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Liquid chromatography

Liquid chromatography is an analytical or preparative technique that isolates components from complex mixtures. The mobile phase, in this case a liquid, flows through the stationary phase, which can be a column packed with resin or another matrix, such as electrospun cellulose fibers. Chromatography helps purify and analyze biomolecules based on characteristics such as shape, size, charge, molecular structure, and binding affinity to the stationary phase.

Reverse phase chromatography

Reverse phase chromatography separates proteins and peptides with differing hydrophobicity based on their reversible interaction with the hydrophobic surface of a chromatographic medium. Due to the nature of the reversed phase matrices, the binding is usually very strong. Reverse phase chromatography is often used in the final polishing of oligonucleotides and peptides and is well-suited for analytical separations, such as peptide mapping.

Reverse phase chromatography FAQs

Here are answers to some frequently asked questions about reverse phase chromatography.

Which column is used in reverse phase chromatography?

Reverse phase chromatography uses columns packed with a non-polar, hydrophobic resins such as polystyrene/divinyl benzene particles.

How does a reverse phase column work?

Samples bind as they are loaded onto a column. Binding may be modulated by the use of organic solvents and other additives (ion pairing agents). Conditions are then altered so that the bound substances are eluted differentially. Elution is usually performed by increases in organic solvent concentration, most commonly acetonitrile. Samples, which are concentrated during the binding and separation process, are collected in a purified, concentrated form.

What is the difference between reverse phase chromatography and hydrophobic interaction chromatography?

Reverse phase chromatography and hydrophobic interaction chromatography (HIC) are both based on interactions between hydrophobic patches on the surface of biomolecules and the hydrophobic surfaces of a chromatography medium. However, in practice, the techniques are very different. The surface of a reverse phase chromatography medium is usually more hydrophobic than that of a HIC medium. This leads to stronger interactions that, for successful elution, must be reversed using non-polar, organic solvents such as acetonitrile or methanol. HIC media offer an alternative way of exploiting the hydrophobic properties of biomolecules by working in a more polar and less denaturing environment.