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HPLC user





As a HPLC user you should understand the basic principles of chromatography ( see e.g. the "Novice" section). Furthermore you should be familiar with the most important concepts that is used in chromatography. You also need to have a basic knowledge about the HPLC-equipment and how it is working.


The three most important concepts that are used in chromatography are:


The Retention Factor (Capacity Factor)


Since the separation is based on adsorption, the quantitative measure of the tendency of a particular type of molecule to adsorb to the stationary phase is important. The measure used for this is the retention factor (often also called the capacity factor).


Peak Width


Due to the nature of the chromatographic equipment a dilution of the injected sample solution occurs when the sample molecules migrates through the column. As a consequence the zone containing the sample molecules broadens continually during its passage through the column. The detector will register a peak with a certain width. The measure for the peak width is the height of a theoretical plate.  




Resolution is a measure of the quality of the separation between two kind of molecules. It is composed of the two previous concepts, peak width and retention factor of the two components.


How is separation achieved?


Retention in a chromatographic column is achieved because the retained molecule adsorbs reversibly to the stationary phase. On a molecular level this means that it spends some time in the stationary phase.  The movement of the molecules is a mixture of a random molecular movement and the movement caused by the flowing mobile phase.


Separation is achieved because different types of molecules will spend different periods of time on the stationary phase surface.


Simulation of the separation process


The principles are illustrated in the following animated simulation of small circles moving in an erratic manner and randomly stick to the column walls for a certain time.


In the animation a mixture of 200 red and 200 blue circles will be separated. The adsorption time to the wall at the for the blue is 0.1 s and for the red 0.5 s.


Press the inject button to start the animated simulation.




There are other sites that is of interst to you. At LC resources you find a very good tutorial about basic HPLC theory as well as about the equipment used. Animations of HPLC pumps, the injector and detector is found here. A narrative animation of the injector used in HPLC is found in a site from Sam Houston State University.


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