columns, materials, technique, and understanding, trouble-free HPLC
operation is still not guaranteed. Fortunately, our ability to anticipate, diagnose,
and solve HPLC problems is now more informed and systematic. One of our three
authors (JWD) has been especially active in this area.
Different readers will use this book in different ways. An experienced worker
may wish to explore topics of his or her choice, or find an answer to specific
problems. For this audience, the Index may be the best starting place. Beginning

readers might first skim Chapters 1 through 7, followed by 9 through 10, all of
which emphasize reversed-phase HPLC. The latter sequence is similar to the core of
the basic HPLC short courses developed by the authors. After this introduction, the
reader can jump to chapters or sections of special interest. Other readers may wish
to begin with topics of interest from the Contents pages at the front of the book or
at the beginning of individual chapters. The present book has been organized with
these various options in mind.
This third edition is highly cross-referenced, so as to allow the reader to follow
up on topics of special interest, or to clarify questions that may arise during reading.
Because extensive cross-referencing represents a potential distraction, in most cases
it is recommended that the reader simply ignore (or defer) these invitations to jump
to other parts of the book. Some chapters include sections that are more advanced,
detailed, and of less immediate interest; these sections are in each case clearly
identified by an introductory advisory in italics, so that they can be bypassed at the
option of the reader. We have also taken pains to provide definitions for all symbols
used in this book (Glossary section), along with a comprehensive and detailed index.
Finally, attention should be drawn to a ‘‘best practices’’ entry in the Index, which
summarizes various recommendations for both method development and routine use.
We very much appreciate the participation of eight collaborators in the
preparation of the present book: Peter Schoenmakers (Sections 9.3.10, 13.10), Mike
Swartz (Chapter 12), Tim Wehr (Sections 13.1–13.8), Carl Scandella (Section 13.9),
Wolfgang Lindner, Michael L
¨
ammerhofer, and Norbert Maier (Chapter 14), Geoff
Cox (Chapter 15), and Ron Majors (Chapter 16). Their affiliations are as follows:
Peter Schoenmakers University of Amsterdam
Mike Swartz Synomics Pharma
Tim Wehr BioRad Corp.
Carl Scandella Carl Scandella Consulting
(4404 91st Avenue NE

Bellevue, WA 98004)
Wolfgang Lindner,
Michael L
¨
ammerhofer,
and Norbert Maier
University of Vienna
Geoff Cox Chiral Technologies
Ron Majors Agilent Technologies
xxxiv PREFACE
We also are indebted to the following reviewers of various parts of the
book: Peter Carr, Tom Chambers, Geoff Cox, Roy Eksteen, John Fetzer, Dick
Henry, Vladimir Ioffe, Pavel Jandera, Peter Johnson, Tom Jupille, Ron Majors,
Dan Marchand, David McCalley, Imre Molnar, Tom Mourey, Uwe Neue, Ravi
Ravichandran, Karen Russo, Carl Scandella, Peter Schoenmakers, and Loren Wrisley.
However, the authors accept responsibility for any errors or other shortcomings in
this book.
L
LOYD R. SNYDER
J. J. (JACK)KIRKLAND
JOHN W. DOLAN
Orinda, CA
Wilmington, DE
Amity, OR
GLOSSARY OF SYMBOLS
AND ABBREVIATIONS
T
his section is divided into ‘‘frequently used’’ and ‘‘less-frequently used’’ sym-
bols.’’ Most symbols of interest will be included in ‘‘frequently used symbols’’.
Equations that define a particular symbol are listed with that symbol; for example,

‘‘Equation 2.18’’ refers to Equation (2.18) in Chapter 2. The units for all symbols
used in this book are indicated. Where IUPAC definitions or symbols differ from
those used in this book, we have indicated the corresponding IUPAC term (from
ASDLID 009921), for example, t
M
instead of t
0
.
FREQUENTLY USED SYMBOLS AND ABBREVIATIONS
A the ‘‘weak’’ component in a binary-solvent mobile phase (A/B); in
RPC, the A-solvent is water or aqueous buffer; also, ‘‘type-A’’ silica
(older, more acidic silica)
ACN acetonitrile
B (%B) the ‘‘strong’’ component (and its %-volume) in a binary-solvent
mobile phase (A/B); in RPC, the B-solvent is an organic, such as
acetonitrile; also, ‘‘type-B’’ silica (newer, less acidic silica; Section
5.2.2.2)
CSP chiral stationary-phase
CV coefficient of variation (equivalent to %-relative standard deviation);
also, column volumes (Section 13.9)
C
8
,C
18
Reversed-phase column-packing designations, indicating length of
alkyl ligand bonded to the particle
d
c
column inner diameter (mm)
d

