What is High Performance Liquid Chromatography (HPLC)

High performance (pressure) liquid chromatography is an improvement of the classical method of liquid or partition chromatography. In liquid chromatography, a solute (or solutes) is partitioned between two immiscible liquids.

In other words, both the mobile phase and the stationary phase are liquids.

ColumnSubstances such as silica gel in a finely divided form (150 pm to 200 pm) are packed in a glass tube or a column.

One of the liquids is immobilized or fixed on the surface of a solid and this fixed liquid acts as the stationary phase.

The components of a mixture which is poured down such a column get partitioned between the stationary liquid phase and the mobile phase.

However, this method is time consuming and tedious. This is because of the low flow rates of the liquid mobile phase which moves down the column under the influence of gravity alone. Attempts made to increase the rate of flow of the mobile phase by using a high pressure and decreasing the column length resulted in increased plate heights and a decrease in separation efficiency.

In order to increase the flow rate of the mobile phase, two conditions have to be satisfied.
1. The column packing should consist of small, spherical and uniformly sized particles (about 60 gm) so that the packing has the optimum homogeneity and density.
2. The stationary liquid phase should be in the form of a thin uniform layer.
These conditions are satisfied in high performance liquid chromatography.

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