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.
The main applications of HPLC are:
Qualitative analysis: HPLC as a qualitative tool has very limited applications. It is not possible by HPLC to positively identify a compound unless some pure compound is also available. A small quantity of the pure sample of the substance, which is supposed to be present in the analytical sample, is added to the sample. If the assumption about the presence of that compound was correct the height of the peak due to that compound will rise. and if the assumption was wrong, a new peak will appear on the chromatogram. The components separated are generally identified by mass spectrometry or infra red spectroscopy.
Quantitative analysis: Quantitative analysis is accomplished by measuring the peak heights and peak areas and comparing these with a calibration curve. Different known amounts of a substance are mixed with the carrier gas and the mixture passed through the chromatographic column. The peak height is drawn. This is the calibration curve. If several compounds are present in the analytical mixture, a separate calibration curve is drawn for each compound. The peak height is length of the perpendicular drawn from the top of the peak to the base line. The concentration can also be calculated by measuring the area under the peak forming curve. This is called the peak area. The peak area is found by multiplying the peak height by half width of the peak (width measured at the middle of the peak height.
Separation of mixtures: High performance liquid chromatography is an efficient, highly selective method of separation. Small sample sizes can be separated. The only condition is that a suitable immiscible solvent pair must be available. Generally, the more polar of the two is made the stationary phase. In HPLC, separation is carried out at room temperature. Therefore, thermally unstable substances, which cannot be separated by GLC, can be separated. The method is also applicable for the separation of inorganic ions.