The Chromatography of Steroids

457 Pages·1961·8.047 MB·English

by  I. E. Bush, R. K. Callow, P. N. Campbell and S. P. Datta (Auth.)

OTHER TITLES IN THE SERIES ON PURE AND APPLIED BIOLOGY
BIOCHEMISTRY DIVISION
Vol. 1. PITT-RIVERS AND TATA— The Thyroid Hormones
Vol. 3. ENGEL—Physical Properties of Steroid Hormones
BOTANY DIVISION
Vol. 1. BOR—Grasses of Burma, Ceylon, India and Pakistan
Vol. 2. TURRILL (Ed.)—Vistas in Botany
Vol. 3. SCHULTES—Native Orchids of Trinidad and Tobago
Vol. 4. COOKE—Cork and the Cork Tree
MODERN TRENDS IN PHYSIOLOGICAL SCIENCES DIVISION
Vol. 1. FLORKIN—Unity and Diversity in Biochemistry
Vol. 2. BRÄCHET—The Biochemistry of Development
Vol. 3. GEREBTZOFF—Cholinesterases
Vol. 4. BROUHA—Physiology in Industry
Vol. 5. BACQ AND ALEXANDER—Fundamentals of Radiobiology
Vol. 6. FLORKIN (Ed.)—Aspects of the Origin of Life
Vol. 7. HOLLAENDER (Ed.)—-Radiation Protection and Recovery
Vol. 8. KAYSER— The Physiology of Natural Hibernation
Vol. 9. FRANÇON—Progress in Microscopy
Vol. 10. CHARLIER—Coronary Vasodilators
Vol. 11. GROSS—Oncogenic Viruses
Vol. 12. MERCER—Keratin and Keratinization
Vol. 13. HEATH—Organophosphorus Poisons
Vol. 14. CHANTRENNE—The Biosynthesis of Proteins
Vol. 15. RIVERA—Cilia, Ciliated Epithelium and Ciliary Activity
Vol. 16. ENSELME—Unsaturated Fatty Acids in Atherosclerosis
PLANT PHYSIOLOGY DIVISION
Vol. 1. SUTCLIFFE—Mineral Salts Absorption in Plants
Vol. 2. SIEGEL— The Plant Cell Wall
ZOOLOGY DIVISION
Vol. 1. RAVEN—An Outline of Developmental Physiology
Vol. 2. RAVEN—Morphogenesis: The Analysis of Molluscan Development
Vol. 3. SAVORY—Instinctive Living
Vol. 4. KERKUT—Implications of Evolution
Vol. 5. TARTAR—Biology of Stentor
Vol. 6. JENKIN—Animal Hormones
Vol. 7. CORLISS—The Ciliated Protozoa
Vol. 8. GEORGE—The Brain as a Computer
Vol. 9. ARTHUR— Ticks and Disease
Vol. 10. RAVEN—Oogenesis
Vol. 11. MANN—Leeches (Hirudinea)
THE CHROMATOGRAPHY
OF STEROIDS
BY
I. E. BUSH
M.A., PH.D., M.B., B.CH. (Cantab.)
BOWMAN PROFESSOR OF PHYSIOLOGY
THE UNIVERSITY OF BIRMINGHAM
PERGAMON PRESS
OXFORD · LONDON · NEW YORK · PARIS
1961
PERGAMON PRESS LTD.
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Copyright
©
1961
PERGAMON PRESS LTD.
Library of Congress Card Number 61-9097
Set in Imprint 11 on 12 pt. and printed in Great Britain by
J. W. ARROWSMITH LTD., BRISTOL
TO MY FATHER
who, after all, started me off
ACKNOWLEDGEMENTS
I am grateful to the Editors of the Biochemical Journal for
permission to publish Figs. 3.2,5.3 and 6.11 : to the Editors of
Helvetica Chimica Ada and Dr. R. Neher for permission to
publish Figs. 3.22 and 3.23 : to the Editors of the Journal of
Endocrinology for permission to publish Figs. 3.3 and 4.3 :
and to Messrs. J. & A. Churchill, Ltd., London, and Pro-
fessor C. J. O. R. Morris for permission to publish Fig. 3.8a.
I am also grateful to the following for many useful dis-
cussions, and for their generosity in providing unpublished
material in advance or for drawing my attention to special
features of their publications. Any errors of interpretation
are, however, my own responsibility.
