Pitfalls and Errors of HPLC in Pictures E-Book

Veronika R. Meyer

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch
• Veröffentlichungsjahr: 2013

Seitenzahl: 153

Beschreibung

The third edition of this popular problem-solving guide for this widely-used method includes eleven completely new examples and several updated ones, adding up to 100 contributions about pitfalls and errors in HPLC. Each example is presented on a double page with the text on the left-hand and a fi gure on the right-hand side, true to the motto 'a picture says more than a thousand words'. In addition, the author presents essential fundamentals as well as helpful strategies, such as equipment tests or quality assurance processes. New in this edition * Variability of the standard deviation * Infl uence of the acid type and concentration in the eluent * Water as an unintentional additive in the mobile phase * Inadequate purity of mobile phase water * Incomplete degassing * Inadequate stabilization of the extraction solvent * Tailing of phosphate compounds in the presence of steel * Different detection properties of diastereomers * Detector overload in ELSD * System suitability test * From repeatability to reproducibility A must-have resource for all users - showing how to use HPLC efficiently and obtain reliable results.

Leseprobe

Table of Contents

Further Titles for Chromatographers

Title Page

Copyright

Preface

Introduction

Part I: Fundamentals

1.1 Chromatography

1.2 Chromatographic Figures of Merit

1.3 The Resolution of Two Peaks

1.4 Reduced Parameters

1.5 The Van Deemter Curve

1.6 Peak Capacity and Number of Possible Peaks

1.7 Statistical Resolution Probability: Simulation

1.8 Statistical Resolution Probability: Example

1.9 Precision and Accuracy of an Analytical Result

1.10 Standard Deviation

1.11 Variability of the Standard Deviation

1.12 Uncertainty Propagation

1.13 Reproducibility in Trace Analysis

1.14 Ruggedness

1.15 Calibration Curves

1.16 The HPLC Instrument

1.17 The Detector Response Curve

1.18 Noise

1.19 The Playground Presented as an Ishikawa Diagram

1.20 The Possible and the Impossible

Part II: Pitfalls and Sources of Error

2.1 Mixing of the Mobile Phase

2.2 Mobile Phase pH

2.3 Adjustment of Mobile Phase pH

2.4 Influence of the Acid Type and Concentration in the Eluent

2.5 Water as an Unintentional Additive in the Mobile Phase

2.6 Inadequate Purity of Mobile Phase Water

2.7 Inadequate Purity of a Mobile Phase Solvent

2.8 Inadequate Purity of a Mobile Phase Reagent

2.9 Incomplete Degassing

2.10 System Peaks and Quantitative Analysis

2.11 Sample Preparation with Solid Phase Extraction

2.12 Inadequate Stabilization of the Extraction Solvent

2.13 Poor Choice of Sample Solvent: Peak Distortion

2.14 Poor Choice of Sample Solvent: Tailing

2.15 Sample Solvent and Calibration Curve

2.16 Impurities in the Sample

2.17 Formation of a by-Product in the Sample Solution

2.18 Decomposition by the Sample Vial

2.19 Artifact Peaks from the Vial Septum

2.20 Formation of an Associate in the Sample Solution

2.21 Precision and Accuracy with Loop Injection

2.22 Injection Technique

2.23 Injection of Air

2.24 Sample Adsorption in the Loop

2.25 Extra-Column Volumes

2.26 Dwell Volume

2.27 Elution at t0

2.28 Classification of C18 Reversed Phases

2.29 Different Selectivity of C18 Reversed Phases

2.30 Different Batches of Stationary Phase

2.31 Chemical Reaction within the Column

2.32 Tailing of Phosphate Compounds in the Presence of Steel

2.33 Recovery and Peak Shape Problems with Proteins

