Question

A type of chromatography known as reverse phase chromatography uses a non-polar stationary phase instead of a polar stationary phase. How do you predict this will alter R_f values of the molecules shown above? Explain.

This is a qeustion for the Chromatograph lab.

Answer 1

A normal-phase chromatography employs a hydrophilic (polar) stationary phase and a non-polar mobile phase while a reverse-phase chromatography employs a non-polar stationary phase and a polar mobile phase.

The retention factors of compounds in chromatography are dependent on their solubilities in the mobile phase and their affinities to the stationary phase. An increased solubility and a decreased affinity causes the compounds to elute first and so have a high Rf, closer to 1 while compounds with low solubility and an increased affinity elute later, with R_f closer to zero.

In a normal-phase chromatographic separation, the use of a non-polar solvent and a polar stationary phase ensures that hydrophobic (non-polar) compounds elute first or have a high R_f value, while polar compounds have lower R_f. This is because the non-polar molecules will dissolve better in the mobile phase (like dissolves like) and cannot establish the polar interactions the stationary phase provides, thereby giving them a low affinity and high solubility.

In a reverse-phase chromatographic separation, the exact reverse happens. Polar substances dissolve better in the polar mobile phase and have low affinity to the non-polar stationary phase. This causes the polar compounds to elute first, having higher R_f values while non-polar compounds, with their low solubilities and higher affinities elute later, with lower R_f.

So, in theory, the Rf values of compounds in normal-phase chromatography and reverse-phase chromatography will be the opposites of each other and mathematically will be equal to 1 - R_f or R_f -1.