What is the difference between normal phase and reverse phase high-performance liquid chromatography?


2 Answers 2


Normal phase

Normal phase HPLC systems are similar to the flash-column chromatography that you might be familiar with.

A silica stationary phase is eluted with a non-polar solvent such as hexane, or a fairly non-polar solvent mixture such as 2-propanol in hexanes. In normal phase chromatography, only organic solvents are used.

In the normal phase, polar molecules elute slowly, and non-polar (greasy) molecules elute quickly.

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Reverse phase

Reverse phase is essentially the opposite of normal-phase.

A non polar stationary phase (often silica in which the free hydroxyl groups are end-capped with something greasy, C18 chains are common but many many variants are possible) is eluted with a polar solvent such as acetonitrile/methanol, or a fairly polar solvent mixture (acetonitrile water mixtures are common, or methanol water mixtures).

In the reverse phase, polar molecules elute quickly, and non-polar (greasy) molecules elute slowly.

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Normal phase HPLC means the stationary phase is polar and the mobile phase is non-polar; reversed phase means the stationary phase is non-polar and the mobile phase is polar.

See HPLC Separation Modes (from Waters, a manufacturer of chromatography equipment and supplies) for more insight.


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