Practical Aspects of Modern High Performance Liquid Chromatography: Proceedings, December 7-8, 1981, Berlin (West) [Reprint 2011 ed.] 9783110845082, 9783110088922

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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-DoktorIng.-Herbert-Knauer-GmbH

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 "Reversed 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 nonmetals, 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 Krause role of HPLC in pharmakokinetics W. Improvement of column performance in HPLC using a special inlet port W. Lamer, I. Molnar Dissoluti on rate determination of low dose oral contraceptives using automated HPLC, with column switching technique A. Hiihn

197 213 227

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 estrogen-receptor-test as specific detection system H. G. Grohmann, W. J. Stan Methods of detection in modern HPLC rt. Baumann

307 315

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 OLIGONUCLEOTIDES IN REVERSED PHASE CHROMATOGRAPHY*

Ziad El Rassi and Csaba Horväth Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520 USA

Introduction High performance l i q u i d 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 c l a s s i c a l technique has been ion exchange chromatography.

Anion exchange resins or DEAE-cellulose were

used for the separation of r e l a t i v e l y small species ( 2 - 4 ) , whereas l i q u i d anion exchangers such as RPC-5 were employed to separate large oligonucleotides (5-7).

Improved resolution was achieved by using microparticulate

s i l i c e o u s - a n i o n exchangers, such as P a r t i s i l 10-SAX (2).

Size exclusion

chromatography was also used in the separation of short unprotected o l i g o nucleotides (8).

Recently reversed phase chromatography with ocadecyl-

s i l i c a as the stationary phase has emerged as an eminently e f f i c i e n t 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 i s to examine the effect of eluent composition as far as pH, organic co-solvent and the nature of the buffer ions i s concerned on the retention behavior of oligonucleotides and thus to f a c i l i a t e the selection of optimum conditions for their separation. * VzcUcaXtd to PAO^eA-ioA. George Μ anecke, {,ολ. (ιίύ 4-ixtu

Practical Aspects of Modern H P L C Copyright © 1982 by Walter de Gruyter &. Co., Berlin · New York Printed in Germany

faßth

bifithdo.y.

2 Experimental Materials.

(Ap) 2 A, (Ap)3A and (Ap)4A as well as (Up) 2 U, (Up) 3 U,

and

( U p K U were purchased from PL Biochemicals (Milwaukee, Wis., USA). was from ICN Pharmaceuticals (Cleveland, Ohio, USA).

S -UMP

5 -CMP, 5 - AMP and

5 - G M P were obtained from Schwarz/Mann (Orangeburg, NY, USA).

All other

mono- and oligonucleotides used in this study were purchased from Sigma (St. Louis, MO, USA).

The names and abbreviations of mono- and oligo-

nucleotides studied are listed in Table I.

Tri ethyl amine, H 3 P0 lt , NaH 2 P0 4 ,

Na 2 HP0 u , CH3COOH, CH 3 C00Na, ^ S O ^ , acetonitrile and methanol (both HPLC grade).isooctane and CCI4 were supplied by Fisher (Pittsburgh, PA, USA). Distilled water was prepared with a Bransted distilling unit. Instruments and Columns.

The liquid Chromatograph consisted of a Model LC

250/1 pump Kratos-Schoeffel (Westwood, NJ, USA),Model 7010 sampling valve with a 20yl loop and a Model 770 variable wavelength UV-detector (KratosSchoeffel).

Chromatograms were obtained with a Schlumberger (Benton-Harbor,

MI, USA) Model SR-204 strip chart recorder. tored at 260 nm.

The column effluent was moni-

The column was kept at room temperature.

Octadecyl-

Spherisorb was prepared from 5-pm Spherisorb silica according to Koväts et at. (13) and the carbon load of the stationary phase was 14% w/w.

Some

experiments were carried out with a column packed with 10-um LiChrosorb RP-18.

The dimensions of the octadecyl-Spherisorb and the LiChrosorb

RP-18 columns were 150 χ 4.6 mm and 250 χ 4.6 mm, respectively.

Columns

were packed by using a slurry of the column material in CC1 4 that was pumped with isooctane into the column (14). Procedures.

The effect of pH on retention was studied by using 0.1 Μ

sodium phosphate buffer at pH 2.12-3.0 and 6.0-7.0 as well as 0.1 Μ sodium acetate buffer at pH 4.0-5.5. phate was used.

In some experiments tri ethyl ammonium phos-

In all experiments the ionic strength was maintained

constant by addition of Na2S0i+.

Sodium nitrate was used to measure the

mobile phase hold-up time on the assumption that with eluents containing electrolytes this ionized tracer is not excluded.

3 Table I.

List of mono- and oligonucleotides investigated in this study.

Abbreviation 5' -CMP 5'-AMP 5' -GMP 5' -UMP 3' -CMP 3'-AMP 3'-GMP 3'-UMP ApA ApC ApG ApU CpA CpC CPG CpU GpA GpC GpG GpU UpA UpC UpG UpU (Ap)2A (Ap) 3 A (Ap) 4 A (Up)2U (Up)3U (Up)4U ApApC ApApG ApApU ApCpC ApGpU ApUpG ApUpU

Name Cytidine-5' -monophosphate Adenosine-51-monophosphate Guanosine-51 -monophosphate Uridine-5' -monophosphate Cytidine-3' -monophosphate Adenosine-3'-monophosphate Guanosine-3' -monophosphate Uridine-3' -monophosphate Adenylyl (3'->-5' )adenosine Adenylyl (3' -+51 )cytidine Adenylyl (3'->-5') guanosine Adenylyl (3*->-5')uridine Cytidylyl (3' ->5' ) adenosine Cytidylyl (3' ->5' )cytidine Cytidylyl (31 ->-5' ) guanosine Cytidylyl (3' ->5' )uridine Guanylyl (3* ->5' ) adenosine Guanylyl (31->-5') cytidine Guanylyl ( 3 ' ) g u a n o s i n e Guanylyl (3'->5')uridine Uridylyl (3' ->5' ) adenosine Uridylyl(3· ->-5' )cytidine Uridylyl (31-+5')guanosine Uridylyl (3' ->-5> )uridine (Adenylyl (3'->5' )) 2 adenosine (Adenylyl (3' ->5' )) 3adenosine (Adenylyl (3* ->5' )) ^adenosine (Uridylyl (3' -+51 )) 2 uridine (Uridylyl (3' ->5' )) 3 uridine (Uridylyl (3' ->5' )) 4 uridine Adenylyl (31 ->5' ) adenylyl (3' ->5' ) cytidine Adenylyl (3'->-5' )adenylyl (3'->-5' )guanosine Adenylyl (3' ->5' )adenylyl (3'-»-51 juridine Adenylyl (3' ->-5' ) cytidylyl (3' -+51 ) cytidine Adenylyl (3'-^5' )guanylyl (3* ->5' )uridine Adenylyl (3' ->5* )uridylyl (3' )guanosine Adenylyl (3'-»-5' )uridylyl (31 -»-51 )uridine

4 Results and Discussion Effect of pH.

In reversed phase chromatography the retention of ionogenic

species such as mono- and oligonucleotides is dependent on the pH of the eluent and the acid dissociation constant of the eluites (15).

In order

to assess the effect of eluent pH on the retention of oligonucleotides experiments were performed with sixteen dinucleotides

(diribonucleoside

3' ->5' monophosphates) and the major monoribonucleotides by using a column packed with octadecyl-Spherisorb.

The eluent contained either 0.1 Μ sodium

phosphate or acetate and, in the case of dinucleotides, also 5% v/v of acetonitrile.

The ionic strength was 0.34 in all experiments.

The interpretation of the results requires the knowledge of the pertinent acid dissociation constants.

The pK

values of some 3'- and 5'-nucleotides a are presented in Table II and it is seen that the pK a of the amino groups

Table II. pK values of ribonucleoside monophosphates. The data were taken from Ref. 16 except those with an asterisk which are from Ref. 17. PKai a

Nucleotide

PKa a

l

primary phosphate

PKa, a

2

amino group

3

secondary phosphate

5'-CMP

-

4.5

6.3

5' -AMP

-

3.8

6.2-6.4

5' -GMP

0.70*

2.4

6.1

5'-UMP

1.02*

-

6.4

3' - CMP

0.80*

4.3

6.04

3' - AMP

0.89*

3.6-3.7

5.92

3' -GMP

-

2.21

5.92

3' -UMP

-

-

5.88

is slightly higher for nucleoside-5'-phosphates than nucleoside-3'-phosphates (18).

This is expected because 51 - phosphate is located closer to

the amino group than the 3'-phosphate in such molecules (19). nucleotides studied here, of. available.

For oligo-

Table I, such data is unfortunately not

These compounds do not have e terminal phosphate, only a

5 phosphate bridge in the position 3'-+5', and we may assume that the pK

a value of this group is approximately one as that of primary phosphates in nucleotides.

In most cases the amino groups in the oligonucleotide mole-

cules are expected to have pK a values similar to those of the amino functions in the corresponding nucleoside-3'- or-5'-monophosphates. Retention factors of 3'- and 5'-nucleotides, the constituents of the oligonucleotides, were measured at pH 3.0 and 5.0 and the results are shown in Table III.

It is seen that 5'-nucleotides are less retained at both pH

than 3'-nucleotides under otherwise identical conditions.

This may be ex-

Table III. Retention factors of 3'- and 5'^nucleotides at two different pH. The eluents used were 0.1 Μ sodium phosphate (pH 3.0) or acetate (pH 5.0) buffers of the same ionic strength, 1=0.34.

Nucleotide

pH 3.0 k'

51 -CMP 5' -AMP 5' -GMP 5» -UMP 3' -CMP 3' -UMP 3' -AMP 3'-GMP

0.95 2.45 3.10 3.50 2.10 3.50 5.55 7.00

plained by the slightly lower pK that in 5'-nucleotides.

pH 5 k' 0.60 5.00 1.50 1 .90 1.45 1.90 14 .40 3.70

of the amino group in 3'-nucleotides than

Moreover, the nonpolar surface for binding in

reversed phase chromatography is expected to be smaller with 5'-phosphates than with 3'-phosphates as the ionized phosphate in the 5' position may shield the methylene group of the ribose moiety. In agreement with the results by Zakaria et al.

(20) 5'-nucleotides are

retained longer at pH 3.0 than at pH 5.0, except for 5'-AMP which has higher retention factor at pH 5.0. leotides is similar as shown in Table III.

Retention behavior of 3'-nucThe faster elution of both the

3'- and 5'-monophosphates of guanosine and uridine at pH 5.0 than at pH 3.0 is believed to be due to the dissociation of the secondary phosphate in this pH range.

The amino group in 3'- and ö'-GMP'shas a pK

of 2.2 and 2.4

6 respectively and therefore is largely undissociated whereas the uridine in 3'-and 5 - UMP'shas no group that is protonated at this pH.

In the case of

3'-and 5'-CMP 's. the retention is expected to increase when the pH of the eluent goes from 3.0 to 5.0 because the electronic charge in the molecule decreases due to deprotonation of the amino group that has a pK 4.3 and 4.5, respectively.

value of a As seen in Table III, however, the retention

decreases instead of increasing.

This anomalous behavior may be due to a

pH mediated conformation change that has been observed by spectroscopic means (21-23) and could result in a lower binding surface area in the molecule with increasing pH of the eluent.

Irregular retention behavior due

to conformation changes in the eluite upon changes in eluent composition have been found in other applications of reversed phase chromatography as well (24).

Whatever is the nature of the conformation change, it affects

retention in such a way that it overcompensates the effect of protonation of the amino group.

The increase in the retention of both 3'- and 5'-AMP's

when the pH of the eluent changes from 3.0 to 5.0 as shown in Table III is attributed to deprotonation of the amino group in the adenosine moiety that has a pK of 3.6 and 3.8, respectively, a Retention factors of dinucleosides3 1 -»• 5'monophosphates obtained in the pH range from 2.0 to 7.0 is shown in Figs. 1 and 2.

Retention of all oligo-

nucleotides containing adenosine increases with the pH in the range investigated.

Since the pK

value of the phosphate bridge in the dimer is

around 1, this moiety is almost fully dissociated at pH 3.0.

Therefore,

the retention of the dimers in the pH range investigated should depend mainly on the protonation of the amino group in the nucleoside moiety. Indeed, the observed retention behavior can be readily interpreted if the amino group in the adenosine moieties of ApA has the same respective pK values as that in 3'- and 5'-AMP's, i.e., 3.6 and 3.8.

a As the ionization

constant of the amino group in 3'- and 5'-GMP's is smaller than that of the corresponding adenylic acids, the dependence of the retention factor of GpA and ApG on the eluent pH is essentially determined by the protonation and deprotonation of adenosine moiety in the mixed dimers.

As uridine has

no ionogenic functions in the pH range investigated, the retention of UpA and ApU is controlled by the change in the charge on the amino group of the adenosine moiety.

In the case of CpA and ApC the retention behavior is

7

pH Fig. 1. Plots of the retention factor of dinucleotides against the pH of the mobile phase. Eluent: 0.1 Μ sodium phosphate or acetate buffer containing 5% v/v acetonitrile.

Fig. 2. Plots of the retention factor of dinucleotides against the pH of the mobile phase. Conditions are the same as in Fig. 1.

8 governed by the adenosine moiety since it is more hydrophobic than the cytidine moiety.

The inflection point of the sigmoidal curves in Figs

1 and 2 for all dinucleotides containing adenosine is located at pH 3.84.0 which correspond to the pK

a

value of the adenosine moiety in the mixed

dimers. Dinucleotides containing guanosine but no adenosine exhibit slight increase in retention when the pH of the eluent increases from 2.0 to 3.0 as illustrated in Fig. 2.

The observed behavior is expected from the deprotona-

tion of the guanosine moiety in this pH range which has a pK

a

of approxi-

mately 2.2. Retention behavior of dinucleotides containing uridine should in pH range 2.0 to 7.0 depend only on the dissociation of the other residue because the uridine moiety is essentially neutral and the phosphate group is fully dissociated.

Indeed as seen in Fig. 2 the retention of UpU does not change

significantly, but the retention of UpA and ApU increases with the pH of the eluent and the retention of GpU slightly increases when the pH from 2.0 to 3.0.

changes

This difference is attributed to the relatively small

p« a value of the amino group in the guanosine moiety with respect to that in the adenosine. increasing pH.

The retention of CpU and UpC practically invariant with

This observation is attributed to a pH mediated configura-

tion change similar to that observed with mononucleotides. The relative retention order of mixed dinucleotides over the pH range from 2.0 to 7.0 is A p O C p A , GpC>CpG, GpU>UpG, ApU>UpA, UpC>CpU and ApG>GpA. There is one exception:

GpA>ApG at pH

ΝΙΗΙΤ»

Fig. 4. Combined separation of model proteins. The highmolecular-weight substances transferrin and bovine serum albumin were separated from the low-molecularweight substances on the SEC column; the process is shown in Fig. 4 A, conditions as in Fig. 2. They were then retained on the RP-300 column and eluted. The reversed phase chromatogram is shown in Fig. 4 B, conditions as in Fig. 3.

115

Fig. 5. Combined separation of model proteins. The lowermolecular-weight substances were subject to separation from the high-molecular-weight substances on the SEC column, Fig. 5 A. They were then retained on the RP-300 column and eluted with the gradient described in Fig. 3 - Fig. 5 B. Other conditions as in Figs. 4 A and B. which separate the protein from the membrane matrix. With further purification of the protein mixture and isolation of particular proteins it is often desirable to remove these substances from the mixture. The results of the analyses of the membrane proteins, solubilized by the detergent NP-40 are shown in Fig. 6 according to the molecular size of the proteins. NP-40 has an absorption maximum at 220 nm and can be determined together with proteins. On the LiChrosorb-DIOL column this detergent will be retarded

116

Fig. 6.

Ν Ρ 40

SEC of membrane proteins, which were solubilized with the detergent NP-40; column: Lichrosorb-DIOL from Knauer.

ο

CM CM

Other conditions as in Fig. 2.

0.768 -

0.512 -

0.256 -

ο.ο _L 10

MINUTES

under the given conditions and can be removed from the proteins without any difficulty (cf. also Fig. 6).

117

If the detergent NP-40 is not removed from the protein mixture, it will be washed out by gradient elution on the RP-300 column together with the protein, as shown in Fig. 7. A chromatogram of a protein mixture, from which the detergent has previously been removed on the DIOL-column, is shown in Fig. 8. In order to verify the macromolecular content of this fraction, the eluate from the RP-300 column was evaporated and once more analyzed on the SEC column (Fig. 9). As it can be seen from these data, the molecules proved to be the expected macromolecules, with a maximum of about 70,000 daltons. Corresponding results of analyses of membrane proteins which were solubilized by octyl-glucose are shown in Figs. 10 and 11.

u Fig. 7. RP chromatogram of the membrane proteins which were solubilized with the detergent NP-40. The detergent was not removed from the mixture; conditions as in Fig. 3.

