Question

Using Silica or Alumina in packing the column results in “Normal” chromatography. What is reversed-phase chromatography and why is it useful?

Answer 1

Reversed-phase chromatography is so named because it is the reverse of the normal-phase mode. The stationary phase contains non-polar and hydrophobic surface instead of polar functional groups like silanol groups in silica. Solute retention is mainly a result of hydrophobic interactions between the solutes and the hydrophobic stationaryphase surface.

Polar mobile phases usually water mixed with an organic modifier such as methanol, acetonitrile or tetrahydrofuan are used for elution. Optimization of the mobile phase can often be achieved by using a mixture of two or more organic modifiers.

Solutes are normally eluted in order of increasing hydrophobicity and increasing the content of the non-polar component in the mobile phase decreases the retention of the solutes.

Reversed phase chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Molecules that possess some degree of hydrophobic character, such as proteins, peptides and nucleic acids, can be separated by reversed phase chromatography with excellent recovery and resolution.