HPLC Made to Measure: A Practical Handbook for Optimization - Hardcover

Hardcover

ISBN 10: 352731377X ISBN 13: 9783527313778

Publisher: Wiley-VCH, 2006

Synopsis

The only topical HPLC book to focus on optimization, this volume addresses the needs of HPLC users who wish to constantly improve their methods, in particular in terms of throughput, accuracy and cost-effectiveness.
This handbook features contributions from such bestselling authors as John W. Dolan, Michael McBrien, Veronika R. Meyer, Uwe D. Neue, Lloyd R. Snyder, and Klaus K. Unger, as well as from scientists working for major companies, including Agilent, AstraZeneca, Merck, Schering, Tosoh Biosep, VWR, and Waters. It covers essential aspects of optimization in general, optimization in different LC-modi, hyphenated techniques and computer-aided optimization. The whole is rounded off with a section of user reports.

About this title

About the Author

Following his PhD thesis in 1983, Stavros Kromidas worked as a sales manager for Waters Chromatography . From 1989 to 2001 he was Managing Director of Novia GmbH, a consultancy company for analytical chemistry, and he has been an independent consultant since then. He has been working in the field of HPLC since 1978 and has given lectures and training courses since 1984. He is the author and co-author of numerous articles and several successful books.

From the Back Cover

Alongside developing new ones, the optimization of existing methods is a key task in the HPLC laboratory. And a task that nowadays has to be solved increasingly fast and cost-efficiently.
This handbook provides well-founded assistance in better meeting this challenge. Internationally renowned authors cover both the general basics and strategies in optimization, as well as the specific aspects involved in such different methods as RP-HPLC, NP-HPLC, micro- and nano-HPLC, and hyphenated techniques, such as LC/MS. Nor are such topics as column selection and chiral separations left uncovered. Some of the contributions present applications using common optimization software, such as DryLab or ChromSword. All of the chapters concentrate on the essentials and are written in a practically oriented style, while their self-contained structure allows for targeted references.
Throughout the text, the authors offer concrete, practical tips as well as pertinent background information, together with insights into the optimization methods of seven major international companies from various sectors.
The whole is rounded off with real-life reports from experienced users coming from the different areas of application, in particular the life sciences, such as proteomics or drug development.

Excerpt. © Reprinted by permission. All rights reserved.

HPLC Made to Measure

A Practical Handbook for Optimization

John Wiley & Sons

Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All right reserved.
ISBN: 978-3-527-31377-8

Chapter One

Fundamentals of Optimization

1.1

Principles of the Optimization of HPLC Illustrated by RP-Chromatography

Stavros Kromidas

First of all, some questions will be discussed, which should reasonably be answered before beginning method development. Subsequently, we will treat the principal possibilities for improving the resolution in HPLC. It follows a discussion about efficiency and the "right" sequence of such measures for the isocratic and the gradient mode. There is a particular focus on strategies and concepts for developing a method and checking peak homogeneity.

The last section will show ways to achieve other aims than "better separation": "make it faster", "raise sensitivity" or "save money". The chapter ends with a conclusion and an outlook.

1.1.1 Before the First Steps of Optimization

For economic reasons, one really ought to address the following questions prior to commencing the development of a method or the optimization of a given separation. What do I want? In other words, what is the true intention of the separation?

What do I have? That is to say, what relevant information about the analytical purpose and the samples is available?

How should I do it? Do I have all what I need, and is what I want to do really possible?

At first glance, these questions might appear too theoretical or even over-critical. Nevertheless, careful consideration of the actual aims and realistic possibilities for solving an analytical problem would seem to be important at the outset. An early discussion with my boss, a colleague or my client - if you are short, even with yourself - can later prevent a good deal of trouble, time expenditure, and last but not least costs. This time-saving can be considered a good investment.

As regards the first question: "What do I want?"

If it is at all possible, at the outset the following or similar questions should be answered:

Do I need a method for the accurate quantification of this toxic metabolite, or is the aim that the authorities just accept my method?

What is most important in this case: short analysis times, durable columns, robust conditions, or simply optimal specificity?

Must the relative standard deviation [S.sub.rel] be no higher than 2%? What loss of quality would be incurred if [S.sub.rel] were to be 2.5%? Is there actually a correlation between the cost of the analysis and real improvement in the quality of the product?

In other words, is the aim just to meet the requirements in this specific case or is the real "truth" at stake, i.e., are formal aspects or analytical questions in the foreground? This question should be consciously and truthfully answered because of the possible consequences.

How difficult it can be to stand by meaningful and well-considered decisions without being regarded as outlandish or as a troublemaker has been documented elsewhere. Where possible, one should question all aspects. Unconventional questions frequently result in simple solutions.

As regards the second question: "What do I have?"

Information on the sample makes the design of a suitable method easier.

Some examples:

What is written in the report of colleagues from the chemical development department on the light sensitivity and the sorption properties on glass surfaces of a new drug?

