Reverse Phase Chromatography (RPC)

Reverse Phase Chromatography is a special, yet powerful column chromatography that involves the use of hydrophobic adsorbents. Normal phase chromatography normally uses hydrophilic stationary phase. This technique uses the hydrophobic adsorbent, which are the aliphatic chains that are attached to the silica beads.

Brief Introduction

The gel bead is surrounded by a layer of lipophilic aliphatic chains of carbon, which are the ligands. The sample is applied to an aqueous solvent. The peptides that are adsorbed to the gel bead are eluted out from the column by decreasing the organic solvent concentration of the column, thus creating an increasing organic solvent gradient. The elution of peptide from the column is in the trend of increasing hydrophobicity. The peptide with the lowest hydrophobicity would get eluted out first.

Advantage

1. High resolution
   
2. Speed
   
3. High capacity, which is the best method to concentrate samples
   

 

Disadvantage

1. Solvents
   

   The organic solvents are toxic, which have the possibility of denaturing the labile proteins.
   

2. Silica-based
   

   Hence, it is unstable under alkaline condition.