ο

5

10 MINUTES

15

118

MINUTES

1.28 -

Fig. 8.

—·

Τ

RP chromatogram of the same membrane proteins as in Fig. 7. Here the detergent was previously removed on the DIOLcolumn? conditions as in Fig.3

0.6Ί -

Fig. 9. SEC chromatogram of the membrane proteins eluted from the RP column (the experiment concerned is shown in Fig. 8); conditions as in Fig. 6. 0.0

_L 5 MINUTES

119

Fig. 10. RP chromatogram of the membrane proteins which were solubilized with octyl-glucose. The detergent was not removed from the mixture; conditions as in Fig. 3.

10 MINUTES

By the use of octyl-glucose a separation of the highly hydrophobic proteins from the membrane matrix cannot be achieved (cf. Figs. 8 and 10). When the slope of the gradient was lowered from 5 % B/min to 2 % B/min, the separation gave better results. When 0.2 % octyl-glucose was added to both eluents, a further improvement in the separation was observed

substantial

(cf. Fig. 11).

Conclusions

By the results presented in this paper HPLC has been shown to

120

Fig. 11. RP chromatogram of the same membrane proteins as in Fig. 10. Octyl-glucose was previously removed on the DIOL-column (cf. also Fig. 6). Gradient slope 2 % B/min; 0.2 % octyl-glucose were added to both eluents; other conditions as in Fig. 3.

be a rapid and effective tool in the separation of complex protein mixtures such as the membrane proteins. By aqueous SEC the removal of low-molecular-weight substances from the mixture has been achieved. Successive reversed phase chromatography resulted in a specific separation of the higher-molecular-weight substances according to their hydrophobic!ty. Using the column switching technique, a steady and extremely fast control of the molecular size in routine biochemical

121

work has been

achieved.

I m p r o v e m e n t of the s e p a r a t i o n has b e e n m a d e p o s s i b l e by adding a detergent

(0.2 % o c t y l - g l u c o s e

in this case)

to b o t h e l u e n t s

of the g r a d i e n t run.

References 1. W. K r e i s e l , Β.Α. V o l k , R. B ü c h s e i & W . Proc. N a t l . A c a d . Sei. U . S . A . 77, 1828 2. R. T a u b e r , C.-S. Park & W. Eur. J. Cell Biol. 27, 31

Reutter: (1980)

Reutter: (1982)

3. I. M o l n a r P r o c e e d i n g s of the S y m p o s i u m on P r a c t i c a l A s p e c t s of M o d e r n HPLC

(I. M o l n a r ed.), W a l t e r de G r u y t e r , B e r l i n

· New York

1982. 4. D.M. N e v i l l e : J. B i o p h y s . B i o c h e m . C y t o l . 8, 415

(1960)

SEPARATION OF PROTEINS BY SIZE EXCLUSION AND REVERSED PHASE HIGH PRESSURE LIQUID CHROMATOGRAPHY

Walter Schwarz, Jochen Born, Heinz Tiedemann Institut für Molekularbiologie und. Biochemie der Freien Universität Berlin, D-1000 Berlin 33 Tmre Molnar Dr. Herbert Knauer GmbH, D-1000 Berlin 37

SummaryProteins and peptides (M r 500-50 000) were separated by size exclusion chromatography (SEC) on glycerolpropyl derivatized silica particles of different pore size (6-10 run) and 50 % formic acid as eluent. A combination of size exclusion (SEC) with reversed phase chromatography (RPC) enhances the resolving power. Different octyl derivatized silica gels were compared. In RPC proteins are strongly retarded on the hydrophobic column packings in diluted formic acid and eluted with a linear gradient of 1-propanol.

Introduction The efficient and rapid separation of complex mixtures of proteins is still a difficult task. Proteins can be separated due to size differences after incubation with sodium dodecylsulfate on Polyacrylamide gels (1). The method gives excellent results for analytical purposes. Some proteins tend however to form polymeres even in the presence of dodecylsulfate. Moreover the complete recovery of proteins from analytical Polyacrylamide gels is difficult. In preparative polyacryl-

Practical Aspects of Modern H P L C Copyright © 1982 by Walter de Gruyter & Co., Berlin · New York Printed in Germany

124

amide electrophoresis the resolution of proteins is often insufficient. The separation of proteins according to size by SEC has b e e n performed on polysaccharide beads as column packing (2). Although adsorption of proteins on polysaccharide gels is small, the separation of proteins of different size is rather inefficient. Microparticulate, rigid, porous silica packings allow higher pressure and flow rates, but on the other hand they do adsorb certain proteins to a considerable degree. The interaction of silica gels with proteins in aqueous media is reduced when a layer of diol-groups is covalently linked to the particles to create a hydrophilic electroneutral surface ( 3 , 4). By choosing derivatized silica particles of different pore size as column packings peptides and proteins could efficiently be separated in the range from 10 000 to 170 000 Daltons by high performance SEC using 50 % formic acid as eluent. Proteins with limited solubility in aqueous solutions could be separated in this system without adsorption to the column packing (5)· Very efficient separation of peptides has b e e n achieved 1976 by Molnar and Horvath. In opposite to SEC, where separation is based on differences in the biopolymer diffusion velocity, they used a novel chromatographic technique, called "reversed phase chromatography" (RPC), utilizing hydrophobic, or more generally, solvophobic interactions (6). Peptides, which were forced by an aqueous buffer eluent onto the surface of a chemically modified octadecyl silica gel as column packing, were gradually eluted b y an acetonitrile gradient (7)» which had an approximately linear elution strength (8), allowing a reasonable prediction of peptide retention ( 9 ) · Proteins of higher molecular weights were recently separated on reversed phase particles of large pore diameter (10). HPLC has b e e n applied to biologically active proteins as the interferons (11). We were interested in the isolation of embryonic inducing factors, which change the differentiation

125

of amphibian gastrula ectoderm so that endoderm and mesoderm derived tissues or neural tissues are formed (12). The final purification of these factors, which are present in the tissue in very small amount, proved to be difficult by conventional purification techniques, such as Sephadex chromatography or SDS-polyacrylamide electrophoresis. Preliminary experiments have shown that HPLC can successfully be applied to these proteins which are stable in acidic solution and which renature when they are transferred back to aqueous solution (5)· To find the most suitable conditions for the separation of these proteins we have compared the separation of test proteins on different derivatized silica particles and with different eluents. We wanted further to measure the recovery of radioactive labelled proteins under the conditions of HPLC chromatography.

Materials said Methods Commercially available stationary phases: Zorbax BRC8 (6 pa; pore size 7-8 nm) was purchased from DuPont

(Frankfurt/M.,

FRG), LiChrosorb Si 60 (5 pm; pore size 6 nm), LiChrosorb RP-8 (7 pm; pore size 10 nm), LiChrospher Si 100 (5 p i and 10 pm; pore size 10 nm), LiChrospher Si 300 (10 pm; pore size 30 nm) and LiChrospher Si 500 (10 pm; pore size 50 nm) are manufactured by E. Merck (Darmstadt, FRG). Glycidoxypropyltrimethoxysilane

(1,2-epoxy-3-propoxypropyltrimethoxysilane;

Dynasylan Glymo) was purchased from Dynamit Nobel AG (Troisdorf, FRG), octyltrichlorosilane and octyldimethylchlorosilane from Wacker-Chemie (Burghausen, FRG). Formic acid (p.A. Merck) and 1-propanol (p.A. Merck) were quartz d e s t i n ed before use. Preparation of bonded phases: To bind glycerolpropyl groups covalently to the silica gel, the particles were washed with HCl, 50 % formic acid and water and then treated with glycid-

126

oxypropyltrimethoxysilane at pH 3·5 as described for glass beads (4-). The derivatized material was extensively washed with water, 50 % formic acid, water, acetone and ether on a sintered glass filter and dried. The derivatization procedure was repeated once more to derivatize all silanol groups as completely as possible. From small particle sized gels (5 fim) the fines were removed by 1 g sedimentation. To prepare hydrophobic particles for reversed phase chromatography, n-octyl-groups were covalently bonded to silica gel. LiChrospher Si 300 was washed with 6 Ν HCl and water until neutral reaction and then with acetone and methanol to remove the water. The dried silica gel (vac. desiccator) was then treated with octyltrichlorosilane or octyldimethylchlorosilane as described (10) with the modification, that the derivatized silica gel was not treated in a soxhlet extractor, but extensively washed on sintered glass filters with toluene, acetone, methanol and dried in a vacuum desiccator. High pressure SEC/RPC-chromatography: The prepared packing materials were packed into 25 χ 0.46 cm columns (Knauer, Berlin, FRG). Up to ten columns, which could be packed with materials of different pore sizes were connected. It is important, especially at low sample loading not to use glass fiber filters at the column connections, because a large percentage of protein was adsorbed to the glass filter. When columns were packed with 5 um material, stainless steel sieves of 3 pm pores were used at the column fittings. The packed columns were stored under methanol. The device for gradient elution with reversed phase columns is shown in Fig. 1. Linear gradients were formed with 2 HPLCpumps (typ 52.00, Knauer) and a gradient mixer (Knauer) on the high pressure side. For injection of the probes a Rheodyne ventil 7120 and for detection of the proteins at 280 n m a spectrophotometer (type 87-00, Knauer) were used. To avoid gas buble formation in the detector cell a pressure of 1-3 bar

127

was applied on the cell b y sawing a small hole in the capillary tubing which was then covered with a silicon tubing. All eluents were degassed by vacuum and short ultrasonic treatment prior to use.

puffer ^

Pump I

Injection Valve Gradient Former

/Buffer II

Pump II Column

Fraction Collector

Fig. 1.

Photometer

Flow diagram for reversed phase chromatography.

Radioactive Labeling of Proteins: Soy bean trypsin inhibitor was radioactively labelled b y reductive methylation of the protein amino groups (13) with formaldehyde and

plijNaBH^.

The reaction was carried out under mild conditions, so that only about 10-20 % of the amino groups were derivatized (14-). 1 mg trypsin inhibitor was dissolved in 0.1 ml 0.1 Μ borate buffer, which contained 0.2 % lithium dodecylsulfate (pH 8.6), cooled in an ice bath and added to 5 mCi freeze dried (from 5 pi 0.1 Ν NaOH) and precooled p H ^ N a B H ^ (specific activity 5 Ci/mmol; Amersham Buchler, Braunschweig). After addition of 3 pi formaldehyde (0.5 nil 35 % formaldehyde Merck p.A. + 1.4- ml ^ 0 ) and incubation for 10 minutes at 0 °C, a second 3 pi aliquot of formaldehyde was added and the mixture was incubated for further 10 minutes in ice. The reaction was stopped with 10 pi 1 Ν HCl and 5 |ul lysine-HGl (20 mg in 1 ml 0.1 Ν HCl). After standing for 30 minutes in ice, the protein was pre-

128

cipitated "by the addition of 40 |ul 80 % trichloroacetic acid (TCA) (freshly prepared from TGA (Merck) p.A.)· The sediment was washed twice with 20 % TCA which contained 0.5 % NaCl and five times with ethanol/ether (1:1). The sediment was then dissolved in 0.05 Μ phosphate "buffer containing 0.2 % sodium dodecylsulfate and applied to a column (15 x 0.4 cm) of Sephadex G 25 (Pharmacia, Freiburg/Br., FEG) which was prewashed with the same buffer. The column was eluted (0.33 ml/ min), the protein peak precipitated with TCA, the sediment washed with TCA and ethanol/ether (1:1) and suspended in water and freeze dried. Cytochrome c (horse) was purchased from Merck, serum albumin (bovine) from Gibco, carboanhydrase (bovine erythrocyte) from Serva (Heidelberg, FRG) and soy bean trypsin inhibitor from Sigma (Taufkirchen, FEG). The proteins were dissolved in 100 % formic acid, diluted to the appropriate formic acid concentration and 10-50 pi injected into the system. Proteins were determined by the biuret method. The radioactivity was measured in aliquots in 2 ml Quickscint 212 (Zinsser, Frankfurt/M., FRG) in 4 ml scintillation vials. A Beckman scintillation counter was used.

Results and Discussion Size Exclusion Chromatography (SEC) Previous experiments have shown that proteins in the molecular weight range of 10 000 to 170 000 can be separated by SEC with glycerolpropyl derivatized LiChrospher Si 300 as column packing and 50 % formic acid as eluent. For most proteins a 'linear relationship exists between the elution volume and the logarithm of their molecular weight under these condi-

129

tions (5)· To increase separation efficiency, columns were packed with small particle sized gels (5 pi particle diameter) which were modified to glycerolpropyl LiChrospher Si 100 (Fig. 2). A very sharp separation of proteins in the

0

S)

40

60

80

100 120 Minutes

Fig. 2. Chromatography of 33 μg carboanhydrase CI, M r 29 500), 33 μg trypsin inhibitor (II, M r 21 500) and 33 |ug ribonuclease (III, M r 12 640) on glycerolpropyl derivatized LiChrospher Si 100 (5 p i ) at 20 oq. Eluent: 50 % formic acid. Column: 500 χ 4.6 mm. Flow rate: 0.06 ml/min. Pressure: 22 bar. The last peak (methanol) marks the total liquid volume accessible to small molecules. molecular weight range of 10 000 to 50 000 was obtained on short columns. The loading capacity is however smaller as with derivatized LiChrospher Si 300 or Si 500. The molecular weight range could be extended to smaller peptides when

130

glycerolpropyl derivatized LiChrosorb Si 60 alone (Pig. 3a) or in combination with derivatized. LiChrospher Si 100 (Fig. 3b) were used as column packing. Ill

gig- 3a. Fifi. ?b. Chromatography of aprotinin (I, Mr 6 500), melittin (II, M r 2 840) and. the peptide tyr-gly-gly-phe-leu (III, M r 556) on glycerolpropyl derivatized LiChrospher Si 100 (5 jum) and glycerolpropyl derivatized LiChrosorb Si 60 (5 pm) at 22 °C. Eluent: 50 % formic acid. a) Column: LiChrosorb Si 60 (5 pm) 1 000 χ 4.6 mm. Flow rate: 0.25 ml/min. Pressure: 100 bar. b ) Column: LiChrosorb Si 60 (5 |um) 1 000 χ 4.6 m i + LiChrospher Si 100 (5 pm) 1 500 χ 4.6 mm. Flow rate: 0.20 ml/min. Pressure: 290 bar. Reversed Phase Chromatography (RPC) Proteins can efficiently be separated by combination of size exclusion chromatography with reversed phase chromatography (15). Mixtures of formic acid and 1-propanol were used for

131

gradient elution in reversed phase chromatography. This system allows a direct recording of proteins by spectrophotometry at 280 n m and gives a somewhat better separation than the formic acid (or acetic acid)/pyridine buffer/1-propanol systems, which have been used in RPC of proteins (11). Silica gels of different origin and pore size with covalent1y bound n-octyl groups were tested with the same mixture of proteins using the same gradient elution profile. The least efficient separation was obtained on Zorbax BRC8 (Fig. 4a).

Fig. 4a. Reversed phase chromatography of 30 |ug horse cytochrome c (I), 30 P-g soy bean trypsin inhibitor (II) and 30 pg bovine erythrocyte carboanhydrase (III) at 22 °C. Column: Zorbax BRC8 (6 jum) 250 χ 4.6 mm. Solvent A: 15 % formic acid. Solvent B: 30 % formic acid/1-propanol (1:1; —). Slope of linear gradient: 5 % B/min. Flow rate: 2 ml/min. Pressure: 100-180 bar. Trypsin inhibitor is eluted together with carboanhydrase. These proteins are well separated on LiChrosorb Si 100-RP-8

132

(Fig. 4b). The Separation is improved when a spherical gel

Fig. 4-b. Reversed, phase chromatography of 30 jug horse cytochrome c (I), 30 pg soy bean trypsin inhibitor (II) and. 30 |ug bovine erythrocyte carboanhydrase (III) at 22 OC. Column: LiChrosorb Si 100-RP-8 (7 p i ) 250 χ 4.6 mm. Solvent A: 15 % formic acid. Solvent B: 30 % formic acid/1-propanol (1:1; —). Slope of linear gradient: 5 % B/min. Flow rate: 2 ml/min. Pressure: 100-180 bar. with larger pores (LiChrospher Si 300-RP-8) is used (Fig. 5)· The separation of the test proteins could be improved further when the slope of the gradient was lowered. Derivatization with monochlorodimethylalkyl-silanes is supposed to result in better surface coverage and less residual silanol groups on the surface of the silica particles (16). LiChrospher Si 300 or Si 500 silica particles were therefore derivatized with either n-octyltrichlorosilane or n-octyldimethylchlorosilane. No difference in the separation of our

133

Fig. 5. Reversed phase chromatography of 30 p g cytochrome c (I), SO ug serum albumin (II), 30 )J.g trypsin inhibitor (III), 30 ug carboanhydrase (IV) at 22 °C. Column: LiChrospher Si 300-RP-8 (10 jam) 250 χ 4.6 mm derivatized with octyldimethylchlorosilane. Solvent A: 15 % formic acid. Solvent B: 30 % formic acid/1-propanol (1:1; —). Slope of linear gradient: 5 % B/min. Flow rate: 2 ml/min. Pressure: 180-350 "bar. test proteins was however observed when silica particles derivatized with the two reagents were compared under otherwise identical conditions. Recovery of Radioactive Labelled Proteins in RPC The applicability of chromatographic methods depends not only on the resolving power, but also on the rate of recovery of proteins. When soy bean trypsin inhibitor was chromatographed on columns of very different length (length ratio 1:6.25)

134

packed with LiChrospher Si 300-RP-8, the peak areas of the eluted proteins are the same (Fig. 6a and Pig. 6b).

