High performace Liquid Chromatography

Basis of the separation process

As explained in the introductory section, chromatographic separation process based on the difference in the surface interactions of the analyte and eluent molecules.

Let us consider a separation of a two component mixture dissolved in the eluent. Assume that component A has the same interaction with the adsorbent surface as an eluent, and component B has strong excessive interaction. Being injected into the column, these components will be forced through by eluent flow. Molecules of the component A will interact with the adsorbent surface and retard on it by the same way as an eluent molecules. Thus, as an average result, component A will move through the column with the same speed as an eluent.

Molecules of the component B being adsorbed on the surface (due to their strong excessive interactions) will sit on it much longer. Thus, it will move through the column slower than the eluent flow.

Usually a relatively narrow band is injected (5 – 20 ul injection volume). During the run, the original chromatographic band will be spread due to the noneven flows around and inside the porous particles, slow adsorption kinetics, longitudinal diffusion, and other factors. These processes together produce so called band broadening of the chromatographic zone. In general, the longer the component retained on the column, the more broad its zone (peak on the chromatogram).

Separation performance depend on both component retention and band broadening. Band broadening is, in general, a kinetic parameter, dependent on the adsorbent particle size, porosity, pore size, column size, shape, and packing performance. On the other hand, retention does not depend on the above mentioned parameters, but it reflects molecular surface interactions and depends on the total adsorbent surface.

Retention parameters

The easiest way to find the chromatographic retention is to measure the time between the injection point and maximum of the detector response for correspondent compound. This parameter usually called “retention time”. Retention time, tR is inversely proportional to the eluent flow rate.

The product of retention time and eluent flow rate, so called “retention volume”, is more of a global retention parameter. Retention volume, VR represent the volume of the eluent passed through the column while eluting a particular component.

Component retention volume VR could be split into two parts:

  1. Reduced retention volume is the volume of the eluent that passed through the column while the component was sitting on the surface.
  2. Dead volume is the volume of the eluent that passed through the column while the component was moving with the liquid phase.

The second part is equal to the volume of the liquid phase in the column (dead volume, Vo), and it will be the same for any component eluted on this column.

Retention volume is independent of the flow parameters for the particular run, but it depend on the geometrical parameters of the column. VR will be different for the same compound eluted on the different columns packed with the same type of adsorbent.

The more universal and fundamental retention parameter is the ratio of the retention volume and dead volume (k).

k = VR/Vo

Historically, a slightly different retention parameter, called “capacity factor” (k’) was introduced by the analogy with the liquid partitioning theory and widely accepted in chromatographic practice.


Capacity factor is dimentionless and independent on any geometrical parameters of the column or HPLC system. It could be considered to be a thermodynamic characteristic of the adsorbent-compound-eluent system.

Constant Pressure Pumps

The primary advantages of constant pressure pumps are simplicity and freedom from pulsations, resulting in smooth baselines. The most simple of these are usually inexpensive, easy to operate, and easy to maintain. They suffer from several disadvantages, however. Flow rate must be monitored carefully and constantly, especially when performing either qualitative or quantitative analysis. Flow rates can and do change! This can happen if the solvent viscosity changes due either to a temperature or composition change.


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