Can I contact these colleagues quickly? That is to say, can I get relevant information with a minimum of effort?

There may be information about similar separations in the past, which were not pursued further, in an internal database (which is perhaps rarely updated and even more rarely accessed).

May I quickly calculate the p[K.sub.a] value of the known main component in the sample with appropriate software (see Chapter 1.4)?

Has a colleague in a neighboring department worked in the past with similar compounds and might therefore be able to provide valuable insights?

As far as possible, all means of communication with colleagues should be pursued to gather information. At times it may be helpful not to make this public.

As regards the third question: "How should I do it?"

One should assess the feasibility of the proposed work absolutely unconditionally.

Some examples:

Can I convince my boss that it is useful from the overall company point of view to discuss in advance with the later routine users the design of the method and additional details? If fear of loss of know-how or questions of budget or other psychological and social barriers make impossible de facto a discussion with "the others", it is a bitter reality that one must accept.

On the other hand, is it worth fighting for a change of the following well-known and accepted situation? A deadline is fixed and therefore a validation must be finished in two weeks. Later, the burden of subsequent, substantial costs for a repeat of the measurements, complaints, out-of-spec situations, etc., which inevitably result because an analytical method can hardly be validated within two weeks under real conditions, is not placed on "us" but on quality control, and as testing costs they have been accepted since decades in the absence of overall considerations. The reader may imagine the consequences, or viewed more positively, the possibilities for improvement.

Is it really worthwhile in the case of the development of a routine method, which shall be applied all over the world, to opt for a polar RP-phase because of the frequently observed higher selectivity, even if one has to expect problems with the charge-to-charge reproducibility? Might a hydrophobic, more rugged column with a lower but still sufficient selectivity the better choice?

Is it useful to demonstrate my analytical "knowledge" by further trimming the relative standard deviation of a method being used in diverse plant laboratories to a value of 0.7%?

Realities - and opinions are also realities - which determine the success or failure of an analytical activity should, wherever possible, influence the design of the method. It is useful if the number of meetings can be reduced to "a cup of coffee" or "lunch often together". The point is to improve the communication and this can turn out to be easier in a less formal situation.

In conclusion, two basic preconditions for successful method development may be noted:

1. Expert knowledge exists or can be loaned or sold.

2. The analytical possibilities correspond with the requirements, and it is possible to talk about them.

In the author's opinion, a clear definition of requirements, unequivocally formulated, understandable goals for all involved persons, shortcuts to information, and a critical estimation of possibilities/risks are more important, not only in analytics, than obtaining exemplary results such as low detection limit, correlation factors around 0.999, [S.sub.rel] smaller than 1%, or 30% less expensive equipment.

1.1.2 What Exactly Do We Mean By "Optimization"?

Optimization of a separation is principally directed by the following goals:

to separate better (higher resolution),

to separate faster (shorter retention time),

to see more (lower detection limit),

to separate at lower cost (economic effort),

to separate more (higher throughput).

The three first-named goals may be most important, and of these the improvement of the resolution is the prime concern. Therefore, we will treat this topic before we start to deal with the other aspects. Preparative HPLC is not the subject of this book.

Preliminary Remarks

The theory of chromatography is fundamentally valid for all chromatogaphic techniques. Therefore, basically the same principles are pursued. However, it is evident that the priorities and the weighting of the specific steps are very different, for example in GPC and in -LC-MS(MS). In the following, the possibilities for optimization are presented and proposals for the most popular liquid-chromatographic technique, RP-HPLC, are given in short form.

The characteristics of the different modes are treated in Chapters 2.1 to 2.7. It is assumed that the reader is familiar with the chromatographic rules and the theory of HPLC, so these are not treated in detail, but where necessary some technical terms are briefly explained.

The immediately following remarks relate to isocratic separations.

1.1.3 Improvement of Resolution ("Separate Better")

Resolution (R), in simplest terms, is the distance between two neighboring peaks at the base of the peaks. An increase in this distance is what every chromatographer routinely strives for.

The corresponding equation is:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

where N is the plate number, a measure of the performance or the efficiency of the column.

The number of plates is in effect a measure of the widening of the substance band because of diffusion effects. The basic question is whether the molecules of the analyte that reach the detector are contained in a small or a large (peak) volume, i.e., will one get sharp or broad peaks?

Strictly speaking, one should distinguish between the theoretical and the effective plate number. The theoretical plate number is the number of plates of an inert component (see below) and therefore a characteristic and constant value for a column under defined conditions. The effective plate number is the number of plates of a specified retained component, and the retention factor (see below) enters into the calculation. Today, however, this distinction is not made everytime; one speaks only about plate number. In the most cases, the theoretical plate number is calculated, but of retained substances. In this context, it should be made clear that the plate number depends on a lot of factors, e.g. the injection volume, the temperature, the composition of the eluent, the flow rate, the retention time, the analyte, and last but not least the equation used for the calculation, i.e. peak width at the peak base, at 10% or at 50% peak height. Therefore, the comparison of literature values of plate numbers is inherently difficult.