Fig. 6a.

Fig. 6b.

Comparison of the amount of protein eluted (peak areas) with different amounts of silica gel used as column packings (columns of different length). 60 iug trypsin inhibitor was applied to each column. Solvent A: 15 % formic acid. Solvent B: 30 % formic acid/1-propanol (1:1; —). Gradient: 10 % B/min, for 5 min; 0 % Β for 5 min; 10 % B/min, for 5 min. Column packing: LiChrosorb-RP-8 (7 pn). a) Column: 40 χ 4.6 mm. Flow rate: 2 ml/min. Pressure: 15-30 bar. b ) Column: 250 χ 4.6 mm. Flow rate: 2 ml/min. Pressure: 90-180 bar. This shows that the proteins are not adsorbed irreversibly to this column packing under the conditions of reversed phase chromatography. However glass fiber filters are a major source for protein adsorption and should therefore not be used. When 30 |ug trypsin inhibitor, which was labelled with tritium by reductive methylation (s. methods) was applied to

135

a 4.6 mm. diameter reversed phase column equipped with three glass fiber filters about 10 % of the protein was adsorbed to these filters. The percentage of adsorption is much higher with smaller sample loadings. About 90 % of the protein was adsorbed to glass fiber filters when only 3 ug trypsin inhibitor was applied to the reversed phase column. When this amount of protein was applied to a size exclusion column equipped with glass fiber filters and the column eluted with 50 % formic acid, about 30-4-0 % of the protein was adsorbed to the filters. The structural features of proteins which determine their elution pattern in reversed phase chromatography are at the present still unknown. In the formic acid containing eluents all ionizable groups are protonated, so that the charge of the proteins depends only on the basic ammonium-, guanidiniumand imidazolium groups. Cytochrome c which has b y far the highest content of lysine and arginine is eluted first. Other features which do significantly contribute to the pattern of elution are the content of hydrophobic amino acids with longer aliphatic or aromatic side chains and on the other hand the carbohydrate content of proteins. The evaluation of all structural features requires the investigation of many more proteins, and will be subject of further investigations. This investigation was supported b y the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 29; Embryonale Entwicklung und Differenzierung).

References 1. 2. 3·

Weber, K., Osborn, M.: J. Biol. Chem. 244, 4406-4412 (1969). Porath, J., Flodin, P.: Nature 18£, 1657-1659 (1959). Unger, K.K., Becker, Ν., Roumeliotis, P.: J. Chromatogr. 125, 115-127 (1976).

136

4.

Regnier, F.Ε., Noel, R.: J. Chromatogr. Sei. 14, 316-320 (1976).

5.

Schwarz, W., Tiedemann, Η., Tiedemann, Η.: Mol. Biol. Rep. 8, 17-20 (1981). Horvath, C., Melander, W., Molnar, I.: J. Chromatogr. 125, 129 (1976).

6. 7. 8.

Molnar, I., Horvath, C.: J. Chromatogr. 142, 623-640 (1977). Molnar, I.: HRC & CC 4, 276-279 (1981).

9.

Molnar, I., Schöneshöfer, Μ.: HPLC in Biochemistry, ed. Lottspeich, Henschen, Hupe, Verlag Walter de Gruyter & Co., Berlin 1982. 10. Lewis, R.V., Fallon, Α., Stein, St., Gibson, K.D., Udenfriend, S.: Anal. Biochem. 104, 153-159 (1980). 11. Rubinstein, Μ., Rubinstein, S., Familletti, Ph.C., Miller, R.S., Waldman, A.A., Pestka, S.: Proc. Natl. Acad. Sei. U.S.A. 26, 640-644 (1979). 12. Tiedemann, H.: In: Organizer, a milestone of a half century from Spemann, 0. Nakamura a. S. Toivonen eds., Elsevier/North-Holland, Amsterdam · Oxford · New York (1978). 13. Means, G.E. , Feeney, R.E.: Biochemistry 7, 2192-2201

(1968).

14. Asahi, K.-i., Asashima, Μ., Geithe, H.P., Born, J., Tiedemann, Η., Tiedemann, H.: in press (1982). 15· Molnar, I., lecture, presented at the Symposium on practical Aspects in Modern HPLC, Berlin, Dec. 1981. 16. Roumeliotis, P., Unger, K.K.: J. Chromatogr. 149, 211-224 (1978).

HIGH

PERFORMANCE

STUDIES

Maria

OF THE

Kehl,

LIQUID

CHROMATOGRAPHY

FIBRINOGEN

Friedrich

AS

APPLIED

TO

THE

STRUCTURE

Lottspeich,

Agnes

M a x - P l a n c k - I n s t i t u t für B i o c h e m i e , München, Germany Federal Republic

Henschen

D-8033

Martinsried

bei

I n t r o d u c t i on During have

the

changed

dation the

last y e a r s

tion

of

being

considerably.

method

methods

for

have

the

protein been

automated

the m o s t

techniques

gradation

of

niques

It

0.5

to

for

poses

high

excellent as

a model

The

plasma

One

is

monomer

the

introduc-

Edman

and

Begg

analysis

contribution.

5 nmol

of

that

Thus, r a p i d

generated, protein

or

which

sensitive

and

the

sequencing

techniques.

For

chromatography by

(2)

highly

material

purification,

be d e m o n s t r a t e d

described

permit

peptide

these microscale

liquid

may

by

first

degra-

(1),

been

reaction

but

(Αα,Ββ,γ) 2 fibrinogen

in

two

B, a r e

into

that

then

plays the

cleavage

Hereby

A and

changed form,

fibrinogen

in t h e

thrombin.

brinopeptides nogen

Edman

improved

identification

protein

consists

enzyme

manual

continuously

have

This

the

analysis

derou-

tech-

isolation, all

proves using

these to

be

puran

fibrinogen

protein.

coagulation. cess

since

structure was

performance tool.

protein

analysis

equally

f r a g m e n t a t i o n .and

Even

sequence

is o b v i o u s

demand

in

sequence

important

sensitive tinely.

methods

of

very the

small

thrombin

soluble

and

fibrin,

polymerises

role

complex

peptides,

released of

a central

to

the

(α,β,γ) 2 fibrin

blood

coagulation fibrinogen

the is

first

insoluble + 2A

the fi-

of

fibri-

in

the

clot.

+ 2B

fibrinopeptide

Practical Aspects of Modern H P L C Copyright © 1982 by Walter de Gruyter &. Co., Berlin · New York Printed in Germany

pro-

by

so-called

structure

which an

in

138 The up

complete of

three

amino pairs

molecular

weight

However,

studies

of

interest,

related sumed

that

these e.g.

for

the

molecular

a giant

molecule.

In

report

this

which

are, t h e

Separation

of

γ-chain and

of

have

been

Bß-chain The

the

of

one

sition

52

to

and

i.e.

chains, from

the

The

γ-chain nalysis,

on is

the

dues

being

fore

larger

chemically

far

of

be

the

Edman

of

(Fig.l)

shortest 461

and

being

γ-chain

carry

may

be

the

by

the

presence

large

for

a complete

during

have

to

enzymatically

be and

lione fibri

with

amino

amino

610

the

acids

to

subdivided

amino side

very

Li-

8 Μ

amino

in

fragments

re-

po-

form

chroma(7).

The

sequence acid

degradation. by

chains, acid

pure

urea

re-

chains,

exchange

direct

70

acid

asparagine in

of

sequenator

the

the

fibrinogen

ion

in

identifiable

411

three

on

60

such

Fibrinogen

isolated

of m a x i m a l l y

with

Performance

carbohydrate

fibrin(ogen)

in

with

finally

High

Aa-chain

located

search

degradation.

Human

the

residue.

first

(PTH)

by

the

protein

error

then

and

contains

of

the

as-

performance

demonstrated

fibrinogen

been

acid

an

high

the γ - c h a i n ,

still

systematic

localise

Fragments

Bß-chains

proteins

to

(3,4,5).

are

has

in

amino and

applying

the γ - C h a i n

the

sequence

or

i.e.

Peptide

too

of

will

products

CM-cellulose by

a quick

the

fibrinogen-

It

errors

a single

how

human

S-carboxymethylated

tography

of

found.

made

has

elucidated

inherited

by

is

and

molecule

phenylthiohydantoin

the γ - c h a i n (6).

caused

conduct

(HPLC)

containing γ-

are

exchange

fibrinogen thus

this

disorders

advantages

Large

is

sidues.

from

end

of

which

chains

been

of

Chromatography:

sidues the

of

peptide

recently

structure

error,

released

identification

has

fibrinogen,

number

how

the

of

a large

the

arises

fibrinogen

nopeptides

The

the

chromatography

the

000,

disorders

by

problem

quid

340 of

sequence

non-identical

because

The

of

of

coagulation

structure

quid

acid

cleaving sequenced

a-

resiTherethem sepa-

139 1

CT

10

CT

20

30

Τ

40

50

70

CB 80

100

CT 110

Y V A T R D N C C I L D E R F G S Y C P T T C G I A D F L S 31

CHO

Τ

Τ

60

T Y Q I K V D K D L Q S L E D I L H Q V E N K T S E V K Q L

61

Τ

Τ

CT TCB90

I K A I Q L T Y N P D E S S K P N M I D A A T L K S R K M L

91

CB Τ

120T

E E I M K Y E A S I L T H D S S I R Y L Q E I Y N S N N Q K

121

Τ

Τ

130

140T

150

I V N L K E K V A Q L E A Q C Q E P C K D T V Q I H D I T G

151T

T160

Τ

K D C Q D I A N K G A K Q S G L Y F I

181

190

Τ CT

170

Τ

180

K P L K A N Q Q F L V

200

Τ Τ

210

Y C E I D G S G N G W T V F Q K R L D G S V D F K K N W I Q

211

Τ

220

230

Τ

240

Y K E G F G H L S P T G T T E F W L G N E K I H L I S T Q S

241

Τ

AI

CT

250

CT

260

CB

Τ

270

P Y A L R V E L E D W N G R T S T A D Y A M F K V G P E

271

Τ

CT

Τ

280T

290

300

320 Τ

330

A D K Y R L T Y A Y F A G G D A G D A F D G F D F G D D P S

301

Τ

310CB

D K F F T S H N G M Q F S T W D N D N D K F E G N C A E G D

331

CB

G(S)G H(W)M

Τ 340

350

Τ

360

N K C H A G H L M G V Y Y Q G G T Y S K A S T P

361

370

391CT

400

Τ

CT

CB380rT

CB Τ

390

Ν G Y D Ν G I I (W)A T(W)K T R W Y S M K K T T M K I Τ

I P F N

410

R L T I G E G Q Q H H L G G A K Q A G D V

F i g u r e 1. Amino a c i d sequence o f human f i b r i n o g e n Ύ - c h a i n . CB d e n o t e s cyanogen bromide c l e a v a g e s i t e s , Τ t r y p s i n c l e a v a g e s i t e s , CT t r y p s i n c l e a v a g e s i t e s a f t e r c i t r a c o n y l a t i o n , CHO denotes a c a r b o h y d r a t e s i d e c h a i n . rately

in

order

to

obtain

the

complete

primary

structure

infor-

mation. Cyanogen

bromide

chain

1.5 ml of

in

cyanogen

bromide

gen bromide

cleavage. in

= 1 : 5 )

About

70% f o r m i c 1.5 ml o f for

15 h.

50 mg o f

human f i b r i n o g e n

a c i d was t r e a t e d 70% f o r m i c

acid

The m i x t u r e

with

250 mg o f

(protein

was then

γ-

: cyano-

diluted

140

with

the

tenfold

evaporator superfine Trypsin the

cleavage. from

10 mg

of w a t e r cium

then

of w a t e r ,

directly

or a r e v e r s e d

pools

lised;

and

volume

For

adjusted

with

cleavage

of a r g i n y l

was

then

tri ethyl a m i n e of t r y p s i n ,

pH was

pH 2.5 w i t h

rectly Total

injected amino

in 5.7

16 h at 3 7 ° C . T h e acetic

acid.

on a r e v e r s e d

acid analysis.

Ν hydrochloric

G-50

lysyl

G-50 were

in 2.5 ml

added

and

then

=

the

calchlo-

digest

adjusted

A p a r t of t h i s m i x t u r e phase

bonds lyophi-

treated with TPCK

left about

rotary

to pH 8 . 5 , 0.1 mg of were

for

and

dissolved

ro-(N-tosyl-1-phenylalanyl)-methane, at pH 8.5

on a

on a S e p h a d e x

on S e p h a d e x

material

and 0.1 mg

in v o l u m e

column.

the g e l f i 1 t r a t i o n

of p e p t i d e

chloride

fractionated

phase

the

reduced

was

to di-

column.

Lyophilised

samples were

acid

at 1 1 0 ° C

for

to s e p a r a t e

cyanogen

bromide

hydrolyzed

24 h in e v a c u a t e d

tu-

bes. HPLC

conditions.

I. S y s t e m

used

brinogen γ-chain on S e p h a d e x Instrument

and the

corresponding

cleaved

pools

from

human

fi-

gelfi1tration

G-50. Milton

Roy H P L C

a mixing Roy

pumps

chamber, all

gradient master;

constametric controlled a Waters

U6K; a fixed wavelength LKB

and a P h i l i p s

Column

Hewlett

Buffer

A: 0.1 Μ s o d i u m B:

100%

all

Packard

dihydrogen

being

206

model nm,

from

recorder. (lOy).

phosphate

(uvasol, Merck,

filtered

III,

Milton

injector

0.4 χ 25 c m , R P - 8

acetonitrile

solvents

by a

detector,

two-channel

I and

pH

2.1,

Darmstadt),

and d e g a s s e d

before

use. Gradient

10% 100%

Fl ow

Β for

10 m i n

Β in 100

2 m l / m i η.

isocratically,

min.

then

from

10

to

141 R e c o r d i ng

0.2

Temperature

ambient.

II.

System

used

filtration

on

to

absorption

separate

Sephadex

trypsin

G-50

Column

Hibar

Buffer

A:

RT

units

of

scale

at

cleaved

pools

from

human

250-4,

0.025

full

fibrinogen

LiChrosorb

Μ ammonium

phori c acid

to

B:

Μ ammonium

40%

0.05

phosphoric (uvasol,

acid

0

Fl ow

1.5

m 1 / m i η.

R e c o r d i ng

0.4

absorption

Temperature

ambi ent.

100%

Β

in

the

gel-

(5μ),

with

(Merck)

ortho-phos-

6.0

to

Merck,

Gradient

to

pH

nm.

γ-chain.

RP-18

acetate

206

pH

acetate 6.0,

with

60%

ortho-

acetonitri1e,

Darmstadt). 100

min.

units

full

scale

at

206

nm.

Results

When

human

cleaved and

by

charge

γ-chain,

which

cyanogen

bromide,

(Table

been

to

separate

This

separation

according tion is

takes

type

of

contrast,

with

the

the this

HPLC type

material better

fragments The

of

established

gelfi1tration

as

the

Even

under

peptides

optimised

Furthermore,

re-fractionate

the

obtain

separate

(Fig. elute the

pure

pools

size

strategy

conditions

to

r e s i d u e s , is

different

by

size.

to

methionine

fragments partial,

time.

eight

obtained.

only

has

2). mainly separa-

peptides

on

a

it

diffe-

column. the

with

chromatography

the

separation of

nine

is

HPLC-method

comparison

are

these

a long

necessary

rent In

to

1)

contains

proved

to

on be

gelfi1tration is

finished

chromatography

( 1/10

of

that

used

purified

in

a shorter

the most

for time

min.

smaller the

G-5C

with

phase

efficient

(Fig.2),

in 60

are:

reversed

(Fig.

