Elution Modes in HPLC

 

Elution Modes in HPLC

 

In adsorption chromatography, solvent molecules compete with solute molecules for sites on the stationary phase. Elution can be described as a displacement of solute from the stationary phase by solvent.

 

Elution Modes in HPLC

1.Isocratic elution–Performed with a single solvent or constant solvent mixture (i.e The composition of the mobile phase kept constant throughout elution). 

2.Gradient elution–Continuous change of solvent composition to increase eluent strength (i.e The composition of the mobile phase varied during elution). 

The main purpose of gradient elution is to move strongly retained components of the mixture faster, but having the least retained component well resolved.

Starting with the low content of the organic component in the eluent the least retained components are allowed to be separated. Strongly retained components will sit on the adsorbent surface on the top of the column, or will move very slowly.

When the amount of organic component increased then strongly retained components will move faster and faster, because of the steady increase of the competition for the adsorption sites.

Gradient elution also increase quasi-efficiency of the column. In the isocratic elution, the longer a component is retained, the wider its peak. In gradient elution, especially with the smooth gradient shape without a flat regions, the tail of the peak is always under the influence of the stronger mobile phase when compared to the peak front. Thus, molecules on the tail of the chromatographic zone (peak) will move faster. This will tend to compress zone and narrow the resultant peak.

Performance of the gradient elution is strongly dependent on the instrumentation. Two main points the chromatographer needs to know about his instrument:

1.How large the volume between the component mixing point and column inlet is. For the low-pressure gradient systems this volume usually correspond to the pump volume, and about 2 - 3 ml.

2.How well does the system mix eluent components. If the system does not mix components well then it will supply for the certain time one component then another and so on. Chromatographic performance of such system will be very low especially for the least retained components.