The theory of HPLC

The separation and purification of compounds by HPLC relies on a number of physical parameters which can be related to one another by theoretical considerations. To carry out successful HPLC separations it is important to have a reasonable understanding of these parameters and their interrelationship in order overcome problems which can arise in the practical use of HPLC. Firstly, to carry out efficient separations it is necessary to have an understanding of what constitutes the difference between a good and a bad column (Fig. 2.1). Elution of a component from a good column occurs as a sharp, narrow band representing minimal dilution from the original sample concentration. Conversely, a bad column will cause considerable dilution, resulting in the elution of a broad band and consequently little resolution between different components. 

It is not only the stationary phase and its packing within the column that determines the quality of the overall chromatographic system. For example, the valves, tubing and detector may retain unnecessarily large volumes of the mobile phase similarly the column end piece design may not generate an even liquid distribution any of these parameters can cause considerable band broadening. 

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It is not the purpose of this chapter to go into the theory of HPLC, which is adequately covered in other texts, but to describe the applications of the method. This section will concentrate on analytical applications because semipreparative HPLC has been described in Section 1.3.2. Analytical HPLC finds use in the quantitative determination of plant constituents, in the purity control of natural products, and in chemotaxonomic investigations. 

This book describes the. hplc method and explains and illustrates its use. Each chapter deals with a different aspect of the method, beginning with an overview and ending with a detailed summary. Throughout, an attempt has been made to focus on questions related to the assay of the activity of an enzyme rather than its purification. More detailed discussions on the theory of hplc and on its use for purification, particularly for the purification of proteins, will be found in the references at the end of each chapter. 

Although many have contributed to the theory of HPLC, only the summary by Kaizuma, H., Myers, M.N. and Giddings, J.C., J. of Chromatog. Sci., 8, 630 (1970) will be discussed now. Refer to the van Deemter section in Chapter 20, p. 217, to help you compare the theories of gas-liquid chromatography (GLC) and HPLC. In order to get the best separations, band broadening must be held to a minimum. The major factors that contribute to this are summarized in equation 19-1. 

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 various theoretical and practical aspects of the use of HPLC methods have been recently discussed in exquisite books, such as the application of HPLC-MS in drug analysis [59], the theory of chromatography [60], the fundamentals of chromatography [61, 62], the practice and theory of ion-chromatography [63], problem solving in HPLC [64], the... 

As the pH of the mobile phase markedly influences the retention of ionizable compounds, it can be assumed that the separation capacity of RP-HPLC for ionizable analyses can be modified by changing the pH of the mobile phase. The theory of effect of pH gradient on the performance of RP-HPLC systems has been recently elaborated. The basic equation describing the dependence of the retention of the solute on the gradient of pH or organic modifier is ... 

Experimental values correlated well with the predicted ones proving that the model is a valuable addition to the theory of ion chromatography [98], Many synthetic dyes are sulphonated derivatives of complicate organic molecules. Because of their strong adsorption capacity their separation under traditional RP-HPLC conditions is sometimes difficult. Anion chromatography offers an advantageous alternative for the analysis of this type of synthetic dyes. 

The primary object of this book is to provide the HPLC practitioner with a handy guide to the use of HPLC for analyzing pharmaceutical compounds of interest. This means familiarizing the practitioner with the theory, instrumentation, regulations, and numerous applications of HPLC. This handbook provides practical guidelines using case studies on sample preparation, column or instrument selection, and summaries of best practices in method development and validation, as well as tricks... 

The great versatility of HPLC is evidenced by the fact that all chromatographic modes, including partition, adsorption, ion exchange, chromato-focusing, and gel exclusion, are possible. In a sense, HPLC can be considered as automated liquid chromatography. The theory of each of these chromatographic modes has been discussed and needs no modification for application to HPLC. However, there are unique theoretical and practical characteristics of HPLC that should be introduced. 

The goal of this serial publication is to provide up-to-date accounts of various topics in HPLC. The individual chapters will cover subjects of particular interest in this rapidly growing field. Throughout the successive volumes, the coverage of applications, instrumentation, and theory will be balanced, although the contents of some volumes may focus on one or the other of these subjects. As the field evolves and the horizon of HPLC expands, future volumes are expected to present full accounts of the advances in HPLC and to unfold the perspective required for exploiting its full potential. 

Many attempts to correlate the analyte structure with its HPLC behavior have been made in the past [4-6], The Quantitative structure-retention relationships (QSRR) theory was introduced as a theoretical approach for the prediction of HPLC retention in combination with the Abraham and co-workers adaptation of the linear solvation energy relationship (LSER) theory to chromatographic retention [7,8],... 

In the late 1960s, emphasis was placed in developing LC as a complementary technique to GLC. In the early 1970s, the evolution and application of chromatographic theory, coupled with the technical improvements and advancements made in instrumentation and separation media, resulted in the development of HPLC and later capillary gas and liquid chromatography. ... 

J.H. Knox Theory of HPLC, Part II Solute Interactions with the Mobile Phase and Stationary Phases in Liquid Chromatography . In C.F. Simpson (ed.), Practical High Performance Liquid Chromatography. Heyden and Son, Chichester 1976. 

At the beginning of this article, we referred to current confusion as to the. basis of HPLC separations of macromolecules. We believe that the material reviewed here and interpreted in terms of conventional chromatographic theory largely resolves this question. That is, we have shown that conventional theory leads to a comprehensive, yet detailed picture of macromole-etde HPLC—one which can be tested by quantitative comparisons of exper-iinental data versus predictions of this model. So far, these comparisons seems to support a normal chromatognqihic process in most cases. 

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