Reverse Phase chromatography – Methods and Principle

Reverse Phase chromatography separates proteins and peptides based on the difference in hydrophobicity, Reversible interaction with the hydrophobic chromatographic matrix/medium results in the separation. As the sample is loaded, Binding to the chromatographic matrix takes place, altering the conditions results in elution of the protein or peptide from the matrix. The reverse phase nature of column matrix causes strong binding, usually harsh conditions (organic solvents or ion pairing agents) are required to elute the protein out from the matrix.

<! --adsense-- > In Reverse Phase high purity can be achieved. Since harsh conditions are used proteins and enzyme with final tertiary structure need to be maintained may not suitable for purification using RPC since the protein or enzyme activity may lose during the process.

RPC used is often used in the final purification of oligonucleotides, peptides and proteins. It is ideal for analytical separations such as peptide mapping.

Reverse Phase HPLC separation: Overview

Select suitable Hydrophobic Ligand

Ligand is the material on which proteins will interact with and separate based on the hydrophobicity. Commonly used ligands in RPC are C4, C8, and C18 n-alkyl hydrophobic ligands. Select suitable ones based on their degree of hydrophobicity required for the separation.

Highly hydrophobic molecules bind tightly to strong hydrophobic ligand like C18.

Suitable matrix which gives high resolution and purity need to be selected, screening of RPC media can be done to achieve this, trying from low hydrophobicity to high hydrophobicity screening is usually done.

Sample volume and binding capacity

RPC is a binding technique which is independent on sample volume. Total binding capacity depends on the matrix used and the conditions provided for the binding. Matrix should be selected in such a way that one should get high resolution with high sample loading volume.

Sample and column Preparation

As like any other column chromatographic separation process, sample should be free of particulates, this can be achieved by filtering the protein sample with no or low binding filters. Sample with particles can lead to clogging and results in poor resolution and or damage to the system due to back pressure generation and other factors.

Columns of RPC need to be equilibrated with enough column volumes of equilibration buffer so as to give perfect binding condition for the proteins.

Storage

Column need to be stored as per the specifications of the column supplier.