[alpha]: Separation factor, formerly selectivity factor.

[alpha] is a measure of the capability of a chromatographic system (chromatographic system: the actual combination of the stationary phase, the mobile phase, and the temperature) to distinguish two given compounds.

The [alpha] value is the quotient of two net retention times, i.e. the quotient of the dwell times of the two components within the stationary phase.

The point is that if this particular chromatographic system is selective for these two compounds, then in principle they are separable. Selectivity, in simplest terms, is the distance between two peaks, from the top of one peak to the top of the other. This is different from the resolution in that for the determination of the selectivity the form of the peak (plate number) is not considered, because [alpha] is only the quotient of two (retention) times. The separation factor depends only on the chemistry; see below on the subject of retention factor.

k: Retention factor, formerly capacity factor k'.

k is a measure of the strength of the interaction of a given compound in a given chromatographic system. It expresses for how much longer a given compound remains on the stationary phase compared to the mobile phase.

The k value is an index like the [alpha] value, and is thus independent of instrumental conditions such as the dimensions of the column or the flow rate. The k value changes only if parameters that have something to do with the interaction are changed, i.e., the chemistry: stationary phase, mobile phase, temperature. As long as these parameters are kept constant, the k value also stays constant, irrespective, e.g., of the flow rate or whether a 10 or 15 cm column is used.

Although the dead time does not appear explicitly in the equation for the resolution, it is useful for the following explanation to briefly deal with this term.

[t.sub.m]: dead time, breakthrough time, front, "air peak": This is the dwell duration of an inert component in the HPLC equipment. A component is designated as inert if it is able to penetrate everywhere without steric hindrance, including, of course, in the pores of the stationary phase, but is not retained there. In other words, the dead time is the time of the presence of any not excluded component in the mobile phase - also in the standing mobile phase (i.e., within the pores), but again there is "no" interaction with the stationary phase. Therefore, the dead time only changes if something "physically" or "mechanically" is altered, e.g. a change in the length or the inner diameter of the column, the packing density, or the flow rate. The dead time is a time, which is independent from the particular compound just analysed. As all components move equally quickly in the eluent, the time that the compounds spend in the eluent makes no contribution to the separation. A separation is only possible if the substances stay in the stationary phase for different lengths of time.

The resolution R - the distance from peak base to peak base - depends only on the following three factors:

the strength of the interaction between the compound and the stationary phase (if the peak comes soon or late), i.e. on the k value,

the ability of the chromatographic system to distinguish between the two components of interest, i.e. the [alpha] value,

if the relevant peaks are sharp or wide, i.e., the plate number.

Consequently, to improve resolution, there are in principle only three possibilities,

namely a general increase in the interaction (k value increases),

an analyte-specific change in the interaction ([alpha] value increases), or

an increase in the efficiency of the separation (N value increases).

1.1.3.1 Principal Possibilities for Improving Resolution

The aforementioned topic is illustrated by way of a hypothetical example; see Fig. 1. Starting with a poor resolution (see Fig. 1, upper chromatogram), what are the principle possibilities for improving the resolution?

Remark: The dead time only changes in case 1 ("[up arrow] [t.sub.m]").

Possibility 1: One ensures that all components - including one possible inert component (increase of dead time, see above) elute later. Because now the dead time increases too a physical process must be responsible. This could be a longer column, a larger inner diameter of the column, decrease of the flow rate. (A larger inner diameter leads to peak broadening which rules it out for all practical purposes).

Possibility 2: The retention time stays largely constant, one seeks only a better peak form. Here, there are somewhat more possibilities: reduction of the dead volume (e.g., thinner capillaries, smaller detector cell), reduction of the injection volume (remark: local overload of the column happens more frequently than one might imagine! Peak broadening caused by the injection is inversely proportional to the injection volume.) at an elution composition with equal solvent strength parameter, replacement of methanol with acetonitrile owing to the lower viscosity of the latter (approximately constant retention time can be expected), using smaller particles, or use of a newer, better packed column. In this context, one should also consider an optimization of the injection step as this also improves the peak form and consequently increases the plate number. The solvent of the sample should be weaker than the eluent; to this end, one uses a little bit more water in comparison to the eluent composition when preparing the sample solution.

In this way, it is possible to increase the concentration of the substance band at the head of the column, and the result is a better peak form. Finally, one should also consider various settings, such as sample rate (sampling time, sampling period), bunching factor, peak width, slit width in the case of a diode-array detector, etc. In this way, the peak form can also be improved measurably, without changing the "real" method parameters such as column or eluent.

(Continues...)

Excerpted from HPLC Made to Measure Copyright © 2006 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim . Excerpted by permission.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
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Edition details

• Publisher: Wiley-VCH
• Publication date: 2006
• Language: English
• ISBN 10: 352731377X
• ISBN 13: 9783527313778
• Binding: Hardcover
• Edition number: 1
• Number of pages: 786
• Editor: Kromidas Stavros