Why Is This

Protic solvents solvate polar functional groups and thereby make them more exposed. The only polar functional groups that can be found on a conventional nonpolar reversed phase are the sUanol groups. If these are made more prominent by solvation, then the undesired ionic interactions with basic analytes come more to the fore and the elution bands become broad and asymmetric. If, however, an aprotic solvent such as acetonitrile is used with these columns, then the lone pair electrons of acetonitrile can form hydrogen bonds with the silanol groups, and these will be shielded from the basic analytes. The elution bands are much narrower and more symmetrical. 

With polar reversed phases, the effect is exactly the opposite. Here, the polar groups (usually nitrogen-containing functional groups) become exposed by the use of protic solvents and thereby interact with the silanol groups. This has the advantage that then the basic analyte cannot interact with these silanol groups. As a result, when protic solvents are used bases elute from polar reversed phases with symmetrical elution bands. 

When acidic analytes are being separated, for maximum selectivity the strongest possible interactions ofthe analytes with the silanol groups ofthe stationary phase are required. Consequently, in this case it makes sense with classical, nonpolar stationary phases to use protic solvents such as methanol as the mobile phase. With polar stationary phases the polar interactions should be suppressed as much as possible, and so here aprotic solvents such as acetonitrile should be used as the mobile phase. 

When both acids and bases are present in a sample, then mobile phases suitable for the bases are normally used, as broad and asymmetric elution bands generally have a more serious effect on the analytical problem as a slightly inferior selectivity. In such cases, as a compromise mixtures of protic and aprotic solvents (methanol and acetonitrile) are sometimes used. 

Furthermore, in the reversed-phase HPLC stationary phases whose surface has been modified with phenyl or pentafluorophenyl groups are also used. These functional groups can only develop their maximum selectivity when they are solvated. Therefore, with these columns it is imperative to use a protic solvent as mobile phase. The use of acetonitrile is ruled out, since its lone-pair electrons interact with the stationary phase and thus the interaction of the phenyl groups with the analyte is at least suppressed, if not completely blocked. 

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