High-Temperature Liquid Chromatography: A User's Guide for Method Development: Volume 13 (RSC Chromatography Monographs) - Hardcover

Thorsten Teutenberg

Hardcover

ISBN 10: 184973013X ISBN 13: 9781849730136

Publisher: Royal Society of Chemistry, 2010

Synopsis

This unique book covers all the practical aspects of setting up an HPLC system for high temperature operation.

About this title

About the Author

Thorsten Teutenberg obtained his doctorate from the University of Bochum in 2004 for research dealing with the development of a specially designed heating system for high temperature liquid chromatography. He worked in the Analytical Department and after completing his thesis he joined the Institute of Energy and Environmental Technology in Duisburg. There, he is responsible for a small working group within the department of environmental medicine / analytics which is focused on method development strategies for high temperature liquid chromatography. Currently, he heads some joint research projects with partners from university and industry to implement high temperature liquid chromatography as a routine method. His special interest is the understanding of the temperature dependence of physico-chemical solvent properties of binary liquid mixtures used for reversed phase HPLC.

From the Back Cover

High-temperature liquid chromatography has attracted much interest in recent years but has not yet recognized its full potential in the chromatographic community. There is a widespread reluctance in industry to use temperature to speed up the separation process, influence the selectivity of a separation or implement novel detection techniques. However, the technology has now matured and could revolutionize chromatography as we see it today. Better equipment, such as heating systems able to generate faster heating rates, is becoming more readily available. Also, columns based on silica gel, which can withstand higher temperatures for an extended period, are now being introduced. Nevertheless, further technological and methodical efforts are needed to establish the method in a regulated environment like the pharmaceutical industry. This is the only text to cover all the practical aspects, as well as the underlying theoretical principles, of setting up an HPLC system for high-temperature operation. It is not intended solely for academics but will also benefit the researcher interested in more practical considerations. The author is a recognized expert and has conducted several studies with partners from industry to validate the method. Many real examples from these studies have been included in the book. The aim is to support practitioners in the creation of their own protocols without the need to rely solely on trial and error. The book starts with a brief definition of high-temperature liquid chromatography before going on to cover: system set up; the heating system; mobile phase considerations; suitable stationary phases; method development using temperature programming; analyte stability, and special hyphenation techniques using superheated water as a mobile phase. In each chapter, experimental data is used to illustrate the main statements and the advantages over conventional HPLC are evaluated. The book concludes with a critical outlook on further developments and applications underlining the necessary advances needed to make high-temperature HPLC more robust.

Excerpt. © Reprinted by permission. All rights reserved.

High-Temperature Liquid Chromatography

A User's Guide for Method Development

By Thorsten Teutenberg

The Royal Society of Chemistry

Copyright © 2010 Thorsten Teutenberg
All rights reserved.

ISBN: 978-1-84973-013-6

Contents

Chapter 1 A Brief Definition of High-Temperature Liquid Chromatography, 1, 
Chapter 2 System Set-up for High-Temperature Liquid Chromatography, 15, 
Chapter 3 The Heating System, 24, 
Chapter 4 Mobile Phase Considerations, 52, 
Chapter 5 Suitable Stationary Phases, 87, 
Chapter 6 Method Development using Temperature as an Active Variable, 114, 
Chapter 7 Analyte Stability, 149, 
Chapter 8 Special Hyphenation Techniques, 158, 
Chapter 9 Critical Outlook and Future Prospects, 182, 
Appendix A Vapour Pressure Data, 193, 
Appendix B Viscosity Data, 197, 
Appendix C Static Permittivity Data, 201, 
Subject Index, 205, 

CHAPTER 1

A Brief Definition of High-Temperature Liquid Chromatography

High-temperature liquid chromatography is really a fascinating topic. Nowadays, there is renewed interest in this technique which has long been talked about.

