High Pressure Liquid Chromatography. Biochemical and Biomedical Applications

208 Pages·1973·3.472 MB·English

by  Phyllis Brown (Auth.)

HIGH PRESSURE
LIQUID
CHROMATOGRAPHY
Biochemical and Biomedical
Applications
PHYLLIS R. BROWN
Department of Biochemical Pharmacology
Division of Biological and Medical Sciences
Brown University
Providence, Rhode Island
@
ACADEMIC PRESS 1973 New York and London
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To my husband, without whose infinite patience,
continuous encouragement, and constant support this
book could never have been written
PREFACE
In the past few years, the development of commercially available high
pressure liquid chromatography systems has opened up new horizons for
the analysis of nonvolatile, reactive compounds. Although gas chro-
matography has proved highly sucessful in organic chemistry, its applica-
tions are limited. Many of the thermally labile, complex molecules, which
are difficult to analyze by gas chromatography, are important in bio-
chemistry and the biomedical field. Other techniques, such as thin-layer,
paper, and column chromatography, while useful, have never been com-
pletely satisfactory. High pressure liquid chromatography overcomes many
of these difficulties and provides excellent resolution with high speed and
sensitivity for separating high molecular weight, polar compounds. More-
over, its applications are not limited to this type of sample; separations
of all compounds that are soluble in a liquid phase can be obtained. This
technique can be useful not only in biochemistry but also in inorganic,
organic, and clinical chemistry.
The purpose of this book is to present simply and concisely basic infor-
mation on high pressure liquid chromatography. It should serve as an intro-
duction for those unfamiliar with this technique as well as a reference book
for those who are currently using it. I sincerely believe that high pressure
liquid chromatography will prove to be an invaluable aid in all biochemical
and biomedical research and hope that this book will encourage its use.
Since no analytical method is perfect, the pitfalls likely to be encountered
are discussed to help newcomers avoid many of the problems experienced
by pioneers in the field. Because my experience has been mainly in the use
ix
X PREFACE
of anion-exchange, high pressure liquid chromatography to separate
nucleotides, most of the examples given are from this area.
Much to my surprise, reviewing the literature I found that the great
majority of articles have been concerned with the development of the
instrumentation and that comparatively little work has been published on
the applications of this technique to biochemical and biomedical research.
In the next few years however, when researchers in chemical, biochemical,
and medical disciplines fully realize the great potential of high pressure
liquid chromatography, there should be a literature explosion such as was
experienced in the field of gas chromatography in the 1960's.
I wish to thank the many people who graciously contributed information,
chromatograms, and assistance in the preparation of this book. I would
like particularly to thank Dr. R. E. Parks, Jr., who appreciated the need
for instrumentation to measure nucleotide pools and anticipated the value
of high pressure liquid chromatography for this type of investigation.
Without his constant encouragement and support, this work would not have
been possible. I would also like to thank Drs. John O. Edwards and Ralph
Miech for their many helpful discussions; Drs. Andrew Clifford, Eric
Scholar, J. Arly Nelson, James Little, Richard Henry, Csaba Horvath, and
Herman Shmukler for their gracious cooperation and for making available
to me data prior to publication; Dr. Dennis Gere for his expert technical
assistance and unstinting help when needed; and Miss Elise Bideaux and
Miss Marijo Newman for laboratory assiatance. To Mr. Jonathan Gell are
extended my special thanks for his technical skills which kept our Chro-
matograph in excellent condition, enabling its continuous operation during
the preparation of this book. I am grateful to Mrs. Mary Bonaventure for
her fine typing job and to Dr. Marie Sauer and the staff of the drafting
office of the engineering department for their excellent work in preparing
the illustrations. I am indebted to Drs. Edwards, Horvath, Little, Clifford,
Henry, and Gere for taking time from their busy schedules to read this
manuscript and for giving me the benefit of their knowledge and experience.
Their many helpful comments, suggestions, and criticisms were gratefully
appreciated. I am also indebted to U.S.P.H.S. Grant 16538-01 for support
of research in this field.
