Technique: reversed-phase

Columns and mechanism

The foundation of RP HPLC separations is hydrophobic partitioning which push the less polar molecules to be more distributed into the oil-like environment of the bonded stationary phase within RP columns, which often consists of octadecyl chains (i.e., C18). Differences between molecules in this distribution enables separation of them. In RP, a rather wide range of column selectivity is available due to secondary interactions with the chemical structures of the stationary phase (e.g., C8, phenyl, biphenyl, fluorophenyl), or with the underlying silica base material and its end-capping.

Mobile phase conditions

Eluents in RP are based on mixtures of water and one or several organic solvents, typically methanol or acetonitrile, plus often a buffer or additive to control the pH and ionic strength. RP retention decrease with increasing organic solvent, and since the separation process works under a wide variety of conditions, elution can be performed from almost pure water to pure organic solvent, depending on the sample requirements. However, to maintain high reproducibility, gradient span in practise leaves a few percent admixture of the other component at each end point, typically while maintaining a constant pH and ionic strength throughout the analysis.

Instrumentation

Instruments for HPLC are generally designed with RP mode separations in mind, and a range of detection options are available including UV light absorption, fluorescence (FL), refractive index (RI), evaporative light scattering (ELSD), charged aerosol (CAD), and mass spectrometry (MS), where the latter combination also has gained its own terminology with the abbreviation LC-MS. The recent few decades of developments within silica particle design and pump performance at ultra-high pressures have resulted in coining of the term UHPLC. This indicates an ability to accomplish separations at higher efficiency, with higher sensitivity, at higher speed and thus process a larger number of samples per time unit, but the underlying separation processes are identical to classical HPLC.

Learn more

To learn more, browse the application examples below, return to the knowledge overview, or contact Diduco to discuss your specific analysis.