When liquid chromatography was still young, the sky seemed the limit and the capabilities of liquid chromatography were discussed with great enthusiasm. For a practitioner who was not born during the really "hot" period of chromatography, it appeared that there were no preconceptions about the boundaries of liquid chromatography. It was in the early days of HPLC that Hesse and Engelhardt stated that temperature programming should yield the same results as solvent programming, and even concluded that this procedure would have advantages over solvent gradient elution as the solvents need not be changed. Today, this statement seems to be highly innovative because temperature programming is regarded very difficult to implement in industrial applications, although a number of publications have demonstrated the feasibility of this approach. Since this initial enthusiasm, temperature has long been neglected in liquid chromatography and has not attracted much attention. However, in separation science it is like fashion: the trends of the former decades appear again and sometimes they are presented as if they were totally new. The same seems to be true for this topic, which in fact is not that new. Nevertheless, the instrumentation has improved a lot since the early days of HPLC. Therefore, it is worthwhile revisiting this old but still very new variant of liquid chromatography.

1.1 What is High-Temperature Liquid Chromatography?

Although the question seems to be very trivial, it is not easy to give an answer. Up until now, a definition of this technique does not exist although it has emerged as the topic of many scientific meetings and symposia. I will therefore try to outline what I understand to be high-temperature liquid chromatography. Looking through the literature, a range of terms has been used – some more obvious than others:

• Subcritical water chromatography

• Subcritical fluid chromatography

• Elevated-temperature liquid chromatography

• Superheated water chromatography

• Hot eluent liquid chromatography

• (Ultra) High-temperature liquid chromatography (HT-HPLC)

• Thermal aqueous liquid chromatography (TALC)

• and others.

It seems to be very difficult to select a temperature range and to assign this region to define high-temperature liquid chromatography, as it will be called throughout this monograph. This is immediately clear if we look at the terms given above. Subcritical fluid chromatography directly refers to the mobile phase. But at which temperature range is a liquid subcritical? Many scales are referred to water, which also plays an important role in liquid chromatography. Therefore, if water is taken as a reference, an upper temperature limit of up to 374 °C, which corresponds to the critical point of water, might be considered. Above this temperature, water becomes a supercritical fluid. But how can we distinguish high-temperature liquid chromatography from conventional or room-temperature liquid chromatography? Clearly, the subcritical region extends to the lower temperature range, which is also the domain of conventional HPLC. This contradiction can be solved when we look at the third expression, which is elevated-temperature liquid chromatography. This means that the temperature should be higher than ambient temperature. But when do we exceed the temperature limit beyond which the region of high-temperature liquid chromatography is entered? Is it 40, 50 or 60 °C? In a recent review article on high-temperature HPLC, Heinisch states that "High-temperature liquid chromatography (HTLC) is a term which refers to any separation carried out at temperatures above room temperature (typically within a range from 40 °C to 200 °C) with a mobile phase in a liquid state". Personally, I think that 40 °C is too low to speak about high-temperature liquid chromatography. In my opinion, we should look at the mobile phases we are using. Since I will exclusively talk about reversed-phase liquid chromatography (RP-HPLC) in this book, relevant binary solvent systems which are used in RP-HPLC will be considered.

Again, we could take water as a reference solvent and define the normal boiling point of water as the lower temperature limit for high-temperature HPLC. The normal boiling point of a liquid indicates at what temperature the liquid will turn into a gas at atmospheric pressure. Such a phase transition has to be avoided in the whole chromatographic system because it can lead to the immediate destruction of the column and strong detector noise as will be shown later on. However, two arguments speak against this temperature. First of all, this is a very high starting point, because temperatures above 100 °C are already considered extremely high in some fields of application. The second point is that normally binary mixtures of water and an organic co-solvent are used in RP-HPLC. Typically, a separation is carried out in solvent gradient mode, if very complex samples have to be analyzed containing polar and non-polar compounds. We usually start with a high water concentration and end with a high concentration of the organic modifier. Usually, methanol and acetonitrile are the most widely used organic co-solvents in reversed-phase HPLC. Now let's have a look at the normal boiling-point temperatures of these solvents, which I have listed in Table 1.1, along with some other solvents which might also be used as modifiers.

