What is reversed-phase chromatography?
In reversed-phase HPLC (RP-HPLC), stationary phase is non-polar and mobile phase has a moderately polar characteristic. Silica is most common stationary phase for RP-HPLC. Acetonitrile and methanol are usually used as mobile phases. RP-HPLC operates based on the hydrophobic interactions and allows the measurement of these interactive forces.
What is the principle of reversed-phase chromatography?
All proteins and peptides carry a mix of hydrophilic and hydrophobic amino acids. When mixtures of proteins are loaded into the RP-chromatographic column, their amino acids with high net hydrophobicity tend to bind to those patches to the matrix. Then the polar mobile phase increasing hydrophobicity is used to dissociate the bounds between samples and matrix. When the hydrophobic interaction between the amino acid and the stationary phase is weaker than the interaction between the sample and mobile phase, the protein or peptide releases from the matrix and elutes out of the column. According to the mechanism for RP chromatography, retention time is longer for non-polar proteins, while polar proteins elute more quickly. Besides, adding more water to the mobile phase can increase retention times of all molecules in the sample. Similarly, retention time can be decreased by adding more organic solvent to the eluent.
What is the application of reversed-phase chromatography?
This technique is often and favored used for both peptide and protein separations due to its superior resolution, good repeatability and the compatibility of eluents with MS analysis. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release.
Chromatographic columns for RP
| column | Mw range (Da) | sample | detection wavelength (nm) |
|---|---|---|---|
| ZORBAX XDB-C18 | ≤6,000 | polypeptide | 214 |
| ZORBAX 300SB-C8 | 6,000-120,000 | protein | 280 |
| ZORBAX 300SB-C3 | ≥120,000 | protein | 280 |
Sample preparation for HPLC analysis
- Concentration of protein or peptide ≥1μg/μl
- Samples need desalination before HPLC analysis
