HPLC separations depend on the partition of the sample components between mobile phase and stationary phase. HPLC can be used for analysis of analytes with high vapor pressures. The separations are quite different for different adsorbents or stationary phases. It is best to look at the chemistry of some of the common types of stationary phases to see how the separation takes place.
Reverse phase columns have hydrocarbon chains of 8 or 18 carbons bonded to the solid support. The mobile phase is somewhat polar. The analytes, which are more soluble in the mobile phase (the more polar analytes here), elute faster.
Normal phase columns are frequently silica packing materials with many hydrogen bonding and polar sites. The mobile phase is non-polar. The analytes, which are more soluble in the mobile phase (the more non-polar analytes here), elute faster. Reverse phase has supplanted this technique for most analyses because silica bonds some polar compounds so tightly that the column is difficult to clean. Normal phase is still used for specific applications where nonpolar solvents are desirable and no strongly polar substances are present. For example, the products of a laboratory organic synthesis may be separated and analyzed by this method if only weakly polar components are possible.
Ion exchange columns have ionic sites on the packing material. If the ionic sites are positive, the column is called an anion exchange column because anions interact strongly with the positive sites. The pH of the mobile phase can be adjusted to optimize the separation of analytes. Any positively charged ions or neutral molecules elute rapidly sometimes without any retention. By checking the pKa or the isoelectric point (pI) of a peptide (or protein), the pH of the buffer can be selected for optimum separations. Cation exchange columns are also available for retention of cations. The columns can be washed with increasing concentrations of salt to speed up elution of the components.
Protein and peptide separations [Supelco ] require careful selection of stationary phase and mobile phase buffers.
Size exclusion or gel permeation stationary phases separate large molecules by size. The packing material has a chemically inert surface with holes of different sizes. Large molecules do not fit into most of the holes so they elute quickly. The smallest molecules fit inside most of the holes and elute slowly because they are inside holes most of the time.
For details of instrumentation, theory, and applications refer to these HPLC explanations
Modern columns and equipment is available from several sources. Waters has specialized in HPLC equipment for many years. The polymer application has a nice introduction to size exclusion (SEC or GPC) techniques.
