Basic Concepts of HPLC

Step elution is similar to gradient elution in that the composition of the mobile phase changes during the separation process. However, in step elution the change is not continuous. It involves a sudden change in the composition of mobile phase followed by a period where the mobile phase is held constant. This procedure results in a mobile phase profile that resembles a series of steps. 

Displacement chromatography is commonly used for preparative-scale separations, but, because of its focusing or concentrating effect, it also shows potential on the analytical scale, for example, for the concentration of minor components in complex mixtures.24,25 Operationally, displacement chromatography is similar to the step elution process, except that in the displacement process the mobile phase has a greater affinity for the stationary phase than for the sample components, and therefore the components are eluted ahead of the displacer front. The focusing effect of displacement chromatography is due to the fact that the concentration of the displacer determines the concentration of the product bands.26 

Frontal analysis is a preparative method, used primarily for the separation of one readily eluted component from the other, more tightly held components. The technique is performed by the continuous addition of a sample mixture onto the column. Initially, the component of interest, that is, the component with the least affinity for the stationary phase, will pass through the column while the other sample components are retained to various degrees by the stationary phase. As a result of the continuous sample application, the concentration of bound components steadily builds up at the head of the column. When the column capacity for any given component is exceeded, that component also passes through the column. Therefore, the first component is eluted from the column initially as a pure band and subsequently as a mixture with the next components to be eluted. 

Chromatography is described and measured in terms of four major concepts capacity, efficiency, selectivity, and resolution. The capacity and selectivity of the column are variables that are controlled largely by the column manufacturer, whereas efficiency and resolution can be controlled, 

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This chapter introduces some of the basic concepts and principles of liquid chromatography, providing background on the development of high performance liquid chromatography (HPLC) and briefly describing the basic system components. 

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 ... 

This chapter provides an overview of basic terminology and essential concepts in HPLC including retention, selectivity, efficiency, resolution, and peak symmetry as well as their relationships with key column and mobile phase parameters. The resolution and van Deemter equations are discussed. The concepts of peak capacity and method orthogonality as well as key gradient parameters such as gradient time and flow rate are described. An abbreviated glossary of HPLC terms is listed. 

In the second chapter of this book, you were introduced to the basic theory of chromatography. Here in this chapter, we shall look more specifically at the modes of separation used in HPLC. Already, the concept of a mobile phase and a stationary phase has been established. Here, we further discuss this basic concept and explain the chemical nature of the stationary and mobile phases and how varying these, as well as considering the analytes we have, has an effect on the separations that can be carried out. 

The basic concepts have already been presented in this review. However, the study of single compounds does not require an interface between the chromatographic unit and the MS. The analysis of mixtures by MS relates not only to GC/MS or HPLC/MS . The popularity of GC/MS systems can be examined by the nuihber of relevant publications. These were less than 100 in 1968, rose to a peak of 2000 papers in 1979, dropped to 1500-1750 yearly until 1988, when they rose again to 2000. The use of MSD for HPLC (also called LC/MS) also rose from a few publications in 1975 to a few hundred in 1988. A method, less used for hydrocarbon analysis, SFC (Super-critical Fluid Chromatography)/MS, was initiated only in 1985 and is gaining interest slowly. These data are given by Evershed . Obviously it will be impossible to review all these developments in the use of MSD ", even for the analysis of alkanes and cycloalkanes. 

Thin-layer chromatography has often been considered to be a manual method. With this in mind, users tend to consider this technique to be of low quality and unreliable, thus disregarding its high potential. The instrumental development of new techniques such as HPLC reinforced this idea. Therefore, the TLC concept was reviewed and fully automated to alter this perception of the method. First, the basic performance of TLC had to be improved, in terms of ... 

We will begin by taking a look at the detailed aspects of a basic problem that confronts most analytical laboratories. This is the problem of comparing two quantitative methods performed by different operators or at different locations. This is an area that is not restricted to spectroscopic analysis many of the concepts we describe here can be applied to evaluating the results from any form of chemical analysis. In our case we will examine a comparison of two standard methods to determine precision, accuracy, and systematic errors (bias) for each of the methods and laboratories involved in an analytical test. As it happens, in the case we use for our example, one of the analytical methods is spectroscopic and the other is an HPLC method. 

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