Reversed-phase chromatography (RPC) is a liquid chromatography technology that separates molecules using hydrophobic interactions between solute molecules in the mobile phase and ligands linked to the stationary phase. 
High-performance liquid chromatography using reverse phase It has been determined that RP-HPLC is the best technique for handling physical separations based on HPLC. RP-HPLC has been essential in the quantitative and specific applications of biological molecule separation, including the isolation of proteins, peptides, and nucleotides from a broad range of chemical and biological components.

Because reversed-phase compounds are more flexible than normal-phase adsorbents, they are used in more than 80% of current analytical chromatographic separation procedures. Reversed-phase substances have already been employed efficiently in numerous applications, such as the quantitative separation of medicines, metabolic products, and energetic biochemicals, as well as the retrieval of contaminants from collected samples.

The effectiveness of reversed-phase high-performance liquid chromatography (RP-HPLC) applications in evaluating and purifying peptides, short polypeptides, and pharmaceutical medicines has not been replicated in the observation and purification of bigger polypeptides and globular proteins. This is owing to a decrease of enzyme synthesis and poor yields caused by denatured proteins released during solvent extraction. The reverse-phase chromatography procedure can be summarized as follows: 
1. Enhances polar analysis solubility. 
2. Uses nontoxic solvents. 
3. Provides a means to remove hazardous byproducts. 
4. Active substances mixed in a solvent. 
5. Ensures quick specimen restoration and little solvent evaporation.

The mobile phase in many RPC procedures is a mixture of water and a miscible organic solvent (such as methanol or acetonitrile). Maintaining polarity at a low enough level to allow the solute to dissolve in the mobile phase while keeping it high enough to aid in the chosen molecule's interaction with the stationary matrix is the aim of the organic solvent. Trifluoroacetic acid and other ion-pairing agents may also be employed in certain situations. By raising the concentration of the organic solvent in the mobile phase, the polarity is further decreased after the target molecule has been attached to the column matrix. Gradient elution is the process of changing the quantity of organic solvent in the mobile phase in order to separate a desired molecule.

In preparative separations, reverse phase chromatography provides various advantages over normal-phase techniques. Some of them are listed below:

  • This technique resolves solubility issues that are typical in non-polar normal-phase liquids.
  • Uses fewer toxic solvents.
  • Allows for rapid sample recovery.
  • Removes contaminants and mobile phase additives.

Factors affecting the separation in reverse phase chromatography:

In reverse phase chromatography, the rate of the separation process is influenced by difference parameters. Among them are:

Column:

In reverse phase separations, the resolution of large biomolecules is less affected by column length than that of small organic molecules. Proteins, big peptides, and nucleic acids may all be adequately purified using short columns, and increasing column length has no discernible effect on resolution. Increased column length can occasionally improve the resolution of small peptides (including some).

Mobile phase:

In many cases, the mobile phases in reverse phase chromatography are referred to informally as buffers. Although mobile phase conditions frequently contain strong acids at low pH values and significant concentrations of organic solvents, they have limited buffering capacity. While operating closer to physiological conditions, sufficient buffering capacity should be maintained. Large molecules desorb in a very narrow range of organic modifier concentrations because the partition coefficients of high molecular weight solutes are extremely sensitive to modest changes in the mobile phase composition.

Organic Solvents:

The addition of an organic solvent (modifier) lowers the polarity of the aqueous mobile phase. In reverse phase chromatography, the mobile phase's eluting strength increases as its polarity decreases. The most often used organic modifiers are acetonitrile, isopropanol, and methanol. All three solvents are essentially UV transparent. Because column elution is usually detected with UV detectors, this property is critical for reverse phase chromatography.

Ion Supression:

Protein and peptide retention in reverse phase chromatography can be affected by mobile phase pH since these solutes include ionizable groups. The pH of the mobile phase determines the ionization level. Trifluoroacetic acid and orthophosphoric acid are two popular strong acids used to create the mobile phase for reverse phase chromatography. These acids maintain a low pH and prevent acidic groups in solute molecules from ionizing. The number of strong acid components in the mobile phase can influence the ionization of the solutes.