3).

takes

35

h,

advantages

of

which The

column

amounts

of

peptide

separation)

negligible

In

loss

may of

be pep-

142 Table

1. A m i n o

Acid

γ-Chain Amino

acid

Composition

of

Cyanogen

Bromide

Cleaved

Fragments.

N-terminal YVAT

IDAA 1

sequence LEE I

KYEA

of

the

FKVG

single

QFST

NKCH

fragments KKTT

KIIP

Asp

D

7

Asn

Ν

4

Thr

Τ

7

Ser

S

6

Gl u

Ε

5

Gl η

Q

5

13

Pro

Ρ

3

4

2

Gly

G

2

12

7

3

6

5

Ala

A

3

10

5

1

3

2

Cys

C

4

4

1

1

Val

V

4

7

1

Met

Μ

1

1

1

1

1

lie

I

5

1

1

13

Leu

L

7

1

1

13

1

Tyr

Υ

4

8

3

Phe

F

2

6

7

15

3

Κ

Lys

4

1

1

10

1

3

4

1

1

10

2

1

4

1

11

2

2

3

11

1

2

2

2

6

Η

1

Arg

R

2

Trp

W

His

1

2

1

2

1

3 1

1

1 1

2

1

1

2

3

1

2

2

5

1

1

3

1

78

tide

material. with

the

matography pletely Using

Only

4

1

2

4

1

1

1

3)

170

a single

N-terminal

(Fig.

5

46

peptide

sequence

practically

2

3

26

43

caused

QFST. all

3

the

5

problems,

Thus,

in

peptides

27

i.e.

a single may

be

the

chrocom-

separated.

the as

11

2

2

4

Total

such

8

+

Gl cN

one

10

HPLC

Hibar

conditions LiChrosorb

of

system

RP-18

(5μ)

I,

but

with

or

Zorbax

other

columns

TMS,( D u P o n t ) , s i -

143 milar The

separation

Figures

pools

from

4a, the

patterns 4b,

4c

could

show

Sephadex

be

the

G-50

obtained.

HPLC

chromatography.

original

HPLC-separation

(Fig.

ted.

components

identified

ce

The

and

amino

Figure nogen

4a

G-50

pool

has

N-terminal and

the

the

peptides

N-terminal

sidues,

composition

presents

bromide

phadex the

acid

were

I.

KYEA,

galactose

Figure

4b

the

separation,

size

1).

amino

after

acid

the

startpeak,

Κ11Ρ

were

(27

eluted the

chain

length,

and

Figure

4c

dex

G-50

with

peptides

known

charge

the

Later

which to

shows

the

this

on

of

from

case

might

III

of

the

shows be

rethe

residues

acid

(6).

from

the

sequence

column

very

part

FKVG

The

Se-

intermediate

of

(46

soon

the

residues) elution

a correlation

with

because

parameters

IDAA,

sequen-

observed

accidental

other

with

N-terminal

residues),

gradient.

many

pool

the

with

acid

acid

Se-

N-acetyl-glucos-

isocratic

with

HPLC-separation

separation. sequence LEEIM,

is

with

could

demonstrated

3 could

the

be

The

KKTTM;

material

Fig.

the

however

amino

of

the

amino

peptide

cya-

reten-

including

conformation.

ta-peptide,

be

(43

sequen-

peptide

170

N-terminal

the

peptides

by

in

depend

eluted

NKCH

78

peptides

the

during

the

sequentially

of

is

i.e.

residues),

order

tion

long,

interpre-

from

N-acetyl-neuraminic

with

the

largest

those

the

composed

containing

peptide

residues

chromatography. ces

The

two

longer

than

HPLC-chromatogram

phadex G - 5 0

11

and

way

easily

the

the

contains

chain,

this

the

N-terminal

contains

time

which

side

that

which

retention

mannose,

(Table

the γ - c h a i n , found

of

1).

i.e.

amine,

shows

their

of

YVAT,

carbohydrate

by

(Table

In

be m o r e

of

sequence

sequence

3) m a y

HPLC-separation

It w a s

a shorter

frationations

startpeak

of

pool

contains

when

the

gradient

eluted;

all

later

N-terminal that

recovered

sequence

V from the

QFST.

In

Sepha-

penta-peptide

begins

peaks

the

a second

contain this

all

the

components

from

the

HPLC-separations

peptide

way

it

identified of

pen-

in

the

144

0.10 ι < 0.08

0.06

0.04

0.02

5

10

15

20

25

30

[h]

35

F i g u r e 2. C h r o m a t o g r a p h y o f 5 ing ( 1 0 0 n m o l ) o f c y a n o g e n b r o m i d e c l e a v e d S - c a r b o x y m e t h y 1 a t e d h u m a n f i b r i n o g e n γ - c h a i n on S e p h a d e x G - 5 0 s u p e r f i n e ; c o l u m n , 1 x 1 0 0 c m ; e l u a n t , 1 Μ f o r m i c acid; d e t e c t i o n w a v e - 1 e n g t h , 280 nm.

HKCH

ΚΤΙΛ

F i g u r e 3. R e v e r s e d p h a s e c h r o m a t o g r a p h y o f 5 nmol o f c y a n o g e n bromide cleaved S-carboxymethylated human fibrinogen γ-chain, t h e s a m e s t a r t i n g m a t e r i a l as in F i g u r e 2 ; H P L C c o n d i t i o n s . s e e system I . '

145

F i g u r e 4a. R e v e r s e d p h a s e c h r o m a t o g r a p h y of 1/50 of pool I of the S e p h a d e x G - 5 0 s e p a r a t i o n ; HPLC c o n d i t i o n s , see s y s t e m I.

F i g u r e 4b. R e v e r s e d p h a s e c h r o m a t o g r a p h y of 1/50 of pool III of the S e p h a d e x G - 5 0 s e p a r a t i o n ; H P L C c o n d i t i o n s , see s y s t e m I.

KKTT

F i g u r e 4c. R e v e r s e d p h a s e c h r o m a t o g r a p h y of 1/20 of pool V of the S e p h a d e x G - 5 0 s e p a r a t i o n ; H P L C c o n d i t i o n s , see s y s t e m I.

146

F i g u r e 5. H P L C e l u t i o n p r o f i l e of 1 / 1 0 o f t h e t r y p s i n d i g e s t e d pool I f r o m the S e p h a d e x G - 5 0 s e p a r a t i o n , see H P L C c o n d i t i o n s s y s t e m II; s a m p l e v o l u m e , 0 . 1 0 0 m l ; 0.4 a b s o r p t i o n u n i t s full scale. Sephadex For

the

seemed of

G-50

development to

be

cyanogen

paration

of

very

the

of

well

a difference

In

experiments

pected the

an

of

amino

the

peptide

capacity

profile

of

column

in

26

peaks

by

amino

of

system

II

such

and

the

the

the

were

made

to

-digested

as

great

18

Figure

advantage

could

pure of

the a

be

an

tryp24 to

extest

elution

reversed

mixture

peptides.

being

the

sample

on

ex-

sequence.

with

5 shows

peptide

of w h i c h

containing

pool

separated

The

mean

peptide

a suitable

pool

II.

would

would

separate

I. T h i s

be

a HPLC-system.

to

se-

sufficiently

pattern

which

separation

subsequent are

a known

pool

seemed

column,

systems

in

HPLC-method

initial

the

separation

residue

G-50

the

the

for

peptide,

HPLC-system

the

also

If

attempts

analysis has

, both

a new

acid

trypsin

from

in

Sephadex

using

acid

of

fragments

of

the

phase

for

peptides.

appearance

digest

suited

tryptic

of

tic

system

peptides

exchange first

a fingerprint

bromide

reproducible press

pools .

eluted

identified The

volatile

buffer and

147

F i g u r e 6. H P L C e l u t i o n p r o f i l e o f 1 / 2 0 o f t h e t r y p s i n d i g e s t e d pool I f r o m the S e p h a d e x G - 5 0 s e p a r a t i o n , see H P L C c o n d i t i o n s s y s t e m II; b u f f e r , p h o s p h a t e 0 . 0 5 Μ pH 3 . 0 ; s a m p l e v o l u m e , 0 . 1 ml, 0 . 4 a b s o r p t i o n u n i t s f u l l s c a l e .

therefore two

the

collected

lyophilisation

could

be

obtained

this

buffer

that

for

should

is

the

be

Separation

human

of

different

sidue nus

by

by

in

not

of

an

Shorter : The

position

similar II

error

using

in

analysed

directly

separation

volatile.

Peptides Human

Blombäck

the

be

a

pattern

phosphate

These

(Fig.6)

buffer

results

the γ - c h a i n

after

the

pH

3.0,

indicate, HPLC-method

suited.

Α-peptides.

long,

A

could

system

fibrinopeptides

recognised

sidues

with

search

Chromatography

The

cycles.

however

ideally

peaks

AP-

et The

one

amino

acid

B-peptide,

which

is

14

High

Performance

Liquid

Fibrinopeptides

occur al.

and

a the

residue, amino

in

in

main

carries

three

by

several

1966

(8).

Α-peptide

is

forms

as

There

are

16

phosphate-group AYi.e.

acid

is

amino on

shortened

alanine

residues

(see long,

the at

already three acid

serine the

Table is

rere-

N-termi2).

highly

The sus-

148 ceptible

to

degradation

contaminates C-terminal

fibrinogen

arginine

Arg-B-peptide bin-induced the

may

seem

be

many all

near

show

somewhat system all

human

nogen, nogen

ml

0.15

was

18

h the

of

the the

and

to

sample

was

precipitated

and

is at

all

Brook

were

and

pH

in

protein

peptide-containing

in

a boiling by

they

to

of

a

or HPLC

determine

short

time.

fibrinogens gift

generous

ammonia. 5 NIH

(fibriof

of

Bovine

then

centrifugation. was

of 5 mg/ throm-

units/ml.

waterbath,

liquid

Dr.D.

gift

a concentration

of

are

(Forschungs-Fibri-

the

with

re-

peptide

there

a very

was

supernatant

the

possible

Metz

removed

These

1aborconsuming

generous

concentration

placed

to

the

8.5

i.e.

fibrinogens

was

to

throm-

determination,

abnormal

dissolved

acetate

or

fibrinogen

and

des-

the

applications

simultaneously

normal

such

or

is

the

disturbed,

Although

some it

step

released.

in

time-

always

so-called

often

sites.

report

first

the

themselves

being

which

fibrinogens

defects

by w h i c h

Rouen

a final

the

fibrinopeptide

Stony

Soria)

not

the

Kabi , M ü n c h e n )

Μ ammonium

added

or

this

Human

fibrinogen J.

release

of

described

digestion.

and

bin

and

are

In

off

cleavage

for

in

abnormal

peptides

disadvantage

Louisville

in

the

thrombin

Deutsche

C.

that

fibrinopeptides

Galanakis, Drs.

to

In

partly

indicate

unspecific.

(10)

Thrombin

only

available

the

cleaved

B-peptide

the

methods

being

and

localised

chains

preparations,

formed.

are

to

a carboxypeptidase,

being

A-

peptides

sults

by

After

cooled A

part

subjected

to

H P L C - a n a l y s i s.

HPLC

conditions.

Instrument

Hewlett

Packard

tomatic

sampling

length Column

Buffer

A:

(5μ),(Merck,

0.025

50%

system

column,

ammonium

phosphoric B:

equipped and

with

an

a variable

auwave-

detector.

Prepacked RP-18

1084A

of

Hibar

RT

250-4, L i C h r o s o r b

Darmstadt). acetate

pH

6.0

with

ortho-

acid 0.05

Μ ammonium

acetate

pH

6.0

149 Table Peak

2.

Human F i b r i n o p e p t i d e

number

Components

Designation

Sequence

®

A

+

A D S G E G D F L A E G G G V I j l D S G E G D F L A E G G G V I j t _

+

Z G V N D N E E G F F S A Z G V N D N E E G F F S A I j t _

phosphate

and Ζ p y r o g l u t a m i c

with

ortho-phosphoric

50%

of

acetonitrile

all

solvents

linear from 40

from

12%

not

essential

wavelength full

210

and 28%

Β in

solvents

and

(uvasole,

12% Β t o 34%

acid acid

filtered

Β to

C for

is Detection

Charge

A D | G E G D F L A E G G G V g .

Β

Temperature

HPLC

+

des-Arg-B

Gradient

and

by

AP

AY

©denotes

Separated

and

for

nm a t

Merck,

Darmst.)

degassed. Β in

40

or

min.

oven,

the

30 min this

temperature

separations,

0.025

absorption

units

scale.

Results Preliminary would

not

However,

experiments

be

sufficient

under

column.

directly amino

elution

could

This

of

gradient well

demonstrated

after

composition

behaviour

resolve

be v e r y

is

identified

acid

to

optimised

fibrinopeptides phase

indicated

or

two

that all

isocratic

fibrinopeptides

conditions

all

five

separated

on a

in

7.

Figure

1 yophi 1i s a t i o n

sequence

elution

or

fibrinopeptides

by

comparison

isolated

(8). expected

reversed

The

steps

systems

peaks by

their

with

according

were the

to

clas-

150

η

NORM.

A B-R

"J •X»

Μ •

Β

AP AY

IV

1

2

3

5

4

F i g u r e 7. H P L C e l u t i o n p r o f i l e o f n o r m a l h u m a n f i b r i n o p e p t i d e s f r o m a r e v e r s e d p h a s e R P - 1 8 c o l u m n j g r a d i e n t f r o m 6% to 14% a c e t o n i t r i l e (see HPLC c o n d i t i o n s ) ; r e t e n t i o n times are given in m i n a b o v e t h e p e a k s ; s a m p l e v o l u m e , 0 . 1 0 0 m l , i . e . 2 . 8 n m o l of Α - p e p t i d e s ( A P + A + A Y ) and B - p e p t i d e s (des-Arg-B+B).

sical

procedures.

jection

is d u e

contains

free

paration

and

peaks

all

The

main

AP,

A,

to

elution

products

appear

retention

sidues able

the

acids

order

of

and

B.

the

after

long

Α-peptides

contain

for

calculated

fewer

contain in T a b l e

would

2.

the

the

fibrinogen

by

Meek

and

aromatic

of

Α-peptide

the

later

found

to

be

easily degradation

or

amino it w a s

between times.

acid

re-

predict-

B-peptides.The

acidic

fewer

pre-

products

incubation

(10),

more and

was

amounts

before

The

is m o r e

individual

in-

also

degradation

thrombin

the

after It

B-peptide.

their

elute

aromatic

more

from

small

B-peptide

the

sample.

B-peptide

enzymes,

coefficients

soon

fibrinopeptides

befor

that

shown

As

the

the

or

peaks

example

as

from

human

appears

in

glycine

as m i n o r

for

B-peptides

which

salts

released

as

Α-peptides

and like

contaminating

and

peak,

fibrinopeptides

des-Arg-B

by

des-Arg-B-

acids

arginine,

contain

AY,

first

solvents

amino

attacked

Using

The

amino

acidic

acids,

amino

151

Η r· υ-·

conditions); B - p e p t i des .

The on

sample

advantages

of

where

the

graphed peaks

under

lysis

it w a s

Louisville

by

i.e.

the

one

est:

a)

earlier).

that

at

in

that

the

the

the

From the

This

thrombin because altered

allowed

the

before amino

two

arginine

physiologically,

HPLC-method

normal

one

chemically,

b)

two

and

extra

exchange

of

cleavage the

peptide

because and

at

after

is

blood

When the

sequence due

anato

C-terminus

is

amino

of

now

was

inter-

hydrophilic

reversed

convenient

A-

acid,

great

more

clotting

(e-

A-peptide

were

the

the

place

AP-peptide

and

and

additional

normal

the

A-

chromato-

large

a single

on

of

demonstrated

were

peaks

site,

behaviour

fast

the acid

residue

be

(named

described, the

nmol

fibrinogens.

discovered)

earlier)

histidine.

results

was

before

obvious,

but

replaced

column,

fibrinogen

4.5

could

abnormal

one

min

i.e.

HPLC-system

from

conditions

ml,

detected,

min

1.16

peptides,

which

present

released

of

the

be

1.17

(eluting

, 0.150

abnorma 1 fibrinogen

could

luting

The

the

fibrinopeptides

fibrinopeptides

volume

is

phase disturbed

localisation

of

152

NORM

a)

AP

AY

JL· Μ

ROUEN

j

VJ

r o f i l e_ o f .f i b r i n p e p t i d e s r e l e a s e d o f F i g u r e 9. H P L C e l u t i o n rp...... a) n o r m a l f i b r i n o g e n ; s a m p l e vvoolluummee,, 0 . 1 0 0 m l , b) a n a b n o r m a l M ; sample volume, 0.150 ml, f i b r i n o g e n , i. e. f i b r i nn on gn e^ n nRnO, U" E~ N (see HPLC c o n d i t i o n s ) .

the

error

tion

showed

nopeptide In

in

another

additional low y i e l d

fibrinogen that

was

1 mole

Louisville.