Whereas water has the highest boiling point due to strong hydrogen bonding, the boiling points for the other solvents are much lower. Acetone already starts to boil at 56 °C. For the much more common solvents methanol and tetrahydrofuran, the normal boiling-point temperatures are 65 °C and 66 °C, respectively. This means that from the perspective of the pure components, a much lower temperature limit would be appropriate if the boiling-point temperature is taken as a reference to define the lower temperature limit of high-temperature HPLC. In this case, a lower temperature limit of about 60 °C for high-temperature liquid chromatography would be appropriate. Adjusting the temperature above 60 °C then requires raising the outlet pressure of the column above the atmospheric pressure. Otherwise, a phase transition would be inevitable when a solvent gradient is run from pure water to pure acetone. Therefore, increasing the temperature above 60 °C would mean that the domain of high-temperature liquid chromatography is entered in reversed-phase HPLC.

Now that we have defined the lower temperature limit, we can try to define the upper temperature limit. Again, it is very helpful to have a look on the data presented in Table 1.1. Besides the normal boiling-point temperatures, I have listed the critical temperatures of these solvents. From a purely thermodynamic standpoint a liquid turns into a supercritical fluid once it is above the critical temperature. Most of the organic solvents will become a supercritical fluid around 230 °C to 240 °C, while this temperature is much higher for water. In order to define the upper temperature limit I would consider the temperature at which every solvent or solvent mixture will be in the supercritical state. Here, water clearly limits this region because it has the highest critical temperature of all solvents. Using any binary mixture comprised of water and an organic co-solvent listed in Table 1.1, the critical temperature of these mixtures is always below that of pure water. This means that the upper temperature limit is defined by the critical temperature of water. Therefore, increasing the temperature above 374 °C would mean that the domain of supercritical fluid chromatography has been entered and we have completely exited the domain of high-temperature liquid chromatography.

Now that the domain of high-temperature HPLC has been defined, the question "what is a suitable temperature range for high-temperature liquid chromatography?" needs to be addressed and this will be done in the next section.

1.2 What is a Suitable Temperature Range for High-Temperature Liquid Chromatography?

Although in some fields of application, temperatures as high as 60 °C will not be tolerated and are considered too high to be used, the application of temperatures as high as 370 °C with a pure water mobile phase has been reported in the literature. Even if it is possible to use the complete temperature range for high-temperature HPLC, the question needs to be addressed as to what is the highest temperature which can be used in routine analysis? The requirements to make use of this technique are a stationary phase, which is stable at the highest temperature you would like to apply, and a heating system which is able to generate the desired temperature. Most conventional LC heating systems are only capable of raising the temperature to 80 °C. Although it is possible with every chromatographic system which is equipped with a column oven to enter the domain of high-temperature liquid chromatography, the region cannot be exploited further. Therefore, some instrument manufacturers have developed special heating systems which have an upper temperature limit of about 200 °C. But you also have to consider another very important aspect: the stationary phase. The stability of the stationary phase is a crucial factor, as will be shown in Chapter 5. In the last few years, column manufacturers have created silica-based reversed-phase columns with considerably improved stability. Even when HPLC was still young, the stability of silica-based columns at elevated temperatures was a real problem. It is therefore not surprising that alternative materials were already being examined at the end of the 1980s with polystyrene–divinylbenzene (PS–DVB) phases and then with the pioneering work on metal oxides, such as zirconia and titania by the groups of Carr and others. Since then, many articles have reviewed metal oxide stationary phases designed for high-temperature HPLC. Although silica-based phases have long lagged behind, they are now catching up in terms of stability at high temperatures. In some cases, they are even more stable than their metal oxide-based counterparts. From many recent studies it can be deduced that temperatures as high as 200 °C will not lead to an immediate collapse of the column, and some columns can be used over a reasonably long time without total degradation. What needs to be discussed is the question of what can be regarded as long-term stability, which will be done in Chapter 5.

Although the domain of high-temperature liquid chromatography potentially extends up to 374 °C, the useful temperature range for routine analysis that will be considered in this monograph is currently limited to approximately 200 °C. This is quite reasonable because specially designed heating systems as well as suitable stationary phases both generating and withstanding these temperatures are now commercially available.

