HPLC theory

The process of analyte retention in high-performance liquid chromatography (HPLC) involves many different aspects of molecular behavior and interactions in condensed media in a dynamic interfacial system. Molecular diffusion in the eluent flow with complex flow dynamics in a bimodal porous space is only one of many complex processes responsible for broadening of the chromatographic zone. Dynamic transfer of the analyte molecules between mobile phase and adsorbent surface in the presence of secondary equilibria effects is also only part of the processes responsible for the analyte retention on the column. These processes just outline a complex picture that chromatographic theory should be able to describe. 

HPLC theory could be subdivided in two distinct aspects kinetic and thermodynamic. Kinetic aspect of chromatographic zone migration is responsible for the band broadening, and the thermodynamic aspect is responsible for the analyte retention in the column. From the analytical point of view, kinetic factors determine the width of chromatographic peak whereas the thermodynamic factors determine peak position on the chromatogram. Both aspects are equally important, and successful separation could be achieved either by optimization of band broadening (efficiency) or by variation of the peak positions on the chromatogram (selectivity). From the practical point of view, separation efficiency in HPLC is more related to instrument optimization, column 

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright 2007 by John Wiley Sons, Inc. 

Four major descriptors are commonly used to report characteristics of the chromatographic column, system, and particular separation  

Retention factor (k) is the unitless measure of the retention of a particular compound on a particular chromatographic system at given conditions defined as 

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A.M. Krstulovic and P.R. Brown, Reversed Phase HPLC. Theory, Practical and Biomedical Applications, J. Wiley Sons, New York, 1982. ISBN 0471053694. 

All of the chapters have been written by selected experts in their respective fields, and will undoubtedly provide the readers with an in-depth understanding of HPLC theory, hardware, methodologies, regulations, and applications. 

In recent years HPLC technique has been perfected. A large number of food components may be separated with HPLC. Many excellent books have been published on HPLC theory and thousands of articles have been written on chromatographic separations of food-related components. 

Jacobson, S.C., Ramsey, J.M., Microfabricated chemical separation devices. In HPLC Theory. Techniques and Applications, Wiley, New York, 1998, 613-633. 

Significant number of modern HPLC separation methods are based on gradient elution or continuous variation of the eluent composition during the analytical experiment. Theoretical description of the gradient separation is very complex and, to large extent, nonexistent. It is possible to use basic HPLC theory for phenomenological explanation of gradient retention dependencies. 

HPLC theory is too broad of a topic to be comprehensively covered by one chapter or even by one book. This chapter was intended to create a general understanding of the relationships between different aspects of chromatographic theory, such as analyte retention behavior and retention mechanism. 

The principles and results of HPLC theory that are useful in everyday work have been presented in Chapters 2 and 8. The following is a comprehensive repetition and summary of important equations. Derivations and references are not presented here both can be found in the two chapters mentioned and in many sections of the book (see Index) references can also be found in the paper on which this appendix is based. ... 

The objective of this chapter is to provide the reader with a concise overview of HPLC terminology and concepts. Both basic and selected advanced concepts are covered. The reader is referred to other HPLC textbooks,1-7 training courses,8-9 journals, and Internet resources for a more detailed treatment of HPLC theory and concepts. This chapter has the following sections ... 

A short and not too serious look at the name HPLC is followed by a list of frequently used abbreviations and symbols and some tips for newcomers to the subject, including a checklist to be used before doing an HPLC run. Then comes a brief explanation of some important chromatographic expressions. This is intended as a refresher it cannot replace a study of HPLC theory in an appropriate textbook. The main part of the book is a series of Tips , grouped under three headings ... 

The consequences of multisite attachment include the following. First, the competition model 18,45) can be construed to identify Z with the number of attachments of the solute molecule to the stationary phase in any case Z is expected to be proportional to this number. Consequently, large molecules will have larger values of 5 and Z as compared to small molecules. This is recognized in our version of conventional HPLC theory as applied to macromolecule samples. 

Today, micro-LC is a valuable analytical tool for sample-constrained applications such as proteomics and bioanalysis. The microliter flow rates are ideally suited for direct, splitless coupling with electrospray ionization mass spectrometry (ESI-MS). Many benefits can be demonstrated from well-established HPLC theory, which allows for direct method transfer to micro-LC. 

Pereira L. Porous graphitic carbon as a stationary phase in HPLC theory and applications. J Liq Chromatogr Rel Tech 2008 1(31) 1687—731. 

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