Cromatografia

Guide

Laboratory Chromatography

Laboratory Chromatography Guide
Angelo Talamona

Author Publisher Cover Layout

Angelo Talamona Büchi Labortechnik AG, CH-9230 Flawil, Switzerland NOSE Applied Intelligence AG, CH-8005 Zürich, Switzerland Atelier Güttinger AG, CH-9030 Abtwil, Switzerland

First edition Printed in Switzerland 94175 0105

All rights reserved. No part of thispublication may be reprinted, or reproduced, or utilized in any form or by any electronic or mechanical means – now known or hereafter invented –, including photocopying and recording, or in any information storage and retrieval system, without the publisher’s written permission. © 2005 by Büchi Labortechnik AG, CH-9230 Flawil, Switzerland.

ISBN 3-033-00339-7

“Laboratory Chromatography Guide” – Aclose look at preparative liquid chromatography The present “Laboratory Chromatography Guide” is dedicated to preparative liquid chromatography, a common purification technique in most chemical or life science laboratories. The performance of separations by chromatography is fairly well known in the scientific and industrial communities. Part 1, the “Flash Guide Basics”, gives consideration to thisfact, proceeding swiftly through flash chromatography with an emphasis on speed, reliability and reproducibility of the separation. But there are no rules without exceptions! As usual, problems appear with the most exciting and valuable compounds you want to purify. Therefore, you are personally challenged to understand and solve the purification task as fast as possible. The second part“Preparative Column Chromatography: Theory and Practice” helps you to overcome such drawbacks and leads you back to the shining path of your privileged profession: to understand and explore what modern science offers! We at Buchi, as a leading supplier of high quality laboratory products and responsive services, wish you a lot of challenging and successful work! Dr. Ernst Freydl Büchi Labortechnik AGContents

Part 1

Flash Guide Basics
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Principle of chromatography . . . . . . . . . . . . . . . . . . . Choice of the appropriate stationary phase . . . . . . . Evaluation of the chromatographic system by thin-layer chromatography . . . . . . . . . . . . . . . . Evaluation of the stationary phase . . . . . . . . . . .. . . Selectivity of the solvent . . . . . . . . . . . . . . . . . . . . . Solvent strength . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 14 15

1 2 3 4 4.1 4.2 4.3 5 6

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16 16 16 18 23 26

Injection/Column loading . . . . . . . . . . . . . . . . . . . . . . Gradient elution . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .

Part 2

Preparative Column Chromatography Theory and Practice
Starting point – Definition of the problem Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 32 32 33 34 34 34 34 36 39 39 40 40 40 42 43 43 44 46 46 47 49 49 50 51 53 54 54 55 56 58 58 59 60 61 62 62 63

1 1.1 1.2 1.3 2 2.1 2.2 2.2.1 2.3 2.4 2.5 3 3.1 3.2 3.3 3.4 3.5 3.6 4 4.1 4.2 4.3 4.4 4.5 4.6 5 6 6.1 6.2 6.3 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7

Fundamentals – Thebasic principles . . . . . . . . . . General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adsorption chromatography . . . . . . . . . . . . . . . . . . . . Separation mechanisms in adsorption chromatography Size exclusion chromatography . . . . . . . . . . . . . . . . . . Ion-exchange chromatography . . . . . . . . . . . . . . . . . . Affinity chromatography . . . . . ....