Biochemical Techniques and Instrumentation

2.7.1. Principle

The separation of compounds by a chromatographic column depends on the number of theoretical plates in the column, which is related to the surface area of the stationery phase. The smaller the particle size of the stationery phase, the greater is the retention of the compounds and also greater is the resistance to the flow of mobile phase. The columns with small particle size stationery phase of 5-10 μm diameter that can withstand high pressures is the basis of HPLC.

The separation of compounds is based upon stationary phase of small particle size with large surface area that are packed into short columns of 20-25 cm length or less. The mobile phase is then pumped through the system at high pressures and the separation is achieved in a few min at flow rates of 1-3 ml per min. The pumps operate at the high pressures of upto 50 MPa that are needed to force the mobile phase through the tightly packed column with a steady flow.