Dr. W. Acklin, Dr. H. Dowlatabadi, Dr. L. L. Engel,
Dr. A. Izzo, Dr. A. T. James, Dr. E. H. Keutmann, Dr.
W. Klyne, Dr. M. Lewbart, Dr. S. Lieberman, Dr. A. G.
Long, Professor G. F. Marrian, F.R.S., Dr. R. Neher, Dr.
R. Peterson, Professor U. Prelog, Professor T. Reichstein,
Dr. L. Reineke, Dr. J. J. Schneider, Dr. & Mrs. J. Tait,
F.R.S., Dr. P. Vestergaard, Dr. L. I. Woolf, and Dr.
A. Zaifaroni.
PREFACE
THERE are now many books on chromatography and no excuse is needed
for most of them, dealing as they do with a technique that has revolution-
ized biochemistry. On the other hand, scientists are in danger of being
overwhelmed by the modern plethora of papers and books on increasingly
specialized and esoteric subjects, so that I feel slightly guilty at having
accepted the invitation to write the following chapters which are devoted
largely to one rather special field of application of Chromatographie
methods. My main excuse for doing so is that I have been struck for a
long time by two features of this field which are probably both largely due
to a similar underlying cause: first the small extent to which most practi-
tioners of chromatography in the steroid field have drawn upon the general
theory and background of partition chromatography which was built up
with other families of organic compounds; and second the negligible
extent to which those interested in the general theory of Chromatographie
behaviour have realized that in many ways the steroids represent an ideal
group to study. As a physiologist fumbling my way into the steroid field
and forced to work out my own methods it took me six months to realize
the force of the first aspect; having broken the back of the technical
problem however, the second aspect was lost to view among the many
interesting applications that presented themselves and, because of which,
indeed, the original work had been done. This is probably true of many
biochemists; Chromatographie methods for particular classes of com-
pound had to be worked out primarily in order to tackle problems which
had to be solved. Few had the leisure to enquire into methodology any
deeper than was necessary to obtain or copy a workable method.
Therefore, while I hope that this book may be of interest and practical
use to those who actually use Chromatographie methods in their work
with steroids, its principal aim is to try and remedy the situation described
above. Thus the steroid specialist may at first be surprised by references
to work on fatty acids, flavonoids, sugars and amino acids, which often
demonstrate general principles underlying technical details of work with
steroids far better than the techniques for steroids themselves. Again,
the best example of a general rule must in some cases be drawn from
XI
Xll PREFACE
gas-liquid systems although the latter cannot as yet be used for work with
the majority of steroids.1
In order to preserve a reasonably continuous argument a large number of
topics have been relegated to the Appendices. A further problem was the
enormous extent of the literature and the vast number of different com-
pounds to be considered. If all the compounds, solvent systems, conditions
of operation and RF values were to be tabulated in the usual way over
100,000 items would have had to be listed. Furthermore, such a list would
be of limited use because many of the data, although useful in themselves
are not sufficiently accurate to be compared with data from other labora-
tories. I have attempted to solve this problem by giving a general table of
values from which the RM and hence the RF value of almost any steroid
can be calculated for the aqueous methanol systems (Appendix IV). This
together with the information given in Chapter II should enable anyone to
work out the approximate RF value of a new steroid in other types of
solvent systems with reasonable accuracy when the RF values of a few
standard steroids under local conditions are known. For similar reasons
I have not attempted to cover the whole field descriptively but have drawn
examples mainly from the fields I know best in order to illustrate general
principles. It is not unfair to observe, however, that these fields do in fact
provide the most complete set of results at present on which to base a
general theory. Those who have attempted to assemble Ai?M values from
the literature will realize how often an apparently voluminous set of Rp
values may yield very few reliable ARM values, because one or two key
compounds have not been run in the solvent system under consideration
for reasons of practical convenience.
It is easy to become too scholastic over theories of methodology but it
is equally easy to become the slave of empirical details because of the
absence of a useful general theory. Those looking for cut-and-dried
recipes in this book will be disappointed, but I hope that they will be able
to overcome any resistance they may have to a somewhat theoretical
treatment and be stimulated to apply some of its ideas in practice.