The

quantitative

of

and

1 mole

abnormal

normal

fibri-

released.

abnormal

fibrinogen,

group

peptides

of

of

determina-

of

normal

Α-peptide

the

fibrinogen

appeared release

after and

Rouen

the

the

(Fig.9)

B-peptide.

difference

in

an

The the

153 I

F i g u r e 1 0 . H P L C e l u t i o n p r o f i l e of f i b r i n o p e p t i d e s r e l e a s e d o f an a b n o r m a l f i b r i n o g e n , i. e. f i b r i n o g e n S T O N Y B R O O K ( s e e H P L C conditions)·, sample volume, 0.150 ml.

METZ

B-R

J

v''

F i g u r e 1 1 . H P L C e l u t i o n p r o f i l e of f i b r i n o p e p t i d e s r e l e a s e d oi a n a b n o r m a l f i b r i n o g e n , i . ee. f i b r i n o g e n M E T Z ( s e e H P L C c o n d i ti o n s ) ; s a m p l e v o l u m e , 0 1 5 0 ml

154 retention the

time

between

additional

tively,lead ponded

to

analysis

to

the

the

substitution

retention the

time

was

the

also

Brook

Seems

normal,

mole

clusion

The

(Fig.

that

to

of

at

only

Αα-chain

in

cleavage

site

It

is w o r t h

mination which

of

may

aspects

be

and

One

of

is

thod

for

the

substituted that

important

for

of

of

more

in

po-

clearly

how

shift

advantages

the of

errors.

of

release

fibrinogen

chromatography proves

result

that

allows

of Α - p e p t i d e

is

should

only

the

con-

released be

the

located

indicates

Here

error

no was

arginine

a new

type

of

fibrinopeptide

A

found

the

of

to

the

be

in

thrombin

cysteine.

the

HPLC-method ideally

further

structure-function

This

11)

the

by

is

are

will

analysis the

sequence

site.

The

16, where

and

fibrinopeptide

molecules

(Fig.

released.

fibrinopeptides

for

suited

studies

of

the

for

deter-

kinetics,

physiological

relationships.

Phenylthiohydantoin

Amino

Acids

by H i g h

Per-

Chromatography

the m a j o r

method

abnormal

Metz

the

the y i e l d s

one mole

cleavage

mentioning

Liquid

cing

only

again

structural

look

corres-

residue

residue

shows

the

first

in f i b r i n o g e n .

position

Identification formance

is

the

at

acid

glycine

of

peaks

peptides

shows

the

is r e l e a s e d .

fibrinogen

abnormality

It a l s o

between

(4.56),respec-

later

new

acid

and

min

Amino

The

elucidation

At

in t h e

these

the

amino

(4.31)

29.74

case

calculating

thrombin

B,

This

to q u a n t i f y

10).

genetic

of

importance

because

the

HPLC all,

the

however

error

that

exchange

a peptide.

for

and

hypothesis.

a single

of Α - p e p t i d e

structural close

of

decisive

Stony one

the

HPLC-method of

min

of Α - p e p t i d e s .

a valine.

of

HPLC-method

Using

this

to

12 a g a i n s t

at 2 5 . 8 1

group

confirmed due

and A P - p e p t i d e s

assumption,

a second

hydrophobic sition

peaks

A-

problems

Edman

in t h e

(1,2)

identification

was of

phenylisothiocyanate

to the

find

a fast

reaction

and

end

sequen-

sensitive

products,

me-

i.e.

155 phenylthiohydantoin ready of

been

developed

Zimmerman,

acids,

paration

times. was

such

all

to

Here

this

as

acids.

Pisano

(11).

separating two

or

more

PTH

obtain

commer

cially,

by

the

Most

system

depending

system

too

will

be

in

1980

Lottspeich

some

number for

of

PTH

the

se-

on

long

a1 -

system

show

systems

using

have

gradient

a limited

acids,

or

systems

e.g.

solvent

amino

isocratic

developed

columns

analysis

described

which

(12).

conditions.

I nstrument

Column

Hewlett

Packard

tomatic

sampling

Solvents

1084

A equipped

system

wavelength

detector.

Prepacked,

Hibar

(5p)(Merck,

RT

and

250-4,

with

an

a fixed

au-

254

LiChrosorb

nm

RP-18

Darmstadt).

68.5%

0.01

31.5%

acetonitrile,(p.a.

lyse,

Merck,

+ 0.5%

Μ

sodium

acetate

pH

zur

5.2

Rückstandsana-

Darmstadt).

dichloroethane.

1 . 5 m 1 / in i η

Fl ow Oven

only

common

a simple

Several

purpose,

the

originally

HPLC

amino

and

requiring

of

difficult

for

Appella

disadvantages amino

(PTH)

62°

temperature

Analysis

16

time

Detection

3

limit

C min.

pmol .

Results

Al 1 c o m m o n system tween some

PTH

except these

amino the

two

yield

of

by

pair

of

derivates

difference

accompanied

acids

in

the

is m u c h

are

well

PTH-Gln/PTH-Ser. is

in

practice

retention

PTH-Glu,

PTH-Ser

(Fig.12)

resulting lower

no

times, from

than

as

resolved

To

by

the

distinguish

problem, PTH-Gln

as

t h e r e is

always

de-amidation , a n d

that

of

other

PTH

be-

is

as

the

amino

acids. It

should

be

pointed

out

that

the

HPLC

conditions

are

of

criti-

156 DΕ 2. T h e l o n g e s t s u l f u r chain we f o u n d is

-

S 2 Q - at p r e s e n t , b u t most p r o b a b l y even longer

chains do e x i s t . HPLC at b o n d e d alkane p h a s e s a p p e a r s to b e the only analytical technique to handle a l k y l p o l y s u l p h i d e s . GC s e p a r a t i o n almost e v e r l e a d s to e r r o n e o u s r e s u l t s b e c a u s e p o l y s u l f i d e s with n 2 > 2 a r e thermally u n s t a b l e . GC i n v e s t i g a t i o n s even of d i s u l f i d e m i x t u r e s a r e sometimes questionable since d i s u l f i d e s easily e x c h a n g e their R g r o u p s at elevated t e m p e r a t u r e s .

LC s e p a -

ration at polar a d s o r b e n t s like silica gel or alumina s u f f e r from i r r e v e r s i b l e a d s o r p t i o n and decomposition of p o l y s u l f i d e s . RPLC s e p a r a t i o n s a r e p e r -

171

f e c t l y r e p r o d u c i b l e and permit t r a c e a n a l y s i s as well as p r e p a r a t i v e w o r k . F i g . 6 shows the e t h y l p o l y s u l f i d e s up to ng = 8. T h e p e a k s have a good

Ν « >1 ( / ) ( / ) ! / ) (Η Γ-4 Γ4 LU

LU

LU

U) (/)

F i g . 6 - Chromatogram of diethyl p o l y s u l f i d e s . Column L i c h r o s o r b 5C18 ( 2 χ 10 cm in s e r i e s ) . Eluent 0 . 5 ml/min 90% MeQH + 10% H 2 0 . UV d e t e c t o r at 254 nm shape and can easily be i n t e g r a t e d . T h e l i n e a r i t y of In k 1 with ng is almost p e r f e c t for ng > 3. F o r the smaller members t h e r e is some alternation of the r e t e n t i o n v a l u e s . T h e values for even s u l f u r numbers are somewhat lower, for odd n u m b e r s somewhat h i g h e r than p r e d i c t e d from the linear r e g r e s s i o n . F i g . 7 shows the alternation of r e t e n t i o n for the t e t r a d e c a n e s from C . ^ to ^ 2 ^ 1 2 ' ^ e r e ** a p p e a r s that for ng = 1 to 5 the odd members e x h i bit high r e t e n t i o n , whereas from ng = 9 to 12 their r e t e n t i o n is slightly lower than t h e even s u l f u r number v a l u e s . T h e e f f e c t is fairly

small and is

a t t r i b u t e d to the alternation of permanent dipole moments in p o l y s u l p h i d e s , which are high for even and low for odd n g .

T h e magnitude of the e f f e c t

seems to indicate that v a r i a t i o n s in the dipole moment do not c o n t r i b u t e too much to c h a n g e s in r e t e n t i o n .

172 Fig. 7 R e t e n t i o n indices of t e t r a d e c a n e s ( n g + ης; = 14) v e r s u s s u l f u r atom number n g . Column R C C 5 u C 1 8 . 1 ml/min 95% MeOH + 15% H.,0

1500 Rl

nc+ns=U U00 -

1300

1200

1100

1000

In F i g . 8 t h e retention indices for a l a r g e r system of p o l y s u l f i d e s a r e shown, T h e r e t e n t i o n data were obtained on a R C C 5 u C 1 8 with 1 ml/min 9 5 / 5 MeOH/ Η2*3. F o r all g r o u p s R ^ n

polysulfides t h e r e g r e s s i o n s were c a l c u l a t e d :

Dimethyl-polysulfides:

In k 1 = (j)g

^ + 0g

Diethyl-polysulfides:

In k ' =

β

•—•

•

•

•

•

•

^

Ο

Ο

•

CN r_

•

•

•

•

'

ιη ιχ> Ο -μ β ω (0 Φ 3 •Η Μ Φ > Χ) β Ο υ tO 0) u ω β τ) •γΗ β Μ β m Μ 4-1 β Ο ο -Η 0) +J ω (0 Η εβ -Ρ γΗ β ) ο > α υ β ο u tn Φ υ β β φ β ίΗ 9 m β ω φ Μ

>1 Μ Φ > Ο ο\° υ ω

^

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

+1 +1 ιη CO ιη m

•

•

•

30 ng/ml; A > 10 ng/ml). Again, this expanded procedure, including standard additions method, is only necessary if slight variations within the normal (nonpathological) concentration range are to be determined. Although the results, obtained by the standard additions method, are very satisfying, some uncertainty remains as this method is only valid, provided that the losses during sample handling for the two corresponding samples are equal. This demands an excellent performance of the experiments. In our opinion, a further improvement of precision can be achieved by adding radiotracers (H-3 and/or C-14 labelled NA and A) to the samples before pretreatment is started. After the chromatographic separation the eluate is split and the two streams are fed separately but simultaneously, one into a radiochromatograph and the other into a chemical reaction detector. This procedure would have the advantage of an internal standardization with the same compounds without bringing up any problems concerning recovery rates and run times as were discussed before. In view of this, more experiments are necessary and already proposed. 5. Accuracy, precision, limits of detection For judgement of the accuracy of the described analytical procedure about 150 urine samples were analyzed with four independent courses of analysis. The resulting concentration data were statistically treated with computer programs.

256 A detailed report on the results of this intra laboratory comparison of the methods is in preparation and will be published soon

(148). In advance, it can be summarized that the

results obtained with the above method were confirmed by using HPLC coupled to an electrochemical detector as well as by the radiotracer method. In contrast, the classical THI method (10), which is still used for catecholamine analyses in many laboratories, yields systematically too high concentration values which show a considerable scatter

(149) .

The precision of a single value within a series of analyses was determined to be + 5 % and + 1 5 %

for HPLC coupled to a

chemical reaction detector and HPLC coupled to an electrochemical detector respectively. The limits of detection for the above course of analysis naturally depends on the sensitivity of the fluorometer used. In the present case it turned out to be 100 pg/ml and 70 pg/ml for NA and A respectively. 6. Resolution of the chromatographic

system

As was pointed out earlier, for routine analysis the resolution

(R) of the chromatographic system should be high to-

gether with short separation times. Therefore, in the practical case an optimization of the resolution becomes necessary. This is made possible by the variation of temperature and concentration of the mobile phase. In order to minimize time consumption in the optimization of the system it would be desirable to have a quantitative correlation between the resolution and these chromatographic parameters. Therefore the influence of temperature and mobile phase concentration on the resolution was investigated for the separation of NA and A on RP materials,using aqueous solutions of alkaline Perchlorates and perchloric acid as mobile phases. The experiments were run at temperatures of 0, 10, 20 up to 60 °C. Concentrations of the mobile phases ranged between 0.0025 Μ and 0.5 M. Standard solutions contained 1 pg/ml of NA and

257

0.5 pg/ml of A and for simplification the detection was performed fluorometrically via the native fluorescence (8 μΐ flow cell). For a better evaluation of the chromatograms the chart speed of the recorder was set to 10 cm/min. For calculation of R, t Q values were determined by using the intersecting point method which we have published previously (150) . The decrease of resolution with increasing temperature (T) in-γ · Τ dicates a functional correlation of the type R = a · e For the dependency of resolution on the concentrations (c) of the mobile phases a similar semilogarithmic correlation cannot be claimed. Following the experimental results, the mathematical treatment of the data was performed with the general equation

with a, £3, γ: factors and T: experimental temperature (°C) . Thus, using a multiple regression method, the factors a, Β and γ were calculated and are listed in Table 2. Considering the uncertainties of the factors which depend on the errors of the data points, only a qualitative rating of the fitting results is possible: a) factor a can be correlated to the particle size of the column packings; smaller particles (larger surfaces) result in larger a values, b) factor 3 is also dependent on the particle size; smaller particles result in smaller β values, c) factor γ can be correlated to the chain length of the chemically bound phase; increasing chain length results in larger γ values, and d) a significant influence of the cation (H+, Li + , Na + , K + ) on the factors a, 3 and γ can be excluded. Fig. 5 shows the degree of agreement between the calculated straight lines and the experimental data points for two medium concentrations and three column types. For concentrations

258

>-

tn 0 •Η tö >

0 4-1 to •P (0 Ό rH (0 -P C (1) Β •rH Μ α) α χ 0J Β

ο VH Μ-Ι Ό 0) -Ρ -Ρ >~

Ό C t0 ca. »

ΌΙ ω ^ 0 -μ υ ί0

(Ν dJ ιΗ tfl Eh

a in

CQ

Ln Τ— Ο Ο

ο νο Γ— •

ο

νο OJ Ο Ο

LT) m C\l Ο Ο

ιη ΚΩ CM Ο Ο

CO CN Ο Ο

ιη CN Ο Ο

η

VD ιη τ—

ΓVD τ—

00

CN τ—

Ο • Ο

•

•

•

•

Ο

Ο

Ο

Ο

CN r• (Ν

ιη CN • ΓΟ

(Ν

t0 ε

Ο Η U •Η

τ Ο ΓΗ υ to 2

Ο Η α

259

Fig. 5 Calculated and experimental resolutions as a function of temperature and mobile phase concentration for three column types (mobile phase: HC10J: Nucleosil 5-r 8* 0 . 1 Μ 0 .01 Μ Nucleosil 10- C : 0 . 1 Μ 8 0 .01 Μ Nucleosil 10- C

© © © © © ©

0. 1 Μ 0 .01 Μ drawn lines: calculated from eq. 1 using the factors from table 2; data points: experimental values; dotted line: 6 σ separation 18 :

higher than 0.5 Μ at low temperatures (Τ < 10 °C) and lower than 0.01 Μ at high temperatures (Τ ^ 40 °C) the experimental R values begin to deviate systematically from the fitting curves.

260

Although the resolution is not a fundamental parameter in chromatography and there is no obvious reason why R should obey equation 1, for the practical use of the given chromatographic system and separation problem, this empirical correlation can be useful for the optimization of the HPLC system. 7. Nucleosil - LiChrosorb For fast and sensitive routine HPLC analyses the selection of the optimal stationary phase is essential. Under the here described experimental conditions Nucleosil RP phases showed the best separation behaviour. In contrast to this, the separation time with the tested LiChrosorb RP material was considerably increased together with a reduction in sensitivity and a deterioration of resolution. An improvement of the chromatographic resolution obtained with LiChrosorb can be achieved by decreasing the temperature of the system, however, R remains worse compared to Nucleosil. The influence of the concentration of the mobile phase on the resolution, strange to say, is inverse compared to Nucleosil: lower concentrations result in higher resolutions. Using 0.01 Μ HCIO^ as mobile phase a baseline separation is already obtained at room temperature, however, the sensitivity is unacceptable. 8. A third, unassigned peak A typical chromatogram as obtained in routine HPLC analyses of NA and A in urine samples is shown in Fig. 6. From this it can be seen, that only the two catecholamines in question are detected. All other compounds, which are eluted from the column are suppressed by the chemical reaction system. Thus, in routine analysis, every three minutes an injection can take place. During the recently finished analyses of about 1000 urine samples it turned out, that almost 90 % of the chromatograms looked like the one shown in Fig. 6. However, in the residual 10 % of cases a third peak appeared 3.5 min after the adrenaline peak. The origin of this third peak could not be assigned until now. It is conspicuous that the compound

261 2 0 ~ι

signal

sample

2

[cm]

15 -

standards

10 -

sample

'n A

o

2

1

standards

na

'a «α

5 -

2

iuΗÜI 1°

~r 10

20

F i g . 6 C h r o m a t o g r a m of r o u t i n e d e t e r m i n a t i o n s of NA and A in u r i n e s a m p l e s S t a n d a r d s : 100 ng/ml N A , 40 n g / m l A 1 N A , 1 A : first injection without standard addition 1NA°, a

2NA ,

1A°: first injection with standard

addition

2 a : second injection without standard

addition

2XT„ NA

, 2, : s e c o n d i nJj e c t i o n w i t h s t a n d a r d a d d i t i o n A a a (for e x p e r i m e n t a l c o n d i t i o n s see F i g . 2 B; i n j e c t i o n intervals: '3 min) w h i c h is r e s p o n s i b l e for the t h i r d peak o c c u r e s only in the u r i n e of p a r t i c u l a r i n d i v i d u a l s b u t only u n d e r m o m e n t a r y

un-

k n o w n p h y s i o l o g i c a l c i r c u m s t a n c e s . N e v e r t h e l e s s , by a s l i g h t e x p a n s i o n of the i n j e c t i o n i n t e r v a l to 5 m i n the t h i r d peak a p p e a r s j u s t in b e t w e e n the two n e x t i n j e c t i o n s w h i c h h e l p s to save time

(Fig. 7).

