Practical aspects of modern high performance liquid chromatography : proceedings, December 7-8, Berlin (West)

465 Pages·1983·10.575 MB·English

by  Imre Molnár

Practical Aspects of
Modern HPLC

Practical Aspects
of Modern High Performance
Liquid Chromatography
Proceedings
December 7-8,1981 · Berlin (West)
Editor
Imre Molnar
Walter de Gruyter· Berlin · NewYork1983
Editor
Imre Molnar, Dr. rer. nat.
Institute for Applied Chromatography
Blücherstrasse 22
D-1000 Berlin 61
Germany
This Symposium was organized by
Wissenschaftliche Gerätebau Dr. Ing. Herbert Knauer GmbH, Berlin.
CIP-Kurztitelaufnahme der Deutschen Bibliothek
Practical aspects of modern high performance liquid
chromatography: proceedings, December 7-8,1981,
Berlin (West) / [organised by Wiss.-Gerätebau-Dr.-Ing.-
Herbert-Knauer-GmbH, Berlin].
Ed. Imre Molnar. - Berlin; New York: de Gruyter, 1983.
ISBN 3-11-008892-4
NE: Molnar, Imre [Hrsg.]; Wissenschaftliche-Gerätebau-Doktor-
Ing.-Herbert-Knauer-GmbH <Berlin, West>
Copyright © 1982 by Walter de Gruyter & Co., Berlin 30.
All rights reserved, including those of translation, into foreign languages. No part of this book
may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted
nor translated into a machine language without written permission from the publisher. Printing:
Karl Gerike, Berlin - Binding: Dieter Mikolai, Berlin. - Printed in Germany.
Preface
This volume consists of the publication of selected papers, presented at
the Symposium on "Practical Aspects of Modern High Performance Liquid
Chromatography (HPLC)", organized by Dr.-Ing. Η. Knauer GmbH, held on
December 7th and 8th, 1981 at Schering AG, Berlin (West). The papers
assembled herein cover a range of topics of interest to those involved
in analytical work using High Performance Liquid Chromatography (HPLC).
In contrast to Gas Chromatography, where the mobile phase plays no part
in the retention process, in HPLC the mobile phase plays a dominant role
in separation. In HPLC, optimization of retention and resolution is brought
about by slightly changing the composition of the mobile phase. On the
other hand the stationary phase is rather inactive, especially in "Rev-
ersed Phase Chromatography" (RPC).
This book' demonstrates the dominance of RPC, - this applies to 90 % of all
separation problems. After having used this method for many years, this
technique has become the major analytical tool in research and in routine
laboratory work. RPC is now used not only in biochemistry and genetic
engineering but also in new areas such as the inorganic chemistry of non-
metals, i. e. sulfur, phosphorous, and their compounds. Affinity-HPLC with
bonded silica gels provides another new area of research. Success has also
been achieved with the RPC of membrane proteins.
I would like to sincerely thank those who made this meeting possible: the
authors, Dr.-Ing. Η. Knauer GmbH for their support and Schering AG, Berlin
(West) for their hospitality.
Berlin, October 1982 I. Molnar

