Question

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The differences between Gradient elution in Liquid chromatography and Gradient elution in Gas chromatography??

Answer 1

A gradient method is recommended for samples that cannot be easily separated by isocratic methods because of their wide k range.

Gradient elution is used in chromatography (mainly in reversed-phase but with other modes such as ion-exchange) to modify the separations achieved in a column. It involves a continuous change in the composition of the mobile phase to achieve separation of sample components of widely varying affinities for the stationary phase.

It decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times – all according to the desire for optimum separation in minimum time.

There is a problem which arises in all types of chromatography, when samples of widely differing retention properties are present in the same sample. If the elution conditions are correct for the early eluting compounds, the late ones will remain in the column too long. They will be so broadened that it will be difficult to determine their area accurately. Indeed, they may not elute at all. On the other hand, if we set up the system so that the later eluting compounds spend less time in the stationary phase, the early peaks will come out so quickly that they will not have sufficient time in the stationary phase to allow adequate separation. This problem is called the ‘general elution problem’ and is solved in different ways in different types of chromatography. Usually, some type of "programming" is done. This involves a gradual or stepwise change in one of the operating parameters. The best conditions for the separation of the early compounds are set up. Then these are changed during the run to conditions better for the elution of more retained compounds.

In gas chromatography, the column temperature is raised over time until a temperature favorable for the separation and elution of the later peaks is reached. This is called temperature programming.

Improving Column Efficiency

> Temperature programming:

-Temperature is raised during the separation (gradient)

- increases solute vapor pressure and decrease retention time

Temperature gradient improves resolution while also decreasing retention time.

In liquid chromatography, where retention is more dependent upon strength of the mobile phase, the composition of the mobile phase is changed as a function of time. This is called gradient programming.

The retention can be decreased in liquid chromatography (RP-HPLC) by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.