I would like to take this opportunity of expressing my gratitude to those
who have supported my own work in this field, particularly to Professor
E. D. (now Lord) Adrian, o.M., F.R.S., Sir Charles Harington, F.R.S.,
Sir George Pickering, F.R.S., F.R.C.P., the Medical Research Council, the
Commonwealth Fund and St. Mary's Hospital Medical School. I also
1 See p. 328.
PREFACE xiii
benefited greatly from the hospitality of Dr. L. T. Samuels (Salt Lake
City, Utah) and Dr. L. L. Engel (Boston) and from the early encourage-
ment and advice of Professor C. W. Shoppee, F.R.S., Dr. C. S. Hanes, F.R.S.,
and the late and unique Dr. Konrad Dobriner. Perhaps my greatest debts
however are to Dr. P. R. Lewis (Cambridge) who so tactfully guided my
early footsteps and made me appreciate the value and ubiquity of theory:
and to my early supervisor and present editor, Dr. R. K. Callow, F.R.S.,
for his invariably timely and objective intolerance.
GLOSSARIES
1. SYMBOLS AND TERMS
SOME symbols and terms which are commonly used in discussing chroma-
tography are given below, but many of the more fanciful neologisms and
some well-established terms have been omitted.
Adsorption—The excessive concentration of a solute at an interface. In
chromatography the interface is most commonly that between a liquid or a
gas and a finely divided solid.
Ascending chromatography—Commonly refers to one of the two principal
ways of running paper chromatograms, namely, that in which the paper
dips from above into the mobile solvent phase which ascends through the
paper by capillary forces.
Descending chromatography—Commonly refers to the other principal
method of running chromatograms in which the paper hangs from a trough
containing the mobile phase which passes down the paper by reason of both
capillary and gravitational forces.
Development—Commonly used in adsorption chromatography to refer to
the percolation through a column of a solvent which produces movement
of the solutes adsorbed on the column, as distinct from the original solvent
with which the solutes were transferred to the column. It is sometimes
used in connexion with partition chromatography simply to mean the
running of the chromatogram, but it should be avoided because of its
other uses such as to mean the treatment of paper chromatograms with
reagents giving colour reactions.
Displacement—A change in distribution of a solute between two phases
which is produced by the addition of another solute. Displacement in
adsorption chromatography is due to competition between the displaced
and the displacing solutes for adsorptive points on the solid stationary
phase. Displacement conditions are recognized by the emergence of the
solute suddenly from the column as a relatively sharp front followed by a
constant concentration of the solute in the effluent until the displacer
emerges. The same mechanism however is partially responsible for some
XIV
GLOSSARIES XV
types of chromatography in which this characteristic of displacement
chromatography is not observed.
Effluent—The liquid or gas emerging from a Chromatographie system.
With paper chromatograms this is often referred to as the "run-off".
Eluate—The solution of a substance which is obtained by allowing a
suitable solvent to percolate through a porous medium which originally
contained the solute. This is commonly used in connexion with fragments
cut from paper chromatograms, but is also used to refer to fractions of
effluents and not infrequently as a substitute for effluent itself.
Eluent—The solvent used to obtain an eluate. It thus applies to Chromato-
graphie solvents proper, as well as to solvents used to extract fragments
of paper, etc.
Elution—The removal of a solute from a porous medium by allowing a
suitable solvent to percolate through it. This and the verb "to elute" are
used for the process of removing solutes from a Chromatographie column
in the course of chromatography proper, as well as for the extraction of
small segments of a chromatogram.
Gradient elution—A method of running chromatograms in which the
concentration of one or more components of the solvent system is changed
gradually rather than in discontinuous steps.
Interstitial volume—The volume of fluid or gas in the interstices of a
Chromatographie system. This usually means the volume of the mobile
phase within the system, but is sometimes used to mean the total volume
of the column minus the volume of the supporting material : in this case it
equals the sum of the volumes of the stationary and mobile phases if a
partition system is under consideration. The latter use is avoided in this
book.
Ion-exchange—A process whereby ions of the same charge sign replace
one another in a given phase. In chromatography, the term usually refers
to systems in which the stationary phase is made up of an ionic polymer.
This can either be a synthetic resin or a mineral material such as the
zeolites and specially treated alumina.
Mobile phase—The moving phase of a Chromatographie system, gas or
liquid.
Stationary phase—The stationary phase of a Chromatographie system.
This is made up of the surface of an adsorbent or the liquid or gel held by