Question

1. Explain the basic mechanism of separation in an HPLC column with emphasis on mobile

phase, solid phase, retention time, normal phase, reverse phase and selectivity.

Answer 1

1. High pressure liquid chromatography or high performance liquid chromatography is a improved form of column liquid chromatography used to seperate,identify and quantify each component in a mixture using very less amount of sample volume. The mobile phase is liquid and mixture of solvents whereas the solid phase is densly packed within the stainless steel column.So a high pressure upto 400 atmoshphere is required to flow the mobile phase so that it can illuminated out the sample. A detector is attached after HPLC column used to detect analytes as they elute from the column. A data aquisitor is attached and component which is moving first, will reach at the site of detector and aquisitor stores in in chart recorders or electronic integrator that gives a peak.

Normal phase HPLC : seperate on basis of polarity.

Stationary phase - Polar e.g. silica

Mobile phase - Non polar e.g. hexane, chloroform or mixture of both

So more polar component longer it will retain in column.

Reverse phase HPLC:

Stationary phase - non polar

Mobile phase - polar (water, methanol, acetonitrite or mixture of both)

More the non polar component retain long time in column.

Retention factor(k) is the capacity of a component to interact with the column.More k indicates sample is highly retained and spend a long time with stationary phase.

Selectivity factor is the ratio of retention factor and distingush between sample component. Calculated by distance between apices of two peak.