2.34 Double Peaks from Stable Conformers

2.35 Influence of Temperature on the Separation

2.36 Thermal Non-Equilibrium within the Column

2.37 Influence of the Flow Rate on the Separation

2.38 Influence of Run Time and Flow Rate on Gradient Separations

2.39 UV Spectra and Quantitative Analysis

2.40 UV Detection Wavelength

2.41 Different Detection Properties of Diastereomers

2.42 Fluorescence Quenching by Air

2.43 Detector Overload in UV

2.44 Detector Overload in ELSD

2.45 Influence of the Retention Factor on Peak Height

2.46 Influence of the Flow Rate on Peak Area

2.47 Leaks in the HPLC Instrument

2.48 Impairment of Precision as a Result of Noise

2.49 Determination of Peak Area and Height at High Noise

2.50 Peak Height Ratios

2.51 Incompletely Resolved Peaks

2.52 Area Rules for Incompletely Resolved Peaks

2.53 Areas of a 1 : 10 Peak Pair

2.54 Heights of a 1 : 10 Peak Pair

2.55 Quantitative Analysis of a Small Peak

2.56 Incompletely Resolved Peaks with Tailing

2.57 Integration Threshold and Number of Detected Peaks

2.58 Detector Time Constant and Peak Shape

2.59 Quantitative Analysis in the 99% Range

2.60 Correlation Coefficient of Calibration Curves

Part III: Useful Strategies

3.1 Column Tests

3.2 Apparatus Tests

3.3 Wavelength Accuracy of the UV Detector

3.4 Internal Standards

3.5 A Linearity Test

3.6 Rules for Accurate Quantitative Peak Size Determination

3.7 High-Low Chromatography

3.8 Control Charts

3.9 Verification of the Analytical Result by Use of a Second Method

3.10 Description of Ruggedness

3.11 Rules for Passing on an HPLC Method

3.12 Quality Assurance in the Laboratory

3.13 Standard Operating Procedures

3.14 Method Validation

3.15 Some Elements of Validation

3.16 A Validation Example

3.17 System Suitability Test

3.18 From Repeatability to Reproducibility

3.19 Measurement Uncertainty

3.20 Formal Quality Assurance Systems

Index

Further Titles for Chromatographers

S. Kromidas (ed.)

HPLC Made to Measure

A Practical Handbook for Optimization

2006

ISBN 3-527-31377-X

S. Kromidas

More Practical Problem Solving in HPLC

2005

ISBN 3-527-31113-0

V. R. Meyer

Practical High-Performance Liquid Chromatography

2010

ISBN 978-0-470-68218-0 or 978-0-470-68217-3

S. Kromidas

Practical Problem Solving in HPLC

2000

ISBN 3-527-29842-8

P. C. Sadek

Troubleshooting HPLC Systems

A Bench Manual

2000

ISBN 0-471-17834-9

U. D. Neue

HPLC Columns

Theory, Technology, and Practice

1997

ISBN 0-471-19037-3

L. R. Snyder, J. J. Kirkland, J. L. Glajch

Practical HPLC Method Development

1997

ISBN 0-471-00703-X

The Authors

Dr. Veronika R. Meyer

EMPA St. Gallen

Lerchenfeldstrasse 5

9014 St. Gallen

Switzerland

e-mail: [email protected]

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.

© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN: 978-3-527-33293-9

ePDF ISBN: 978-3-527-65913-5

ePub ISBN: 978-3-527-65912-8

mobi ISBN: 978-3-527-65911-1

oBook ISBN: 978-3-527-65910-4

(A picture says more than a thousand words)

Graffiti on the Baltzerstrasse in Bern

Preface

Errors are a common companion of all human activity, including work in the laboratory. Yet it is a great pity if erroneous results are produced with great effort and by using expensive instruments and demanding procedures. Therefore a book about sources of errors in high performance liquid chromatography, one of today's most widely used analytical methods, is not superfluous. Maybe the topic is not welcomed enthusiastically but I hope I have found a design which encourages reading and thinking.