Finally, I wish to thank my family for their patience, consideration, and
cooperation. Without their understanding, it would have been impossible
to complete this task.
Phyllis R. Brown
ABBREVIATIONS
The following abbreviations are used: AMP, ADP, ATP = adenosine
5/-mono-, 5'-di-, and 5'-triphosphate; cAMP = cyclic 3',5'-adenosine
monophosphate; GMP, GDP, GTP = 5'-phosphates of guanosine; cGMP =
cyclic 2',3'-guanosine monophosphate; UMP, UDP, UTP = 5'-phosphates
of uridine; CMP, CDP, CTP - 5'-phosphates of cytidine; TMP, TDP,
TTP = 5'-phosphates of deoxythymidine; 6 MMPRMP = 5'-mono-
phosphate of 6-methyl mercaptopurine riboside; TGMP = 5'-mono-
phosphate of 6-thioguanosine; XMP = 5'-monophosphate of xanthosine;
IMP, IDP = 5'-phosphates of inosine;UDPG = uridinediphosphoglucose;
UDPGA = uridine 5'-diphosphoglucuronic acid; NAD, NADH = the
oxidized and reduced forms of nicotinamide-adenine dinucleotide; TCA =
trichloroacetic acid; PCA = perchloric acid.
xi
CHAPTER 1
INTRODUCTION
I. Description
In the literature, liquid Chromatographie systems which feature high inlet
pressures and high sensitivity are variously described as high pressure, high
speed, high performance, high efficiency, or high sensitivity liquid chroma-
tography. In all cases, the reference is to liquid Chromatographie systems
which utilize high pressure pumps and sensitive, low dead-volume detectors.
No one title is completely descriptive. In fact, a better description of this
type of separation is liquid chromatography that affords high resolution,
high speed, high efficiency, and high sensitivity at the same time. Although
the various terms will be used interchangeably in this book, I have chosen the
title "High Pressure Liquid Chromatography" for two reasons. First, it was
the term I was introduced to originally and have used continually for the
past three years. It has become an indispensable part of my vocabulary.
Second, the use of pressure to pump the eluent through the column is the
main feature that distinguishes this particular technique from other types of
chromatography. In other modes of modern liquid chromatography high
performance or high efficiency at high speed may be achieved, but for this
method high inlet pressures are a necessity. Many people, when speaking of
liquid Chromatographie systems, are referring only to ion exchange chro-
matography such as is used in the amino acid analyzer or the nucleic acid
analyzer. However, here the term liquid chromatography will be used in the
1
2 1. INTRODUCTION
broader sense and will include liquid-liquid (partition), liquid-solid (ad-
sorption), and gel permeation as well as ion exchange chromatography.
All types of chromatography are based on the phenomenon that each
component in a mixture interacts differently with its environment from other
components under the same conditions. Basically, chromatography is a
separation technique, a differential migration process where the sample
components are selectively retained by a stationary phase. It involves sepa-
ration due to differences in the equilibrium distribution of sample com-
ponents between two immiscible phases: a moving or mobile phase and a
stationary phase. The sample components must be in the mobile phase to
migrate through the Chromatographie system. Since the velocity of migra-
tion is a function of equilibrium distribution, the components which favor
the stationary phase migrate slower than those whose distributions favor the
mobile phase. Therefore, separation of components is caused by different
velocities of migration as a result of differences in equilibrium distributions.
Chromatographie methods are classified either according to the type of
mobile and stationary phases utilized or according to the mechanism of
retention. In referring to a specific type of chromatography, both the mobile
and the stationary phases are stated, with the moving phase always referred
to first. For example, in gas-liquid chromatography, the mobile phase is a
gas and the stationary one a liquid. Even though the liquid phase is bonded
to a stationary support, it is still referred to as a liquid stationary phase. In
the literature, however, if only one phase is mentioned, the mobile phase is
used, such as the shortened terms, gas chromatography or liquid chromatog-
raphy. In the first case the moving phase is a gas and in the second a liquid.