262

Fig. 7 Chromatogram of routine determinations of NA and A in urine samples containing an unassigned compound (X) (for experimental conditions see Fig. 2 B; for indices see Fig. 6; injection intervals: 5 min)

9. Application to plasma samples The low limits of detection of the described course of analysis should make a quantitative determination of NA and A at human plasma catecholamine levels possible. In order to confirm this, plasma samples were deproteinized and centrifuged. Without any further pretreatment the plasma was directly injected into the HPLC system. The chromatogram shown in Fig. 8 reveals, that NA and A were totally separated from each other as well as from the other compounds also eluted. The peak ratio for NA and A is convenient which means that the described procedure can be applied to plasma samples without further optimization. The peaks in Fig. 8 correspond to

263

Fig. 8 Chromatogram of routine determinations of NA and A in plasma samples 1 1 , 2 1 : starting points of the first and second ο ο chromatogram (conditions .as described for Fig. 2 B; injection intervals: 7 min; C ^ = 67 pg abs., C A = 2 2 pg abs.) 6 70 pg/ml and 220 pg/ml for NA and A respectively. Column efficiency is not influenced by strange substances contained in the plasma. Thus, the suggested course of analysis allows the direct determination of NA and A in plasma samples within 7 minutes without time consuming pretreatment.

264 10. D i r e c t d e t e r m i n a t i o n o f N A a n d A i n In the a p p l i c a t i o n

to u r i n e

the time

urine

limiting factor of

described analytical method unequivocally

is s a m p l e

m e n t i n the s e m i - a u t o m a t i c b a t c h p r o c e d u r e . T i m e f o r t h e p a r a l l e l h a n d l i n g o f 12 u r i n e hour. Experiments

for s i m p l i f i c a t i o n

by leaving o u t the w a s h i n g

pretreat-

consumption

s a m p l e s is a b o u t of sample

the

one

pretreatment

steps yielded satisfactory

results,

a f a c t w h i c h h a s to b e c o n f i r m e d f o r h u n d r e d s o f s a m p l e s fore this m o d i f i c a t i o n procedure. eluates make

can be i n t r o d u c e d into

It m u s t be feared,

the column efficiency

frequent regenerations

necessary.

that by injecting is r a p i d l y

m o d e of pH a d j u s t m e n t in sample

standard

less

clean

reduced which

and equilibrations

In this connection,

ted. For example, by adding

the

of t h e

the p o s s i b i l i t y

of

would column

another

t r e a t m e n t is b e i n g

3 m l of tris b u f f e r

be-

investiga-

(pH 8.5)

to

5 m l o f a c i d i f i e d u r i n e t h e f a v o u r a b l e p H o f 8.3 — 8.5 c a n a c h i e v e d w i t h o u t using any indicator. However, l i t y of t h i s m o d i f i c a t i o n

the

applicabi-

for r o u t i n e a n a l y s i s has still

be established.

Some authors have reported on direct

m i n a t i o n s of NA

and A in u r i n e s a m p l e s w i t h o u t s a m p l e

treatment except centrifugation

would make

frequent regeneration and equilibration

In addition,

prethis

as the

will be altered and p l u g g e d after a few injections, irreproducible quench effects and peak

to

deter-

(125). In o u r o p i n i o n ,

proposal m u s t be r e j e c t e d for routine analysis,

be

column

which necessary. over-

lappings will make precise and accurate determinations

im-

possible . However,

the new techniques

very promising

(151,

of c o l u m n s w i t c h i n g

152), although e x t e n s i v e

are necessary before this technique analysis of catecholamines. the field of catecholamine

in HPLC

look

investigations

can be a p p l i e d to

routine

This will promote new efforts analysis.

in

265

Conclusions The presented course of analysis turns out to be very efficient for routine determinations of NA and A in urine and plasma. After pretreatment the samples are injected into a HPLC system coupled to a sensitive fluorometer via a selective continuous flow reaction system. More than 5000 analyses have been performed without any problems. The course of analysis allows a simple, fast, cheap and reproducible determination of catecholamines in body fluids. High precision and accuracy was confirmed by an intra laboratory comparison of the methods with four independent analytical procedures. When using an automatic sample processor for HPLC injection (intervals: 3 min for urine, 7 min for plasma) two technicians can analyze 100 samples per day. Therefore, the presented course of analysis is recommended to all fields of life sciences, when large numbers of urine and/or plasma samples have to be analyzed.

Acknowledgements The authors would like to thank Mrs. P. Deutschmann and Miss G. Baumhoer for excellent performance of the experiments. Special thanks are extended to Dr. W. Brockmann for statistical treatment of the analytical data. We are indebted to Dr. P. Knauth and Dr. F. Klimmer who made the urine samples available to us and to Dr. F. Diel who prepared the plasma samples. Finally we wish to express our sincere thanks for steady support and encouragement to Prof. Dr. Dr. J. Rutenfranz who initiated this study.

266

References 1.

L u n d , Α.: A c t a p h a r m a c o l . 5, 75 - 94

(1949).

2.

A n t o n , A. H., S a y r e , D. F.: P h a r m a c o l . E x p . T h e r . 360 - 375 (1962) .

3.

B e r t l e r , Α . , C a r l s s o n , Α . , R o s e n g r e n , Ε.: A c t a p h y s i o l . scand. 44, 273 - 292 (1958).

4.

C a m p u z a n o , H. C., W i l k e r s o n , J. E., H o r v a t h , S. M.: A n a l . B i o c h e m . 64, 578 - 587 (1975).

5.

C i a r l o n e , A . E.: M i c r o c h e m . J. 2Λ_, 349 - 354

6.

D i a m a n t , J., B y e r s , S. 0.: J. L a b . C l i n . M e d . 678 - 693 (1975).

7.

v. E u l e r , U. S., H a m b e r g , U.: S c i e n c e 110, 561

8.

v. E u l e r , U. S., F l o d i n g , I.: A c t a p h y s i o l . S u p p l . JJ_8, 57 - 62 (1955).

9.

v. E u l e r , U. S., L i s h a j k o , F.: A c t a p h y s i o l . s c a n d . 1 22 - 132 ( 1959) .

45,

10.

v. E u l e r , U. S., L i s h a j k o , F.: A c t a p h y s i o l . scand. 348 - 356 (1961 ) .

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SEPARATION OF CATECHOLOESTROGENS

AND THEIR MONOMETHYL

BY REVERSED-PHASE HPLC WITH TERNARY M O B I L E

PHASES

Ekkehard Kraas, Michael Schütt, Eberhard Zietz and Knuppen I n s t i t u t für B i o c h e m i s c h e E n d o k r i n o l o g i e Hochschule Lübeck D - 2 400 L ü b e c k , G e r m a n y

ETHERS

der

Rudolf

Medizinischen

Summary Procedures

for t h e s e p a r a t i o n of c a t e c h o l o e s t r o g e n s

and

isomeric monomethyl ethers by reversed-phase HPLC have developed using ternary mobile phases.

Systematic

their been

investiga-

t i o n of f i v e O D S - t y p e b o n d e d p h a s e s f r o m d i f f e r e n t

manufac-

t u r e r s e x h i b i t e d s i g n i f i c a n t d i f f e r e n c e s w i t h r e s p e c t to formance and suitability

for t h e s e p a r a t i o n of t h e s e

T h e b e s t s e p a r a t i o n w a s a c h i e v e d o n c o l u m n s of

per-

steroids

ODS-Hypersil

w i t h t e r n a r y m o b i l e p h a s e s of a c e t o n i t r i l e - w a t e r - a c e t i c for 1 7 - o x o - o e s t r o g e n s a n d m e t h a n o l - w a t e r - a c e t i c

acid

a c i d for

17-

hydroxy-oestrogens.

Introduction Catecholoestrogens monomethyl ethers

(i.e. 2 - a n d 4 - h y d r o x y o e s t r o g e n s ) (Fig.1)

have been isolated and

(E^) a n d o e s t r a d i o l

is c u r r e n t l y b e i n g u n d e r t a k e n

metabo-

(E2); i n t e n s i v e

to e l u c i d a t e t h e i r

r o l e i n t h e r e g u l a t i o n of g o n a d o t r o p h i n

secretion

research

potential [1].

For m a n y years our r e s e a r c h has b e e n d i r e c t e d towards the lation,

i d e n t i f i c a t i o n a n d q u a n t i t a t i o n of

and their monomethyl ethers

their

identified

f r o m m a m m a l i a n b o d y f l u i d s as a n i m p o r t a n t c l a s s of l i t e s of o e s t r o n e

and

iso-

catecholoestrogens

in b i o l o g i c a l m a t e r i a l by a v a r i e -

Practical Aspects of Modern H P L C Copyright © 1982 by W a l t e r de Gruyter &. Co., Berlin · N e w York Printed in Germany

276

ty of analytical methods [2-7]. The elaboration of a specific radioenzymatic assay for free catecholoestrogens [8] , and our interest in high-performance liquid chromatography (HPLC) as a prepurification method for steroid hormones prior to radioimmunoassay stimulated a systematic investigation of the HPLC behaviour of catecholoestrogens and their monomethyl ethers. Recently, Shimada and coworkers reported separations of catecholoestrogens with electrochemical detection [9]; the present paper deals with the chromatographic behaviour of the title compounds on different reversed-phase octadecylsilane (ODS) HPLC columns using binary and ternary solvent systems as mobile phases.

Materials and methods Apparatus. The liquid Chromatograph consisted of an HPLC pump (Model 6000 A, Waters Assoc., Milford, USA), an UV-detector with variable wavelength (Model SF 770, Kratos Schoeffel Instr., Germany), an injection valve with a 200 μΐ-ΐοορ (Model 7120, Rheodyne, Berkeley, USA) and a single-pen recorder (Model PM 8202, Philips, Holland). A constant-pressure gas amplifier pump (Model Haskel MCP 110, Dr. Knauer, Berlin, Germany) was used for column packing. Blank stainless steel columns (120 mm χ 4.6 mm I.D.) and directly coupled precolumns (40 mm χ 4.6 mm I.D.) were purchased from Dr. Knauer, Berlin. The ODS columns used in this study are listed with their specifications, suppliers, etc. in Table I. Degassing of mobile phase was performed in an ultrasonic bath (Model R 102 P; 240 W; Bandelin KG, Berlin, Germany). Chemicals and reagents. For self-packing of HPLC columns two different commercially available supports were used: ODS-Hypersil 5 μπι (Shandon Southern Products, Cheshire, Great Britain) and Spherisorb S 5 ODS (Phase Separation, Oueensferry, Great Britain).

277

Oestrone (E^), oestradiol (E2) and oestriol (E^) were gifts from Schering AG (Berlin, Germany). 2-Hydroxyoestrone (2-OHE^, 2-hydroxyoestradiol (2-OHE2), 2-hydroxyoestriol (2-OHE3), 4-hydroxyoestrone (4-OHE^) and 4-hydroxyoestradiol (4-OHE2) were prepared according to Stubenrauch und Knuppen [10]; the monomethyl ethers 2-hydroxyoestrone 2-methyl ether (2-OHE^ 2-Me), 2-hydroxyoestrone 3-methyl ether (2-OHE^ 3-Me), 2-hydroxyoestradiol 2-methyl ether (2-OHE2 2-Me), 2-hydroxyoestradiol 3-methyl ether (2-OHE2 3-Me), 2-hydroxyoestriol 2-methyl ether (2-OHE2 2-Me) and 2-hydroxyoestriol 3-methyl ether (2-OHE3 3-Me) were synthesized by the method of Fishman and coworkers [11,12]. The isomeric monomethyl ethers of 4-OHE 1 , i.e. 4-hydroxyoestrone 4-methyl ether (4-OHE^ 4-Me) and 4-hydroxyoestrone 3-methyl ether (4-OHE^ 3-Me) were prepared by methylation of 4-OHE^ with diazomethane and separation by column chromatography on alumina as described for the preparation of 2-OHE1 2-Me and 2-OHE1 3-Me [13]. 4-Hydroxyoestradiol 4-methyl ether (4-OHE2 4-Me) and 4-hydroxyoestradiol 3-methyl ether (4-OHE2 3-Me) were prepared analogously from 4-OHE2. Methanol (puriss.; Merck, Darmstadt, Germany) was purified by fractionated distillation; acetonitrile

(p.a.; Merck) was used

as obtained. Water was taken from a Milli-Q-system

(Milipore,

Bedford, USA), which was supplied with deionized water. Other chemicals were of analytical grade and were used without further purification. Chromatographic techniques. HPLC columns (Table I) were either purchased or self-packed by the upward slurry technique essentially as described by Becker [14], with isopropanol as high viscosity slurry medium and methanol as pressure fluid. The slurry concentration was approx. 10% (w/v) and the applied pressure was 300-350 bar. Column efficiency (N) was checked by separating a mixture of benzene and toluene using methanol-water (65:35, v/v) as mobile phase with a flow of 1.0 ml/min. The toluene peak was recorded with increased recorder chart speed (320 mm/min) and evaluated by the half peak height me-

278 td ~ ιΗ

β 0 •Η •μ α •Η Μ υ ω ω Ω ^

Ε Ε

•—'

Ω • Η

β 0 •Η ι ω Λ β -μ ω CP Ε β •Η ω α ω Ν -Η Cfl α) rH ο Ε -.Η =1 -Ρ — Μ

β •Η

υ (Ö α β

ω ß Ε Η 0 υ

ε

ι-Η 0 U

u ^ 04

μ 4) rH

ΙΗ

Ο β

0 •Η

-μ (0 υ •Η m •Η υ Φ α. CO Η ω ιΗ Λ id ΕΗ

CO Ω Ο

co Ω Ο

CO Ω Ο

CO Ω Ο

σι • ΓΟ

ο 00

Τ

ο ο η

ο ο r—

Ο CN T—

ο ιη τ—

ο ιη

ο τ—

LO

LO

LT)

ιη

Η td •Η U Μ α) g Ε 0 υ

ι—I (d •Η Ü Μ Ο) ε g 0 υ

ιΗ id •Η υ

η

ω

ω Ό rd Ε ι ΙΗ rH 0) (0

u ο ω 01

•

•

u 0 to cn

ft ft

w

C O Μ Ο) -μ id

Μ

ω -μ td &

ft ft

υ

U)

χ

ε id ss

ft

ω


0) Μ Μ -Η

•

Κ

ι-Η 45 lytical

000 p l a t e s / m w e r e u s e d f o r

ana-

studies.

Water was degassed by ultrasonication

and vacuum

(25 m b a r )

u s e d f o r t h e p r e p a r a t i o n of m o b i l e p h a s e s , w h i c h w e r e shortly before use by Analytical

further ultrasonication

procedures.

free catecholoestrogens ascorbic acid

(1g/l)

degassed

for 3 0 min.

Oestrogens and catecholoestrogen

methyl ethers were dissolved

in e t h a n o l

were dissolved

and acetic acid

and

(approx.

100

in e t h a n o l

(1 m l / 1 )

as

mono-

mg/1);

containing antioxidants,

a n d w e r e k e p t in dark b r o w n v i a l s a t 4 °C. For HPLC analyses portions

(approx.

