When molecules in the mixture are very similar, direct quantification becomes difficult. HPLC is a form of column chromatography used frequently to separate, identify and quantify compounds. It consists of a stationary phase that absorbs the analytes and holds them for a particular time. All the molecules in the mixture will spend different time with different stationary phase and then come out at the end. The time that a molecule takes to travel from one end to the other end of stationary phase is known as Retention time. Hence, all the molecules display their own unique retention time for particular stationary-mobile phase combination. Silicagel is most frequently used as the adsorbent which is packed in a column. HPLC columns are densely packed as packing directly affects the resolution. A detector is kept at the end of the column. As the molecules come out from the end, the detector detects and gives a peak on chromatogram. The peak area calculation quantifies the molecule of interest on the basis of standard values.
