Principle of High Performance Liquid Chromatography (HPLC)
HPLC is an automated version of column chromatography, which involves use of a stationary phase in the form of a column, a mobile phase, complete with a pump and a detector. The sample is injected within the column and mixed with the mobile phase followed by being pumped under high pressure. The analytes in the sample mixture interact with the stationary phase within the column differently depending on their chemical nature. Some might be retained for a longer time as compared to others and will be hence eluted at a later stage. Finally, all the eluted components are recorded by the detector and expressed in the form of a chromatogram. This chromatogram depicts each analyte within the mixture in the form of a peak plotted against the retention time (RT). The area under the curve (AUC) is generally depictive of the concentration of the analyte.
The main advantage of this technique is the platform is open and can be exploited to develop and standardize any protocol of interest. The sensitivity is very high, and with an appropriate choice of mobile phase, column and detector, multiple analytes can be identified and quantified in a single assay run.