There are various types of quantitation methods used in HPLC, each with its own principles and applications. Following are some commonly employed quantitation methods in HPLC.

Area Normalization Method

In this method, % area of each peak is calculated against the total area of peaks in chromatograms. It is the most straightforward and easiest method for quantitation.

External Standard Method

In this method, a calibration curve is prepared using standard solutions with known concentrations of the analyte. The peak areas or heights of the standards are plotted against their known concentrations. The concentration of the analyte in the sample is determined by comparing the sample's peak area or height to the calibration curve.

Internal Standard Method

An internal standard is a known compound added to both the standards and the sample. The internal standard's peak area or height is used to normalize variations in injection volume, column performance, and detector response. The ratio of the target compound's peak area or height to that of the internal standard is used to calculate the concentration in the sample.

Standard Addition Method

This method is useful when the analyte is already present in the matrix. In this method, known amounts of the standard compound(s) are added to the sample, and the mixture is analysed by HPLC. By comparing the peak areas or heights before and after the addition, the concentration of the compound(s) in the sample can be determined.

The selection of the appropriate quantitation method depends on many factors such as the nature of the analyte, sample matrix, standards availability, potential interferences, and desired precision and accuracy. But the basic requirement for all the above-mentioned methods is base-to-base separation of peaks in the chromatogram.