Aqueous, moderately polar mobile phase and non-polar stationary phase characterize RP-HPLC. The interaction of the polar compounds with the polar mobile phase makes this approach of separating the polar compounds particularly effective. An n-alkylsilica-based sorbent is often the basis of an RP-HPLC experimental setup. A gradient of increasing concentrations of an organic solvent, such as acetonitrile with an anionic modifier, is used to elute solutes from the sorbent. It is usually possible to separate complex combinations of proteins and peptides. Performing separations is made simple by adjusting the ionic modifier, operating temperature, solvent gradient slope, and organic solvent composition. Additional characterisation can be done using the separated fractions.
Peptides, proteins, and glycoproteins can all be analyzed and purified using the widely used RP-HPLC technique. The RP-HPLC separation of p-aminobenzoic ethyl ester derivatives of neutral and amino sugars released from glycoproteins can be used to assess the composition and amount of monosaccharides. Furthermore, a study has demonstrated that the RP-HPLC method, which is superior to chromatographic and/or enrichment approaches like HILIC and lectin affinity, has the best coverage for glycopeptide analysis based on the number of detected glycosylation sites and has identified glycopeptides in three model glycoproteins.