The Effect of Stationary Phase Loading on the Performance of a Chromatographic System

The stationary phase content of a column can affect a separation in two ways. The more stationary phase there is in a column, the more the solutes will be retained, the further they will be apart and the greater the separation. Any change in stationary phase, however, will change the retention of all solutes proportionally and, thus, the separation will only increase, if the peak widths remain unchanged. Increasing the amount of stationary phase will usually increase the thickness of the stationary phase film, which, as is shown in Book 7 will increase peak dispersion. It follows that there will be a specific stationary phase loading that provides the best compromise between separation and band dispersion (6) and, thus, provides the maximum resolution. The loading can be quite critical for open tubular columns in GC. Thus, the stationary phase loading cannot be increased indefinitely to separate the peaks as, eventually, the peaks will start spreading to a greater extent than they are being separated.

Increasing the stationary phase load on a GC column (packed or open tube) will allow the sample placed on the column to be increased. A large sample is often necessary in trace analysis to provide sufficient material for detection. Under such circumstances the column may be overloaded giving a very broad asymmetric peak that may obscure the trace materials of interest. This asymmetric dispersion is due to solute-solute interaction in the mobile and stationary phases causing a nonlinear adsorption isotherm. The subject of adsorption isotherms will not be discussed here and it is sufficient to say that the asymmetric dispersion can be reduced by increasing the amount of stationary phase in the column. A larger amount of stationary phase, will, even with a larger charge, reduce the sample concentration in the stationary phase and, thus, the deleterious high sample concentrations are never reached.

The stationary phase loading of a an LC column is not modified the same way as a GC column. This is because, irrespective of the type of support material, the amount of stationary phase in an LC column is primarily determined by its surface area. In addition, the amount of available stationary phase on a bonded phase can be modified by adjusting the molecular size (chain length) of the bonded material.

The chain length of the bonded material can be critical when separating proteins as dispersive interactions between the alkane chains and the dispersive (hydrophobic) groups of the protein can be strong enough to cause structural de-conformation; (i.e., the protein becomes denatured). Reducing the chain length of the bonded material, the dispersive forces can be reduced significantly and the de-conformation diminished. In practice, carbon chains only two or four carbon atoms long are among those most commonly used for separating labile proteins.