How HPLC work
HPLC is known as High performance liquid chromatography it is one of the method which make use of an instrument consisting stationary phase packed in a column through which a mobile phase carrying a mixture of drug molecule passes through with the help of pressure applied by a high pressure pump.
HPLC basically make use of principle of chromatography for separation of drug molecule from mixture,
A sample is prepared in a suitable mobile phase , and injected in to the flow under high pressure this is pumped through column containing solid phase made of polymer , silica , solid in the HPLC column is in granular form , when a mixture of drum molecule pass through , different molecules , ( drug and intermediates , and other substances which are called as related substance (byproducts and intermediates in chemical reaction used in manufacturing of drug molecule ) travel through solid phase (column ) at different speed , hence each molecule in the drug mixture is removed from other end of column at different time interval total time period required for complete removal of a drug molecule is known as retention time.
When a drug molecule comes out from other end of column it is taken by solvent to a detector, where its concentration is found out by detectors spectro photometer, flame photometer, photodiode are used as detector, the concentration is measured, and plotted against time required for elution.
In HPLC quantitative analysis, time required for complete elution of a drug molecule is an important factor it depends up on the concentration of the drug molecule in a given sample higher is the concentration more is the time required, as vice-versa, a graph plotted against amount of drug molecule removed on X axis time and on Y axis concentration.
For quantification aria under the curve is calculated and compared with standard concentration, gives idea about exact idea about the concentration of a drug molecule being analyzed.