10 μΐ)

of t h e s e

w e r e c o m b i n e d a n d e v a p o r a t e d u n d e r a s t r e a m of N 2 ; dues were dissolved the HPLC valve. sitivity

in m o b i l e p h a s e s

(25 μΐ)

2-OHE1

droxysteroids

17-oxo-steroids

and injected

sen-

investigated

sepa-

3 - M e , 4-OHE.j 4 - M e a n d 4-OHE.j 3 - M e ) a n d f o r

17-hy-

(i.e. E 2 ,

lumns

(see T a b l e

(i.e. E 1 ,

in

2-OHE1

4-OHE2

2-OHE2, 4-OHE2,

4-Me and 4-OHE2

3-Me)

I) u s i n g b i n a r y

2-OHE2

2-Me,

and ternary

solvent

T h e s e c o n s i s t e d of v a r i o u s v o l u m e

of m e t h a n o l - w a t e r

or a c e t o n i t r i l e - w a t e r

and methanol-water-acetic acid for the ternary

acid or

solvent

co-

systems fractions

for the b i n a r y

systems

acetonitrile-water-acetic

systems.

T h e m o b i l e p h a s e s w e r e a d j u s t e d to e l u t e (usually 4-OHE^

3-Me for the

2-OHE2

on five different ODS

as mobile phases.

4-OHE2

into

2-OHE.j , 4 - O H E 1 ,

3-Me,

chromatograms

resi-

a.u.f.s.

Separation p e r f o r m a n c e of columns were 2-Me,

the

T h e U V - p h o t o m e t e r w a s r u n a t 280 n m w i t h

s e t a t 0.04

rate runs for

solutions

the

3 - M e for the

17-hydroxy-steroids)

last peak of

the

17-oxo-steroids

within

15-18

and

min.

Results Initially, binary

s o l v e n t m i x t u r e s of a c e t o n i t r i l e - w a t e r

methanol-water were

i n v e s t i g a t e d as m o b i l e p h a s e s

paration of catecholoestrogens

for the

and their monomethyl

or se-

ethers;

280

4-OH Ε, 4-OHE2

CH3O

2-OHE! 2-Μ· 2-OHE2 2-Μ·

2-OHE^ 3-Me 2-0HE2 3-M*

OH 4-OHE^ 3-Me 4-0HE2 3-Me

CH30 4-OHE^ 4-M« 4-OHE2 4-Me

Fig.1. Formulas of primary oestrogens, catecholoestrogens and their monomethyl ethers. (17-oxo-steroids, R : = 0 ; 17-hydroxy-steroids, R : -OH. Abbreviations see text).

281

under these conditions only very poor resolution and tailing peaks were observed on all ODS columns tested in this study. Addition of small amounts of acids to the binary mobile phases as usually employed for suppression of ionization did not improve the separation performance remarkably. On the other side, when ternary solvent systems containing high amounts of acetic acid (9-10% by vol.) were used as mobile phases, significant differences in separation performance were observed despite the fact that all columns were of the ODS-type bonded phase. The results obtained for both groups of steroids (see above) can be summarized as follows: 1)

On μBondapak C^g columns the use of ternary mobile phases did not improve the poor separation patterns for the monomethyl ethers notably; only for the resolution of 2- and 4-hydroxyoestrogens a slight increase of resolution was observed. Fig. 2 shows the chromatograms for the 17-oxoand 17-hydroxy-steroids obtained with ternary mobile phases on this column.

2)

Effective improvements of resolution could be achieved on the four remaining ODS columns (see Table I) using the ternary mobile phases.

3)

For the group of 17-oxo-steroids (see Fig. 1) the most complete separation patterns were obtained on columns of Lichrosorb RP-18 and ODS-Hypersil (Fig. 3b and 4b), although 2-OHE.j and 4-OHE^ were left unresolved even under these conditions; Fig. 3a and 3b demonstrate the effect of acetic acid on resolution and peak form as observed on Lichrosorb RP-18 columns.

4)

For the 17-oxo-steroids all ODS-columns gave better separations with ternary mobile phases of acetonitrile-wateracetic acid in comparison with ternary eluants of methanol-water-acetic acid; the separation patterns observed for both types of mobile phases on ODS-Hypersil are demonstrated in Fig. 4.

5)

For the 17-hydroxy-steroids good separation of E~ and the

282

u

11 12

13

89

10

VJ

w 2

Fig.2.

a

C^g

b

10 12 Κ Time (min)

by

2 = 4-OHE.j ;

by 3

of

Mobile

vol.),

(right):

(50:40:10,

3-Me;

β

i.

6

8

10

12

U

16

Time (min)

17-oxo-

and

17-hydroxy-steroids

on

columns.

(left):

(35:55:10, Fig.2

6

Chromatograms

μΒοηά3ρβΚ Fig.2

4

1.5

= E. ] ;

11

= 4-OHE2

4-Me;

14

= 4-OHE2

3-Me.

1.5 4

methanol-water-acetic

ml/min.

Peak

= 4-OHE.]4Me;

3-Me; 12

acetonitrile-water-acetic

8

acid

ml/min;

Mobile-phase,

vol.),

7 = 4-OHE1

phase,

= 4-OHE2;

= 2-OHE2

2-Me;

acid

i d e n t i f i c a t i o n : 1 = 2-OHE^·

5

= 2-OHE1

9

= 2-OHE2; 13

2-Me;

= 2-OHE2

10

=

6

=

E2;

3-Me;

2-OHE1

283

υU 2

t.

6

8

10

12

14

16

Time(min)

18

Τ 2

1 A

VJ 1 6

1 8

1 10

1 12

VJ 1 U

1 16

1 18

1— 20

T i m e (min)

Fig.3. Chromatograms of 17-oxo-steroids on Lichrosorb RP-18 columns. Fig.3 a (left): mobile phase, acetonitrile-water

(39:61, ν/ν) ,

1.5 ml/min. Fig.3 b (right): mobile phase, acetonitrile-water-acetic acid (32:58:10, by vol.), 1.5 ml/min. For peak identification see Fig.2.

284

monomethyl ethers were achieved on Rad Pak C ^

cartridges

and Lichrosorb RP-18 columns with ternary eluants of acetonitrile-water-acetic acid, although 2-OHE 2 and 4-OHE 2 were left unresolved (Fig. 5). 6)

Complete resolution of the 17-hydroxy-steroids including 2-OHE 2 and 4-OHE., could be performed solely on ODS-Hypersil; exceptionally, in this case a ternary mobile phase based on methanol gave the superior resolution (cf. Fig. 6a and 6b); the elution order of 2-OHE2 3-Me (peak 13) and 4-OHE 2 3-Me (peak 14) usually observed on the other columns was inverted on ODS-Hypersil.

7)

A simultaneous separation of all monomethyl ethers (see Fig. 1) in a single HPLC run was not feasible even under optimal conditions due to the fact that some 17-oxo- and 17-hydroxy-monomethyl ethers displayed very similar retention times. As described for the separation of 2-OHE^ 2-Me, 2-0HE1 3-Me, 4-0HE1 4-Me, 2-OHE2 2-Me, 2-OHE 2 3-Me and 4-OHE 2 4-Me, this can only be accomplished by a combination of Sephadex LH-20 gel chromatography and HPLC, where the gel column preseparates the mixture of monomethyl ethers into two groups of steroids: the 17-oxo- and 17-hydroxy-monomethyl ethers [8].

Applications. The ternary mobile phases developed in this study have found several· routine applications in our laboratories, including e.g. (i)

the separation and isolation of radioactively labelled catechol monomethyl ethers as final step of a radioenzymatic assay for free catecholoestrogens [8];

(ii)

the final work-up and separation of tritium-labelled E^, 4-OHE 2 , 2-OHE 2 and E 2 resulting from incubations of [ 3 H]E 2 with mammalian tissues (Fig. 7);

(iii) the control of chemical microsyntheses; Fig. 8 demonstrates the separation of the isomeric monomethyl ethers 2-OHE3 2-Me and 2-OHE 3 3-Me as obtained from methylation of 2-OHE

with diazomethane in a ratio of approx. 1:1.

285

ι

U

6

8

10

12

Time (mi η)

Η

0

—ι 2

1

U

1

6

1

8

J

1

10

1

12

r~ Κ

Time (min)

Fig.4. Chromatograms of 17-oxo-steroids on ODS-Hypersil columns . Fig.4 a (left): mobile phase, methanol-water-acetic acid (50:40:10, by vol.), 1.0 ml/min. Fig.4 b (right): mobile phase, acetonitrile-water-acetic-acid (35:55:10, by vol.), 1.5 ml/min. For peak identification see Fig.2.

286

8*9

10

h 8

10

12

Κ

16

0

Time(min)

2

Hi

6

8

10

12

14

16

Time (min)

Fig.5. Chromatogram of 17-hydroxy-steroids on columns of Lichrosorb RP-18 and Rad Pak C^g. Fig.5 a (left): column, Lichrosorb RP-18; mobile phase, acetonitrile-water-acetic acid (32:58:10, by vol.), 1.5 ml/min. Fig.5 b (right): column, Rad Pak C^g,· mobile phase, acetonitrile-water-acetic acid (50:40:10, by vol.), 1.0 ml/min. For peak identification see Fig. 2.

287

12

11

11 12

14 9

Μ

13

10

10

W L_J vi \ — 1 1 1 1 1

4

6

8

10

12

1

Η

1—

16

Time(min)

Ii 2

4

6

θ

10

12

14

16

Time(min)

Fig.6. Separation of 17-hydroxy-steroids on ODS-Hypersil columns . Fig.6 a (left): mobile phase, acetonitrile-water-acetic acid (35:55:10, by vol.), 1.0 ml/min. Fig.6 b (right): methanol-water-acetic acid (50:40:10, by vol.), 1.0 ml/min. For peak identification see Fig.2.

288 1

\J

VJ ι —I- —I— —I 6 8 to 12

1 14

1 —I— 18 16

Time (min]

Fig.7. Separation of oestradiol and its metabolites formed in mammalian tissues. Conditions: column, ODS-Hypersil; mobile phase, methanol-wateracetic acid (35:55:10, by vol.), 1.5 ml/min. Peak identification: 1 = E,; 2 = 4-OHE„; 3 = 2-OHE 0 ; 4 = E„.

289

6

8

10

12

14

Time [minj

r 8

10

1 j— 12

14

Time [min)

Fig.8. Separation of 2-OHE^ and its isomeric monomethyl ethers. Conditions: column, ODS-Hypersil; mobile phase, acetonitrilewater-acetic acid (20:70:10, by vol.), 1.0 ml/min. Fig.8 a (left): product mixture after methylation of 2-OHE^ with diazomethane. Fig.8 b (right): same mixture spiked with authentic 2-OHE 3 . Peak identification: 1 = 2-OHE 3 ; 2 = 2-OHE 3 2-Me; 3 = 2-OHE 3 3-Me.

290

To the best of our knowledge this is the first reported separation of these isomeric monomethyl ethers of 2-OHE^.

Conclusion Most reversed-phase HPLC separation are carried out today with binary eluants of either acetonitrile-water or methanol-water, in some cases with addition of small amounts of acids or bases for suppression of ionization. Only in recent time an increasing number of publications have reported applications of ternary mobile phases in order to achieve selectivity effects and resolutions which can not be accomplished with binary eluants [cf. 15-18]. In the present study effective improvements of resolution could be obtained for catecholoestrogens and especially for their isomeric monomethyl ethers on some ODS columns, when ternary mobile phases containing high amounts of acetic acid were employed. Systematic investigations displayed significant differences among five bonded ODS phases from different manufacturers with respect to their performance and suitability for the separation of this class of steroids. The best separations were achieved on columns of ODS-Hypersil using ternary mobile phases of acetonitrile-water-acetic acid for 17-oxo-steroids and methanol-water-acetic acid for 17-hydroxy-steroids.

References 1.

Ball, P., Knuppen, R.: Acta endocrinol. Suppl. 232, 1 (1 980) .

2.

Gelbke, H. P., Knuppen, R.: Acta endocrinol. Suppl. 173, 1 1 0 (1973) . Hoppen, H.-O., Siekmann, L.: Steroids 23^ 17 (1974).

3.

291

4.

Emons, G., Ball, P., Knuppen, R.: Acta endocrinol. Suppl. 208, 119 (1977).

5.

Ball, P., Emons, G., Haupt, 0., Hoppen, H.-O., Knuppen, R.: Steroids , 249 (1978).

6.

Ball, P., Reu, G., Schwab, J., Knuppen, R.: Steroids 33, 563 (1979).

7.

Emons, G., Mente, C., Knuppen, R., Ball, P.: Acta endocrinol. 97 , 251 (1981 ) .

8.

Knuppen, R., Zietz, Ε., Ball, P., Kraas, E.: J. Steroid Biochem., submitted for publication.

9.

Shimada, K., Tanaka, T., Nambara, T.: J. Chromatogr. 223, 33 (1981).

10. Stubenrauch, G., Knuppen, R.: Steroids 28, 733 (1976). 11. Fishman, J.: J. Am. Chem. Soc. 80, 1213 (1958). 12. Fishman, J., Tomasz, Μ., Lehman, R.: J. Org. Chem. 25, 585 (1960). 13. Knuppen, R., Breuer, Η.: Hoppe-Seyler1s Z. Physiol. Chem. 346 , 1 14 (1966) . 14. Becker, N. : GIT Fachz. f. Lab. 22^, 403 (1978). 15. Karch, K., Sebestian, I., Halasz, I., Engelhardt, Η.: J. Chromatogr. 122, 171 (1976). 16. Bakalyar, S. R., Mcllwrick, R., Roggendorf, Ε.: J. Chromatogr. 142, 353 (1977). 17. Roggendorf, Ε.: GIT Fachz. f. Lab. 23, 908 (1979). 18. Gluck, J. A. P., Shek, E.: J. Chromatogr. Sei. J_8, 631 (1980) .

QUANTITATIVE D E T E R M I N A T I O N OF ARYLOXYPROPANOLAFIINES ORGANS OF THE RAT BY ION-PAIR R E V E R S E D - P H A S E

IN PLASMA

AND

HIGH-PERFORPIANCE-

LIQUID-CHROPIATOGRAPHY

H. UJinkler and B. Lemmer Centre of P h a r m a c o l o g y ,

J.U. Goethe-University, Theodor-Stern-Kai 7

D—6000 F r a n k f u r t / W a i n , Federal Republic of Germany

Introduction

Our investigations were focused on the studies of the kinetic

be-

haviour of Q - a d r e n o c e p t o r blocking drugs in plasma and various gans of the light-dark

synchronized rat (1,2). In the

or-

literature

a great number of m e t h o d s were d e s c r i b e d (3-25) to quantify

Q-re-

ceptor blockers in plasma and urine, however, only one or at least two selected drugs were investigated uiith a single method. O - a d r e n o c e p t o r blacking drugs exert their p h a r m a c o l o g i c a l

Since effects

at the level of various organs, such as heart, lung and brain, drug c o n c e n t r a t i o n s in these target organs are of utmost

interest

when studying the dynamic and kinetic behaviour of these

compounds.

Therefore,

it was necessary to develop a fast and sensitiv

method

in order to be able to analyze a great quantity of p l a s m a and tissue samples of different Q-receptor blockers with mainly one

standard

method. The method was described in detail in (26). It is a standard dure to analyze a r y l o x y p r o p a n o l a m i n e s

proce-

(AOPA), since most of the

Q-receptor blockers are compounds of the AOPA type, which differ polarity on behave of the different aryl groups. The

in

Q-receptor

blockers p r o p r a n o l o l , metoprolol and atenolol were chosen as r e p r e sentative compounds on account of the different polarities vary by about three orders of magnitude

(Tab.1). The

differences

in polarity are also reflected by the differences in plasma

Practical Aspects of Modern H P L C Copyright © 1982 by Walter de Gruyter «St Co., Berlin · New York Printed in Germany

which protein

294 binding as well as by the main routes of elimination of these three compounds. Neither urine samples nor metabolites were included in the study, since only atenolol undergoes renal elimination, u/hereas propranolol and metoprolol are metabolized by the liver. Furthermore, the only metabolite of interest, 4-hydroxypropranolol, not contribute to the pharmacological effect after acute

does

intravenous

administration of propranolol.

Tab.1. The molecular structure and some properties of the compounds investigated (partion coefficients from 27). ARYL-OXY-

Ar

PROPANOL

(AOPA)

Η CH3 OH ch 2 -ch-ch 2 Ν -CH CH3

PROPRANOLOL Ar =

Part. Coeff. (oct/buff pH 7.0)

- AMINE

METOPROLOL

h3c-o-ch2-ch2-(Ö)-

ATENOLOL 0

,C-ch2-(h2n

5.36

0.18

0.003

Plasma Prol. Bind. (·/.)

»0

12

0.99).