The branches of chromatography are outlined in Fig. 1-1.
CHROMATOGRAPHY
GAS CHROMATOGRAPHY LIQUID CHROMATOGRAPHY
Ί I T~h I
GLC
GSC LLC LSC ION MOLECULAR
I I EXCHANGE SIEVING
PC TLC
GEL GEL
PERMEATION FILTRATION
Fig. 1-1. Branches of chromatography.
/. Description 3
Therefore, liquid chromatography is that Chromatographie process in
which the moving phase is a liquid which percolates over a stationary bed. If
an open tube or column is used and the technique performed in vertical
columns by gravity flow, the method is referred to as classical or conven-
tional column chromatography. Liquid chromatography can also be per-
formed on an open bed, as in paper or thin layer chromatography. High
pressure liquid chromatography is a liquid Chromatographie technique in
which the solution is pumped through the column at pressures up to and
sometimes exceeding 4000 psi inlet pressure. The stationary phase may be
solid or liquid, but must have a large surface area. If the stationary phase is
solid, the technique is called adsorption chromatography. If the stationary
phase is a liquid, the sample is partitioned between the stationary and mobile
liquid. This process is referred to as liquid-liquid or partition chromatog-
raphy. Normally, the stationary liquid is polar and the mobile liquid non-
polar. This combination is most commonly used. The liquid Chromatographie
method using a nonpolar stationary phase and a polar mobile phase is termed
reversed phase partition chromatography and it has been found useful in
many applications. If the stationary phase is a highly polar, ion exchange
resin, the procedure is referred to as ion exchange chromatography. The
Chromatographie separation takes place by an exchange of ions between the
mobile phase and the resin. Compounds having different affinities for the
resin will be retained on the column for different periods of time.
Another class of liquid chromatography known as molecular sieving,
exclusion chromatography, or gel permeation chromatography (GPC) in-
volves the mechanical sorting of molecules based on the size of the molecules
in solution. Separation is achieved with a porous packing gel which is com-
patible with the mobile solvent.
A. LIQUID-LIQUID CHROMATOGRAPHY (LLC)
In solution or partition chromatography, the sample is partitioned be-
tween the mobile and stationary liquids. The main requirements for this type
of chromatography are that the solutes must be soluble in the mobile phase
and that the mobile liquid not be a solvent for the stationary phase. An
example of high pressure liquid chromatography in which the stationary
phase is a liquid is illustrated in Fig. 1-2A in which the solid support is coated
with 2.0% β,β'-oxydiproprionitrile. Figure 1-2B illustrates reversed phase
partition chromatography. It is interesting to note that one of the most
widely used subgroups in LLC is paper chromatography in which the more
polar stationary phase is held on the cellulose fibers. The mechanism, how-
ever, is not just partition, but more complex.
4 1. INTRODUCTION
i ' i ' i —
0 60 120
TIME (SEC)
(A)
Fig. 1-2. (A) Partition chromatography (liquid-liquid). Instrument: Waters ALC-201
liquid Chromatograph. Column: 50 cm x 2.3 mm i.d.; packed with 37-50 ^m Corasil II. Liquid
phase: 2% /i,/i'-ODPN. Eluent: «-heptane. Flow rate: 3.0 ml/min. Sample: 5 μ\ aniline and
o-aminoaniline. (Contributed by Waters Associates, Inc.)
OH
°V^ 0 *& OH
(B)
Fig. 1-2. (B) Reversed phase liquid-liquid chromatography. Instrument: Varian 4100
liquid Chromatograph. Column: 75 cm x 2.4 mm i.d.; packed with Permaphase ODS. Eluent:
10% MeOH/90% H0. Flow rate: 30 ml/hr. Pressure: 130 psi. Chart: 20 in/hr. Separation: 2μ\.
2
Samples: (1) 0.2 mg adrenosterone; (2) 0.3 mg 19-nortestosterone; (3) 0.4 mg 4-androstene-
3,17-dione; (4) 0.5 mg testosterone. (Contributed by Varian Associates 1971).