HPLC Method Developer

 

This tutorial gives some basic chromatographic theory for those responsible for the HPLC equipment in a laboratory, working with trouble-shooting or develop standard HPLC-methods. For these types of work you need to have a good understanding of the different concepts that are used in chromatography.

 

The main concepts in chromatography are the retention factor ( also called the capacity factor ), the plate height and resolution. Furthermore, you need to understand how they are influenced by different experimental parameters, e.g. the mobile phase composition, the mobile phase velocity and the type of stationary phase. Some of these concepts are illustrated in the following animated simulation.

 

The purpose of the chromatographic process is to separate different types of molecules form each other. From these animations it is clear that in order to achieve this it is important to control two different parameters:

 

- the time the molecule spend in the stationary phase relative to the time they spend in the mobile phase, i.e. the retention factor.

 

- the dilution of the analyte molecules that occurs during the migration through the column, i.e. the band broadening.

 

These two parameters determine the quality of a separation between two types of molecules, i.e. the resolution.

 

Revered phase chromatography is by far the most common technique in liquid chromatography. A very instructive site about reversed phase chromatography is found at hplccourse.com.

 

A good understanding of the separation process in chromatography is therefore required in order to control the band broadening and the retention factor. Professor Yurij Kazakevich provides  a comprehensive text in basic chromatographic theory. Here you can find good presentations of the HPLC equipment too.

 

Other texts that are readable are the presentations of Kenndler and Scott. When you master the fundamentals you might be interested to to go further in certain topics. Dr. S. Levin has a site with a lot of information for e.g. chromatographers. Since reversed phase chromatography is the dominant mode of HPLC this site is focussing on this. If you are intersted in the analysis of inorganic ions by ion chromatography the site by Professor Piers is of interest to you.

 

The separation of large bio-molecules is in many respects a separate field of chromatography. A short introduction to ion exchange chromatography is found in this site.

 

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