In addition, chromatograms of ex vivo metoprolol

brain

samples are shown in Fig.2. It can be seen that the range of drug concentrations studied in spiked samples was also achieved 60-120 min after i.v. application of metoprolol to the rats

PROPRANOLOL

ng/g

0

175

ATENOLOL

γ-

0 Ü ' 4 6 min JL ng/g

BRAIN

r r

I ilt I ι I

0 2 A S min

0

10

(Fig.2,bottom)

*3β

»00

Peak hvlght

1000

·77

BRAIN

JL 20

υ

iV- i JL

50

SO Peak htlghl

100

Fig.3. Representative chromatograms of spiked brain samples. Upper part: spiked propranolol samples (0B77 ng/g); lou/er part: spiked atenolol samples (ΟΙ 00 ng/g; arrows indicate the respective retention times.

100

301 In Table 3 the ranges of drug concentrations and the recoveries obtained for propranolol, metoprolol and atenolol in plasma and the various organs are summarized, fhe recovery for metoprolol in plasma mas 47% and in tissue 56-64%, while for atenolol recovery n/as in the range of 25-37%. For propranolol the recovery in plasma was 70% while it varied from 16% in liver tissue to 42% in heart tissue«

Tab.3. Ranges of drug concentrations and recoveries obtained from spiked plasma and tissue samples for p r o p r a n o l o l , m e t o p r o l o l a n d

PROPRANOLOL Rang· (ng/sample)

organs

Recovery C M *

atenolol

METOPROLOL Range (ng/sample)

Recovery (·«.)*

ATENOLOL Range (ng/sample)

Recovery ("fc)*

plasma

4.4 -

88

70.4 12.5

4.4 -

26

47.2 12.2

20

500

30.6 11.4

liver

8 . 8 - 438

16.1 10.9

8.8 - 440

58.9 10.8

100

2000

30« »0.3

lung

ββ.Ο - 4380

38.4 13.2

8.8 - 440

56.4 2 1.6

100

2000

37.2 « 1.3

muscle

8.8-

351

36.4 13.7

8.8 - 132

58.0 1 1.5

100

2000

37J8 10.4

h»arl

8.8 -

351

41.6 13.4

8.8 - 132

63.6 1 1.6

50

1000

37.1 11.2

8 8 . 0 - 1750

25.3 H . 3

8.8 - 440

57.5 ι 1.8

10

100

28Λ >0.5

8.8 - 220

64.1 ι 3.3

20

500

2SJ >06

brain kidney

'Mton

-

i S E M of 10-12 s a m p l e s

Additional experiments with varying amounts of tissue revealed that the recovery of propranolol uas dependent upon the amount of tissue extracted (Fig.4); thus the limiting factor for the recovery of this highly lipophilic compound is the ratio of tissue weight to volume of perchloric acid. Such a dependence uas not observed for metoprolol and atenolol,

respectively.

302 100η

METO

ϋ 50-

/1012 = 106 photons/s

signal to noise:

10 : 1

ion by fluorescence and absorption

Minimum concentration of 0.1 ng/ml have been detected by fluorescence under favorable conditions.

But it should be emphasized here that increasing the

excitation intensity will not increase the sensitivity of a fluorescence detector proportionally unless the stray light from the excitation source to the photomultiplier is reduced at the same time. in the cell design is of limited value.

Taking isolated steps

To reduce stray light, double mono-

chromators or well-blocked interference filters should be used.

Also,

using laser excitation (HeCd or wavelength-doubled argon-laser pumped dyelasers at the moment) may reduce stray light and simplifies instrument geometry.

In addition to more simple optics, lasers give the possibility of

two photon excitation (1), which greatly enhances selectivity because of

319 different selection rules for one and two photon absorption processes.

As

more molecules do fluoresce than normally is assumed, detection by fluorescence may be widely used.

It is especially sensitive if the fluorescence

quantum yield is high. Thus detection by fluorescence is advisable if sensitivity or selectivity should be increased over detection by UV absorption.

Clearly, restrictions

with respect to the excitation wavelength are the same as for detection by UV absorption. 2.1.4. Other optical detectors. Detectors using optical activity, infrared absorption and light scattering, have been used in special cases where selectivity has been the main goal at the expense of sensitivity. Highly selective and very sensitive are the Thermal Energy Analyzer (TEA) (2) and detection monitoring chemiluminescence. ective to N-nitroso-compounds.

The TEA is extremely sel-

In a catalytic pyrolyzer, the N-NO bonds

are ruptured which yields NO' radicals. vent) and fragments are frozen out. give electronically excited N0£.

Most other compounds (also sol-

The nitrosyl radicals are oxidized to

The light emitted by relaxation to the

ground state (the intensity of which is proportional to the concentration of N0£) is monitored. With chemiluminescence as the physical effect used to detect small concentrations of certain metal ions, e.g. Sn(11), Co(II), Cu(II), the chemiluminescence of luminol is monitored.

It is catalytically enhanced (or

quenched in some cases) by these ions (e.g. 3) .

Although extreme

sensi-

tivity of 1 pg/ml has been reported, the method is experimentally complicated. It should be emphasized that the high sensitivity achieved with the TEA and by using chemiluminescence arises from the fact that due to chemical excitation, there is no stray light nor are there geometrical problems with two separate light beams as in the case of fluorescence. Flame photometric detection has also been used in HPLC for compounds con-

320

taining sulfur and phosphorus.

But because of restricted choice of the

chromatographic system i t scarcely may be used in routine work.

2.2. Remarks on the application of electrical detectors 2.2.1. Electrical conductivity and electrochemical detectors. Electrical conductivity as well as electrochemical reactions can also be used for detection in HPLC.

For both methods commercial instruments are available.

Nevertheless, using these techniques requires a great deal of experience to get full advantage of the highly sensitive and selective methods.

This i s

valid especially for electrochemical detection where, for example, constant flow is required and where the eluate must be absolutely free of oxygen and other disturbing impurities.

Using H^O, I^O/MeOH, MeOH or MeCN or similar

solvents, electrochemical detection may be used i f classical detectors f a i l , either because of very weak UV absorption or because refractometric detection is not sensitive enough.

See Kissinger (4) and references cited

therein. 2.2.2. The dielectric constant detector. Often i t is discussed whether a dielectric constant detector gives improved s e n s i t i v i t y and performance over a refractometric detector.

In principle, both methods measure bulk proper-

ties of the eluate, thus temperature and solvent impurities affect the s t a b i l i t y of the baseline directly. be adequate.

In this respect both systems seem to

In order to compare the s e n s i t i v i t y of both systems, the well

known relation between the dielectric constant ε

and the refractive index

η and the molecular dipole moment μ and p o l a r i z a b i l i t y tx i s compared for radiowaves (1 MHz) and l i g h t for a two component/single phase system, that is for a solution of a compound m with concentration given by i t s mass fraction w in a solvent s with density 9 S .

Μ is the molar mass.

With good

approximation for the aims of this chapter i t is (n 2 -1 )(2n z + 1) 2

3n

s

3ε

ο

(1-w) w -tra + tree Μ s

(1)

321 ( ε -1) (2c +1) 3 ε

Ν

Α 3 3 ε.

(1-W) L—

M

ßM2s)+_(tr

(tr« s +

(2)

S

For more details see Liptay (5) . £q

is the permittivity of the vacuum, ß =

stant k and the temperature T, (n + 1) (2n 3H5

+1)

(kT) ^ with the Boltzmann con-

is Avogadro's number.

w hich

With

is a very good approximation for

(3)

5

(4)

~

nearly all solvents, and with

^

2ε+1 3~e—


jm particle size. Eluent: trichlorobenzene; flowrate: 1,0 ml/min; RI-detector, χ 8. Ferritin

0

10

20

30

40

50

Elution volume, V^(ml)

Fig. 8: Separation of proteins on DIOL-columns with aqueous phosphate buffer as eluent (34).

378 0.2

εC

ο ο CN Ο

r

MYOGLOBIN Horse MW: 17 800

0.1

Ζ
pK HA +2 5 where

[OH* ]

Κμ. =

·

[A

]

^

M A

(21) [HA]

Similarly we can define the capacity factor of a weak base k

b/BH+

i n

protic equilibrium as k k

R/RM+

Β

+ Kk

BH

=

+ ·

[oh+]

— K BH +

ϊ

B / B H

pKB+2 and k bh +

1S

the capacity factor of the protonated base at pH < pKB-2

where K

BH+

= ( [0H

3

]

'

[ 8 ] ) / [BH+]

a

392 6. Reversed-Phase-Ion-Pair-Chromatography

(RP-IPC)

In RPC retention of charged elutes can be augmented by the presence of suitable counter ions, which have a substantial hydrophobic moiety, in the mobile phase. This counter ions or "hetaerons" (28) belong to a group of detergents such as alkylsulfonates or tetraalkylammonium compounds. ι

The basic parameters of retention in RP-IPC, besides those, mentioned above, are the concentration of the hetaeron, the ionic strength and the pH of the eluent and the total hydrophobic surface area of the hetaeron. The influence of these parameter is shown in the following figures (Fig. 18, 19 and 20).

O

20 40 HEXYLSULFATE

60 [mM]

Fig. 18: Dependence of the capacity factor of charged catecholamine derivatives on the concentration of n-hexylsulfate in the neat aqueous mobile phase. Conditions are given in ref. 28.

Usually the increase of hetaeron concentration increases retention as shown in Fig. 18 to a maximum, after which at high hetaeron concentrations a monotonic decrease of retention can be observed. For practical work it

393 is important to adjust the hetaeron concentration for purpose of reproducibility in the range of maximum retention.

Fig. 19: Plots of the capacity factor of adrenaline vs. the hetaeron concentration for various n-alkylsulfates. Conditions are given in ref. 28.

The alkyl chain length of the hetaeron influences also retention as shown in Fig. 19. The maximum value of the function shifts to lower concentrations as the alkyl-chainlength exceeds 7 or 8 carbon atoms. This fact is probably due to stronger adsorption of the hetaeron to the support with longer chain reducing the active hydrophobic surface area of the stationary phase. The kinetics of the adsorption-desorption process with shorter hetaerons are much faster, the maximum of the function k

= f

(hetaeron conc.) is in the range of 4-5 mMol/L. Optimum retention values with alkyl sulfates and alkylsulfonates are available using n-hexyl- to n-octyl-chains. In case of cationic hetaerons, the optimum is with tetramethyl- or tetrabutyl ammonium groups.

394 The degree of retention enhancement in dependence of the alkyl-chain length is given by Horvath et al. as the "enhancement factor", π = B/(k 0

' P),

in which D^ is a normalization factor accounting for the peak profile of the concentration signal before injection.

The value of D^ will therefore depend on

the shape of this profile and on the calculation method σ

ν ο

(2) .

is the broadening in the external volume of the

chromatographic system of an infinitely small sample. This parameter can be regarded as the hydrodynamic equivalent of what is called the impulse response of an electronic

(sub)system.

416

So, eqn. 11 decouples the effect of external broadening and injection volume on the observed external bandbroadening. CTvo .

Moreover eqn. 11 provides a way to establish a linear regression of the experimentally ob-

tained 2 4 1 0 t h e n 3μπι is f a s t e r t h a n 5μπι if 605 < N r e g < 2 4 1 0 t h e n 5μιη is f a s t e r t h a n 10μιη, w h i l s t pressure

3um

limited

if N r e g < 605 t h e n 10μπι is f a s t e s t b e c a u s e 3 a n d 5μπι a r e pressure

limited.

Δρ1ΐΐη

For L = 100mm a n d

= 400 bar:

if N r e q > 7678 t h e n 3μπι is faster t h a n 5μπι if 2126 < N r e g < 7678 t h e n 5μπι is f a s t e r t h a n 10μπι, 3 μ m p r e s sure

limited

if N r e q < 2126 t h e n 10μπι is f a s t e s t a n d 3 and 5μπι are p r e s s u r e limited. In c o n c l u s i o n w e m a y say t h a t if

'standard' c o l u m n l e n g t h s are

a p p l i e d the r e q u i r e d p l a t e n u m b e r for an a n a l y t e p a i r in the sample t o g e t h e r w i t h the i n s t r u m e n t s p r e s s u r e l i m i t , l i m i t the use of v e r y small p a r t i c l e s for h i g h s p e e d a n a l y s e s .

High

s p e e d L C is b e s t d o n e b y a d a p t i n g as g o o d a s p o s s i b l e

the

standard column

(L a n d dp) to the r e q u i r e d p l a t e n u m b e r .

This optimization example was focussing on the kinetic of the s e p a r a t i o n . very practical.

aspects

The p r e r e q u i s i t e k ' j = 2 is of c o u r s e n o t

H o w e v e r , the b a s i c d e s c r i p t i o n still h o l d s .

For any s e p a r a t i o n , N r e q w i l l b e g o v e r n e d by o n e c r i t i c a l a n a l y t e p a i r in the sample a n d the a n a l y s i s time by k' (= c a p a c i t y r a t i o of the last e l u t i n g p e a k ) . a n a l y s i s time =

1Γ 0+k') u o Ζ

Ζ Therefore, (29)

429

Again here N r e q

is limiting u q and therefore analysis time and

pressure drop. On the other hand, it is easily seen from eqn.16 that decrease of analysis time also can be achieved by thermodynamic tion viz. decreasing k' and increasing alpha. dealt

abundantly

optimiza-

These aspects are

in the literature.

4: Dilution and detectabilitv

This subject has been covered in detail in a previous paper (11).

The most significant equations and conclusions are

discussed here. The dilution factor, DF, of a solute after its migration

through

the column can be described as: c0 ^v.j(col)* Do D F1 i = P T = \Π~· cj-max V in) hr

(30)

This definition is the reversal of the commonly used one in the literature, but has been chosen deliberately, so that DF is small for small dilution and

is large for high dilution.

After substitution of eqn. 10, (30) changes to: Co

„VVcol-n + kV-Do

i" cj-max"

(31)

Vjnj-Ncoio.s

where c

is the solute concentration in the sample, c is c ο max the solute concentration at peak maximum at the outlet of the column, D q is the previously described normalization

factor

(section 2) accounting for the peakshape at column outlet (and is

for a gaussian peak) and

is the plate number

generated by the column on injection of an infinitely small sample when no external broadening is present. D q depends on the calculation method as well

The value of

(2).

Dilution of a solute on a column is small for low capacity ratios, large plate numbers and small ratios of column to injection volume.

This last aspect is demonstrated by fig. 5.

430 Here the same injection volume was applied to two columns, both generating approx. 5000 plates.

The tremendous improvement in

peakheight hardly needs comment. However, as already discussed in section 2, the external bandbroadening in the non-separating volume of the chromatographic system, has to be taken into account. As the width of the applied signal

(i.e. the inj. volume) itself also has to be considered

we have suggested eqn. 11 >2 (11)

So, in this description these two sources of broadening are decoupled in contrast to the commonly used description in the literature.

Fig. 3 and ref. 2 demonstrated the proposed

dependence between >-3 φ Φ o \ Q C O -= ( 0 φ ω g C φ Ο φ O Χί ct φ ω ΟΕ § iE & C Ι ω Ν — OD c CO ' t — 'V — Φ ο φ φ — t Ο ο • σ φ Ε φ φ 3 0C • φ 4 — » CO 2 C0 J ε Τ3 ο • + = Φ Φ CD φ C Έ CO Ω. CO Ω.C 13 χ: Ο O CO.Ε Φ : C 0 Φ Ν ι Φ Χ 5 ω 'c Φ CT Φ Φ Q C -Ο Ο CO φ CT 3 C CM '25 w Χ! φ co ώ Ο CO Ι δ Ε ω φ C/) CO (ϊ r c ü cö ~ _ · Ε Ο φ — = :C0 Q x: φ > _Q CTj Φ χ: >~ ο Ε r c C D · — ! = •— φ iS Φ Φ ο 3 ω 5 Ν il Χ3 > ι

1

Φ φ Ε. . L 9? i « ^ φ TD ο C = > CT CO " Τ χ CT C § I s "φ ι- c CO co = τ> Ν φ c φ c 5 C Ε 2 φ -σ *= Ε Ä Χ Φ ^ 5 ® s i C φ φ CO 2 ο ζ CO _Q .3? EL £Ζ •σ Ο Ε ω - 2 '•Η c φ I i 3 C φ .Ε -£ co ü ο ο ™ Φ Ό φ φ CT CC σ> co ; "co ja Ο φ 2 CO :C0~ CO I I > co "σ c ο Ο CO Q. Έ Ε 2 φ Χ co > l l _ •g " φ LÜ Q- μΐ c Φ ^ Ο o f C CD c C Φ "Ο Ο « I CO α> C CO φ CT φ c Φ SZ S CO Φ Ζ C Φ "Ο -D TJ•ο Ξ u2 φ ™ : Ζ 3 Ο t • - 3 x: | S » φ I ?ο Φ Ε ö D CO ι ο Ε OQ co r-,Ο x : -C •Β « CM 5 Ο CO O ρ « C ο CO Ε ^ Q. Q «ί < CO φ = Ε

£

w DE

G F. Lottspeich A. Henschen K.-P. Hupe (Editors)

B.J. Radola D. Graesslin (Editors)

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