High-Performance Liquid Chromatography (HPLC): Stationary Phases

Stationary Phases

In liquid–liquid chromatography the stationary phase is a liquid film coated on a packing material consisting of 3–10 μm porous silica particles. The stationary phase may be partially soluble in the mobile phase, causing it to “bleed” from the column over time. To prevent this loss of stationary phase, it is covalently bound to the sil- ica particles. Bonded stationary phases are attached by reacting the silica particles with an organochlorosilane of the general form Si(CH₃)₂RCl, where R is an alkyl or substituted alkyl group.

To prevent unwanted interactions between the solutes and any unreacted –SiOH groups, the silica frequently is “capped” by reacting it with Si(CH₃)₃Cl; such columns are designated as end-capped.

The properties of a stationary phase are determined by the nature of the organosilane’s alkyl group. If R is a polar functional group, then the stationary phase will be polar. Examples of polar stationary phases include those for which R contains a cyano (–C₂H₄CN), diol (–C₃H₆OCH₂CHOHCH₂OH), or amino (–C₃H₆NH₂) functional group. Since the stationary phase is polar, the mobile phase is a nonpolar or moderately polar solvent. The combination of a polar stationary phase and a nonpolar mobile phase is called normal-phase chromatography.

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an n-octyl (C₈) or n-octyldecyl (C₁₈) hydrocarbon chain. Most reverse- phase separations are carried out using a buffered aqueous solution as a polar mo- bile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5.