In conception, at least, possible problems can be divided into two categories. ‘Errors’ are troublesome opponents of accurate and precise analytical results which, however, can be understood; we need to remember and to anticipate them. In contrast, ‘pitfalls’ are totally unexpected intruders and the secret behind them is difficult to discover. The worst are those which are not detected but which affect the result anyway. Nevertheless, the book does not distinguish between the two types. The readers decide how they classify them. With increasing experience in HPLC it should become easier to avoid the pitfalls.

The third edition could be expanded with new examples and proposals. Many people helped me with examples, hints or ideas on how to improve the text and figures. I want to thank all of them. Special thanks to the publisher who supports the idea of a picture book, not for children but for novices and experts in the analytical laboratory. I hope that the book will be a useful aid in daily laboratory work thanks to intelligible explanations and lucid illustrations.

Veronika R. Meyer

St. Gallen, August 2012

Introduction

This book is not an introductory text to HPLC and also not a troubleshooting guide of the kind “what shall I do if my instrument does not work?”. It does not replace such books but is intended to complement them. Some texts which, according to my personal opinion, are very useful and should therefore be present in the HPLC laboratory are listed on the next page.

Now this book on your desk is a picture book. The figures are at least as important as the texts; sometimes more information can be found in them than could be given in the short descriptions. It is possible, and in principle recommended, to study all the pages in sequence from beginning to end. This method guarantees that one learns about errors which are uncommon and unexpected. On the other hand each pair of pages is limited to one topic, linked to other pages by arrows only, and can therefore be studied in isolation. The index at the end of the book can help you find the right pages when a problem occurs, although it must be stated once again that quick troubleshooting advice is not usually provided.

The book is divided into three parts:

Part I briefly presents some basic facts about HPLC. Many topics may be absent because this is not a textbook, but the matter presented is of utmost relevance in HPLC. Thus the topics discussed should act as reminders and be used for revision. Whoever understands Part I knows a lot about HPLC – more than it seems at first glance.

Part II lists the pitfalls and sources of error. They are in a logical sequence, as far as this is possible, following the flow path in an HPLC instrument, from the preparation of the mobile phase to data evaluation. The list is somewhat arbitrary, and not all errors are of equal importance with regard to their possible consequences. It would, however, be dangerous to distinguish between grave and harmless errors. A minute error can cause much damage under special circumstances.

Part III gives some hints on what can be done to avoid errors. Again this synopsis is very heterogeneous in character. This does not diminish its value, of course.

Incompleteness is an inevitable feature of this book. I am grateful for all hints on other pitfalls and sources of error or on how to avoid them.

Recommended Texts

Veronika R. Meyer

Practical High Performance Liquid Chromatography

Wiley, Chichester

5th edition 2010

John W. Dolan and Lloyd R. Snyder

Troubleshooting LC Systems

Humana Press, New Jersey

1989

Paul C. Sadek

Troubleshooting HPLC Systems: A Bench Manual

Wiley-Interscience, New York

2000

Stavros Kromidas

Practical Problem Solving in HPLC

Wiley-VCH, Weinheim

2000

Stavros Kromidas

More Practical Problem Solving in HPLC

Wiley-VCH, Weinheim

2004

Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch

Practical HPLC Method Development

Wiley-Interscience, New York

2nd edition 1997

Norman Dyson

Chromatographic Integration Methods

Royal Society of Chemistry, London

2nd edition 1998

Part I

Fundamentals

1.1 Chromatography

In chromatography, a physical separation method, the components of a mixture are partitioned between two phases. One of the phases stays in its place and is called the stationary phase, whereas the other moves in a definite direction and is called the mobile phase.

According to the type of mobile phase we distinguish between gas chromatography, supercritical fluid chromatography, and liquid chromatography.

The separation is based upon the different partition coefficients of the sample components between the two phases. It is helpful to divide the chromatographic column into small hypothetical units, the socalled theoretical plates. Within each plate a new partition equilibrium is established. The narrower a theoretical plate, the more equilibrium processes can take place within a column of given length and the more demanding the separation problems which can be solved.

