Biochemical Techniques and Instrumentation

2.7.2.1. Mobile phases

Mobile phase refers to the solvent that is continuously applied to the column or stationary phase. Pure solvents should be used as mobile phases because traces of impurities with absorbance <200 nm present in the mobile phase will affect the column and interfere with the detection system. Dust particles in the mobile phase should be removed as it interferes with detection, clogs the column and damages the pumping system. A microfilter of 1-5 μm size is used before the pump to remove the dust. Mobile phase should also be degassed as it affects the column resolution and causes band broadening. Degassing can be done by warming, stirring vigorously with a magnetic stirrer, applying a vacuum, ultrasonication or bubbling helium gas through the mobile phase reservoir.

There are different types of elution.

a. Isocratic elution

In isocratic elution, compounds are separated using constant mobile phase composition. It consists of single pump that can pump a single solvent or two/more solvents premixed in fixed proportions. This type of elution is simple and inexpensive, but resolution of some compounds is limited.

b. Gradient elution

In gradient elution, different compounds are separated by changing the strength of the mobile phase. Separate pumps are needed to deliver two or more solvents in proportions predetermined by a gradient programmer. Sample is injected while a weaker mobile phase is applied to the system. Strength of the mobile phase is subsequently increased in increments. The resolution and separation are better than isocratic elution.