CONTENTS
Effect of mobile phase composition on the retention behavior of
oligonucleotides in reversed phase chromatography
Z. El Rassi, C. Horvath 1
Quantitative structure retention relationships for oligonucleotides
J. Jacobson, Ζ. El Rossi, C. Horvath 15
Spherical and irregular silica. Does shape influence the selectivity?
H. Müller, Η. Engelhardt 25
The current role of HPLC for the routine analysis of endogenous
compounds in clinical chemistry
M. Schöneshöfer, I. Molnar 41
Analysis of unconjugated cortisone and 6ß-0H-cortisol in human urine
by high performance liquid chromatography
B. Weber, M. Schöneshöfer 63
High performance liquid chromatography of proteines on reversed phase
examplified with human interferon and other proteines: Review and
scope of the method
H. J. Friesen 77
HPLC of membrane bound proteins
Dj.. Josic, W. Reutter, I. Molnar 109
Separation of proteins by size exclusion and reversed phase high
pressure liquid chromatography
W. Schwarz, J. Born, H. Tiedemann, I. Molnar 123
High performance liquid chromatography as applied to the studies
of the fibrinogen structure
M. Kehl, F. Lottspeich, A. Henschen 137
The HPLC of divalent sulfur
J. Möckel, Τ. Freyholdt, J. Weiss, I. Molnar 161
Aspects of affinity chromatography in HPLC
K. Buchholz, A. Borchert, V. Kasche 187
The role of HPLC in pharmakokinetics
W. Krause 197
Improvement of column performance in HPLC using a special inlet port
W. Lamer, I. Molnar 213
Dissoluti on rate determination of low dose oral contraceptives using
automated HPLC, with column switching technique
A. Hiihn 227
VIII
Practical aspects of the routine determination by HPLC of free
noradrenaline and adrenaline in urine and plasma
K. P. Kringe, B. Neidhart, Ch. Lippmann 241
Separation of catecholoestrogens and their monomethyl ethers by
reversed phase HPLC with ternary mobile phase
E. Kraas, M. Schütt, E. Zietz, R. Knuppen 275
Quantitative determination of aryloxypropanolamines in plasma
and organs of the rat by ion-pair reversed phase HPLC
H. Winkler, B. Lemmer 293
HPLC-analysis of estrogen-active anabolica in meat with the estro-
gen-receptor-test as specific detection system
H. G. Grohmann, W. J. Stan 307
Methods of detection in modern HPLC
rt. Baumann 315
Fluorimetric determination of drugs in biological materials by
means of high performance liquid chromatography
P. Haefelfinger 335
Rapid determination of sodium gluconate and glucose in fermentation
fluids
R. Lenz, G. Zoll 355
Retention in practical HPLC
I. Molnar
363
The selection of optimal conditions in HPLC
III. Practical aspects of low volume, small bore, packed
columns in HPLC
Η. H. Lauer, G. P. Rozing 409
List of Symbols used in the Text
435
Chromatograms 439
Author Index
Subject Index
443
EFFECT OF MOBILE PHASE COMPOSITION ON THE RETENTION BEHAVIOR OF OLIGO-
NUCLEOTIDES IN REVERSED PHASE CHROMATOGRAPHY*
Ziad El Rassi and Csaba Horväth
Department of Chemical Engineering, Yale University, New Haven,
Connecticut 06520 USA
Introduction
High performance liquid chromatography (HPLC) has extensively been used
for the separation of oligonucleotides and their derivatives of various
types. Fully protected species were chromatographed on cyano-silica (1)
with dichloromethane which contained a few percent methanol. For chroma-
tography of unprotected oligonucleotides, the classical technique has been
ion exchange chromatography. Anion exchange resins or DEAE-cellulose were
used for the separation of relatively small species (2-4), whereas liquid
anion exchangers such as RPC-5 were employed to separate large oligonucleo-
tides (5-7). Improved resolution was achieved by using microparticulate
siliceous-anion exchangers, such as Partisil 10-SAX (2). Size exclusion
chromatography was also used in the separation of short unprotected oligo-
nucleotides (8). Recently reversed phase chromatography with ocadecyl-
silica as the stationary phase has emerged as an eminently efficient
method for separation of protected and unprotected olgionucleotides having
a wide range of molecular weight and structure (9-11). Thus, reversed
phase chromatography has become the prime technique of separating not only
peptides (12) but also oligonucleotides.
The goal of this study is to examine the effect of eluent composition as
far as pH, organic co-solvent and the nature of the buffer ions is concern-
ed on the retention behavior of oligonucleotides and thus to faciliate the
selection of optimum conditions for their separation.
* VzcU caXtd to PAO^eA-ioA. George Μ anecke, {,ολ. (ιίύ 4-ixtu faßth bifithdo.y.
Practical Aspects of Modern HPLC
Copyright © 1982 by Walter de Gruyter &. Co., Berlin · New York
Printed in Germany