Question

Which parameter do we use in HPLC analysis for identification of the compound? What happens to that parameter, if the compound has a very high affinity towards the stationary phase?

Answer 1

We use the Retention Time parameter for the identification of the compound.

Retention time (RT) is a measure of the time taken for a solute to pass through a chromatography column. It is calculated as the time from injection to detection. The RT for a compound is not fixed as many factors can influence it even if the same HPLC and column are used. These include:

• The mobile phase flow rate
• Column length
• Method of HPLC run

However, keeping these parameters the same, a compound will have fixed retention time.  

If a compound has a very high affinity towards the stationary phase, the retention time would be longer. For example, retention time of benzene in the C18 column will be higher than propanol as benzene will have a higher affinity with the stationary phase of the C18 column.