Reverse-Phase and Hybrid Silica

So far, we have looked at reversed-phase separation using simple solvent mixtures. Many times when we carry out separation of charged or ionizable compounds we run into two problems tailing or poor retention. 

Chromatographic peaks are asymmetric and tend to broaden or tail off on the side away from the injection point. As a result, peaks tend to contaminate longer retaining neighbors. Extreme tailing, which is always due to some type of poorly resolved equilibration within the column, must be dealt with before separations can be achieved. One of the most common causes of tailing is partial ionization, either of the column bed or of the sample in the mobile phase. For instance, at the pKa of an acid, the carboxylate form and the free acid form are present in equal concentrations. If you buffer the mobile phase at this pKa and try to separate this acid from other compounds, the result will be a badly tailing peak as the column tries to separate the two equilibration forms of the acid. 

Another problem is compounds that are too soluble to retain on the column and elute unresolved in the void volume. Modifications of either the sample ionization or of the surface nature can increase retention and, therefore, resolution. In this section, we will study modifications of the column or mobile phase that will allow us to improve our separations. 

A Practical User s Guide, Second Edition, by Marvin C. McMaster Copyright 2007 by John Wiley Sons, Inc. 

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