The figure shows the separation of two compounds. One of these prefers the mobile phase but also enters the stationary phase. For the other compound the preference is the other way round. Thanks to this large difference in their properties the two types of molecule can easily be separated. They are transported through the column by the flow of the mobile phase and thereby reach zones where new equilibria are formed again and again.

In the drawing, such a theoretical plate has a height of approximately 3 1/2 stationary phase particle diameters. This height depends on the packing quality of the column, on the mass transfer properties of the phases, and on the analytes involved. Plate height is a function of the particle diameter of the stationary phase. For good columns, plate heights are equal to ca. 3 particle diameters irrespective of the particle size. A fine packing, e.g. with a 3 − µm phase, gives four times as many theoretical plates as does a 10 − µm packing if identical column lengths are compared. The column with the fine packing can therefore be used for more difficult separation problems.

1.2 Chromatographic Figures of Merit

To judge a chromatogram it is necessary to calculate some data which can be easily obtained. The integrator or data system yields the retention times, tR, and peak widths, w; perhaps it is advisable to determine the peak width at half height, w1/2. In addition the breakthrough time or ‘dead time’, t0, must be known although it can be a problem to measure it unambiguously. In principle, the first baseline deviation after injection marks t0. Then the following data can be calculated:

1.3 The Resolution of Two Peaks

The resolution of two adjacent peaks is defined as

The resolution depends on the separation factor α, the theoretical plate number N, and the retention factor k:

This equation can be expressed in different forms, which are not of interest here. It is important to realize that the resolution is influenced by the three parameters. The separation factor has the largest effect. If a separation needs to be improved it is well worth the effort of increasing α, although it is impossible to give a general proposal concerning how to do this. If the plate number is increased, the effect is only by the factor ; if the column length is, e.g., doubled, and by this also the plate number (at least in principle), the resolution will improve only by . Increasing the retention factor only has a notable influence on resolution if k was small to start with.

The upper figure presents several pairs of peaks separated with varying resolution. The graph below demonstrates how the resolution increases with increasing plate number for three different separation factors.

1.4 Reduced Parameters

The judgement and comparison of HPLC columns is best done with reduced, dimensionless parameters. A test chromatogram is acquired which enables the theoretical plate number, N, of the column to be determined from a suitable peak with low tailing. It is also necessary to measure the breakthrough time, t0, with a refraction index peak or with an otherwise suitable compound (for reversed-phase separations, e.g., with thiourea). The pressure drop, Δp, under the given conditions is noted.

Then the following reduced parameters can be calculated:

1.5 The Van Deemter Curve

It would be best to work at uopt. Practical separations are often performed at higher speed, which gives shorter analysis times and usually only a moderate loss of separation performance. This is, however, only true if mass transfer is fast, which is often not the case with special stationary phases and ion exchangers. Of course it is never advisable to work under conditions left of the van Deemter optimum. In this region the separation performance is very poor and the analysis time is long.

Chromatographic Conditions

Sample:

5 µL of a solution of thiourea, veratrole, acetophenone and nitrobenzene

Column:

4.6 mm × 7.5 cm

Stationary phase:

Zorbax SB C-18, 3.5 µm (reversed phase C18)

Mobile phase:

water/ethanol 3 : 7

Volume flow rate:

0.1 to 1.9 mL min−1 corresponding to u from 0.16 to 3.2 mm s−1

Detector:

UV 268 nm

Optimum:

for nitrobenzene (curve) u ≈ 1.3 mm s−1 (0.8 mL min−1); H ≈ 8.5 µm; for veratrole and acetophenone u ≈ 0.65 mm s−1 (0.4 mL min−1)

Reference: V.R. Meyer

1.6 Peak Capacity and Number of Possible Peaks

If the plate number were constant over the whole range of k values the peak capacity would be defined as:

At constant peak width, w