p
column-packing particle-diameter (μm)
F mobile-phase flow rate (mL/min)
xxxv
xxxvi GLOSSARY OF SYMBOLS AND ABBREVIATIONS
H column plate height (equal to L/N); see also ‘‘less-frequently used
symbols’’ below
HIC hydrophobic interaction chromatography
HILIC hydrophilic interaction chromatography
i.d. column or tubing inner diameter (mm)
IEC ion-exchange chromatography
IPC ion-pair chromatography
k retention factor (same as capacity factor k

); equal to (t
R
/t
0
) − 1
k
∗
gradient retention factor; Equation (9.5)
L column length (mm)
LC-MS liquid chromatography–mass spectrometry
LC-MS/MS LC-MS with a triple-quadrupole mass spectrometer
M molecular weight (Da)
MeOH methanol
MS mass spectrometry
N column plate number; Equation (2.9)
n

c
‘‘equivalent’’ peak capacity, usually referred to as ‘‘conditional’’ or
‘‘sample’’ peak capacity
NPC normal-phase chromatography
P pressure drop across the column (psi); bar or atmospheres = 14.7psi;
megaPascal (MPa) = 10 bar = 147 psi; also, partition coefficient
(Section 6.2)
PC peak capacity; Equation (2.30), Figure 2.26a (isocratic); Equation
9.20, Figure 9.20 (gradient)
pK
a
logarithm of the acidity constant for an acid or base; Equations (7.2),
(7.2a)
R
F
solute fractional migration in TLC; Equation (8.6), Figure 8.8
RI refractive index
RPC reversed-phase chromatography
R
s
resolution; Equation (2.23)
S slope of plots of log k versus φ(d log k/dφ); Equation (2.26)
SEC size-exclusion chromatography
SPE solid-phase extraction
T temperature (
o
C)
t
D
dwell time (min); equal V

D
/F
TFA trifluoroacetic acid
t
G
gradient time (min); Figure 9.10
GLOSSARY OF SYMBOLS AND ABBREVIATIONS xxxvii
t
0
column dead-time (min); also the retention time of a non-retained
solute;equaltoV
m
/F; Equations (2.4a), (2.7)
T-P touching-peak; Figure 15.9b
t
R
retention time (min); Equation (2.5)
type-A older, more acidic silica (Section 5.2.2.2)
type-B newer, less acidic silica (Section 5.2.2.2)
UV ultraviolet absorption
V
D
equipment dwell volume; Section 9.2.2.4
V
m
column ‘‘dead-volume’’; volume of the mobile phase within a column
(mL); Equation (2.7a)
W baseline peak width W; Figure 2.10a
w
s

column saturation capacity (g)
w
x
weight of solute injected (g)
α separation factor; Equation (2.24a)
φ change in φ during a gradient; Figure 9.2g
ε mobile-phase solvent strength in NPC; Equations (8.2), (8.5); also,
dielectric constant
ε
0
value of ε (in NPC) for a pure solvent
φ volume-fraction of the B-solvent (equal to 0.01 × %B)
φ
∗
value of φ during gradient elution for a solute, when the band reaches
the column midpoint
ν reduced velocity; Equation (2.18a)
η mobile-phase viscosity (cP)
LESS-FREQUENTLY USED (OR LESS-COMMONLY
UNDERSTOOD) SYMBOLS AND ABBREVIATIONS
A absorbance
A column hydrogen-bond acidity; Equation (5.3)
AAPS American Society of Pharmaceutical Scientists
AIQ analytical instrument qualification (or validation)
AMT analytical method transfer
AOAC Association of Official Analytical Chemists
APCI atmospheric pressure chemical ionization
API active pharmaceutical ingredient (also atmospheric pressure
ionization)