Reversed-phase chromatography (RPC) is a liquid chromatography technique that involves the separation of molecules on the basis of hydrophobic interactions between the solute molecules in the mobile phase and the ligands attached to the stationary phase.
Concept of reversed-phase
In classic chromatography models, the stationary phase is a polar phase and the mobile phase is a nonpolar organic solvent. The polar constituents dissolved in the mobile phase tend to get attracted to the polar stationary phase, while the nonpolar constituents tend to get eluted along with the solvent.
This forms the basis for separation. This is known as normal phase chromatography. A variety of biological properties ranging from ion exchange to biological affinity have been used for the separation of molecules.
In RPC, alkyl or aromatic ligands are covalently bound to the stationary phase to provide a hydrophobic surface. The mobile phase passing over the hydrophobic stationary phase is a polar solvent containing the solutes.
In this scenario, hydrophobic solutes in the mobile phase tend to get bound to the stationary phase via hydrophobic interactions and thus form the basis of separation. As the principle here is the opposite of what has long been used in classical chromatography, this is known as “reversed-phase” chromatography.
The matrix in reversed-phase chromatography
The chemical composition of the base matrix is important in RPC as it has to be both chemically and structurally stable. Silica and synthetic polystyrene satisfy these requirements and are often used as RPC matrix materials.
The particle size of the beads is based on the specific requirements of the separation. Larger bead size implies larger capacities and potentially lesser pressures. Large-scale preparative processes benefit by using beads of diameter greater than 10 μm; small-scale preparative and analytical separations, on the other hand, benefit with beads sizes in the 3–5 μm range.
The choice of ligand is often governed by the principle that the greater the hydrophobicity of the molecule to be purified, the less is the need for the ligand to be hydrophobic. This principle has also led to the rule that chemically synthesized peptides and oligonucleotides are best separated with C18 ligands and that protein and recombinant peptides are better separated with C8 ligands.
The density of the immobilized ligands on the surface of the beads, the capping chemistry, and the pore size of the beads are further factors to be considered for a successful reversed-phase separation.
The mobile phase in reversed-phase chromatography
Many RPC protocols use a blend of water and a miscible organic solvent (e.g., acetonitrile or methanol) as the mobile phase. The purpose of the organic solvent is to maintain the polarity at a low enough level for the solute to dissolve in the mobile phase and yet high enough to facilitate the binding of the preferred molecule with the stationary matrix.
In some scenarios, ion-pairing agents such as trifluoroacetic acid may also be added. Once the molecule of interest is bound to the column matrix, it is made to dissociate from it by decreasing the polarity further by increasing the concentration of the organic solvent in the mobile phase.
This process of varying the amount of organic solvent in the mobile phase to separate a molecule of interest is called a gradient elution.
Reversed-phase chromatography in practice
Reversed-phase high-performance liquid chromatography (RP-HPLC) has become a powerful tool for the analysis of peptides and proteins for the following reasons:
- Tested stability of the matrix under a variety of mobile phase scenarios
- Reproducibility of separations
- Excellent resolutions achievable for molecules of interest (either closely related or structurally distinct)
- Appreciable recoveries and throughput
- Ease of separations provided by gradient elution
For all these reasons, RP-HPLC has become the method of choice for most HPLC-based separations. RP-HPLC has played a major role in the separation of biomolecules (proteins, peptides, nucleotides) from a wide variety of synthetic and biological molecules, for both analytical and preparative applications.
Because of the versatility of reversed-phase sorbents over normal-phase sorbents, they are used in more than 80% of analytical chromatographic separations performed today. Reversed-phase sorbents have been successfully used in applications such as analytical separation of drugs, metabolites, and active biomolecules as well as extraction of contaminants in environmental samples.
The success of RP-HPLC applications seen in the analysis and purification of peptides, small polypeptides, and pharmaceutical drugs has, however, not been possible to the same extent with larger polypeptides (>10 kDa) and globular proteins, due to loss of enzymatic activity and poor yields because of protein denaturation during the separation process.