It is undoubtedly fine to have defined what can be understood of high-temperature HPLC, but now the question has to be addressed as to why it should be beneficial to increase the temperature.

1.3 Why should High Temperatures be used in Liquid Chromatography?

In today's high speed world, it seems that all that matters is efficiency and throughput, which means a demand to reduce the time required to perform each separation. Indeed, the great majority of publications which have been written about high-temperature HPLC start by explaining the impact of temperature on the separation speed. Clearly, minimizing analysis time is one very important aspect, but it does not provide the whole story of the potential role of high-temperature HPLC. Although it might sound a little curious, temperature can be regarded as a universal parameter in liquid chromatography, even though as many authors have pointed out, it is the most underestimated parameter. Why is this? The answer is again not as straightforward as might be expected. The temperature influences almost every other parameter which can be used to optimize a separation in terms of speed and resolution. This means that temperature can be used to adjust the selectivity of the phase system without changing the mobile or stationary phase. Computer-optimization software has been available for a long time to assist the user in finding the optimal separation parameters, including the gradient slope as well as temperature. In our own laboratory, work is currently carried out to include even temperature programming. For the moment I will not go into further detail about this aspect, because the influence of temperature on all the important parameters related to the optimization of resolution is discussed later in Chapter 6. Instead, I would like to briefly discuss the van Deemter equation, which should be known to every chromatographer and can be written as:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (1.1)

Here, the Height Equivalent to a Theoretical Plate H(u) (HETP) depends on three terms: the band broadening due to eddy diffusion (A-term); longitudinal diffusion (B-term); and the resistance to mass transfer between and within the mobile and stationary phases (C-term) and the mobile phase flow rate (u). Physically, it is often assumed that the A-term does not depend on temperature. However, the remaining Band Cterm are both temperature-dependent. This is because the B-term is directly proportional to the diffusion coefficient, while the C-term is inversely proportional to the diffusion coefficient (DM), which is temperature-dependent:

B [varies] DM (1.2)

C [varies] 1/DM (1.3)

From a purely theoretical standpoint, the goal is always to minimize band broadening and thus minimize H by adjusting the flow rate of the mobile phase to the optimum linear velocity. This is highlighted in a plot of the HETP against the linear velocity of the mobile phase in Figure 1.1.

It is obvious that at velocities higher and lower than the optimum linear velocity there is an increase of the H(u)-curve. However, when the temperature is increased, the profile of this curve changes. The minimum of the H(u)-curve is shifted to higher linear velocities. In addition, there is a much flatter increase of H at flow rates higher than the optimum. Some authors describe this as a "flattening out" of the van Deemter curve. This means that if a separation is carried out at a mobile phase flow rate which is much higher than the optimum flow rate, the loss in efficiency at higher temperatures is less pronounced than at lower temperatures. Please note that because the optimum linear velocity is shifted to higher flow rates, working at low velocities below the optimum flow rate can result in a significant loss of efficiency when the temperature is increased. This means that the flow rate should be high enough to suppress the effects of longitudinal diffusion of molecules. In industry, liquid chromatographic separations are usually not carried out at the optimum flow rate as the van Deemter curve is not recorded if a new method is created. This is practical because many separations are not critical and hence there is no need to adjust the flow rate to the optimum linear velocity. The net benefit of operating HPLC columns at higher temperatures therefore is that the operator need not worry so much about the flow rate as long as it is higher than the optimum linear velocity. However, it needs to be stressed that there is no absolute increase in the efficiency, because it is not possible to lower the minimum of the van Deemter curve. This is often not correctly presented or misunderstood if people speak of an increase in the efficiency by increasing temperature.

(Continues...)

Excerpted from High-Temperature Liquid Chromatography by Thorsten Teutenberg. Copyright © 2010 Thorsten Teutenberg. Excerpted by permission of The Royal Society of Chemistry.
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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2010
• Language: English
• ISBN 10: 184973013X
• ISBN 13: 9781849730136
• Binding: Hardcover
• Number of pages: 220