Liquid Chromatography - Choosing a Bonded Phase

Choosing a Bonded Phase

Different organic moieties can be bonded to all types of silica gel particles including the very popular spherical variety. One of the most important features to consider when choosing a bonded phase is the reproducibility of the product. The packing must obviously achieve the separation that is required, but unfortunately any bonded phase has a limited lifetime that may range from a few hours, if operated at extremes of pH, to several months if operated under mild conditions. It follows, that all columns will eventually need replacing and if a specific analytical procedure has been established on a particular column, then the replacement must have as near identical chromatographic properties as possible. The reproducibility of the column is particularly important in forensic analysis or for analyses that are carried out to ensure adherence to regulatory standards as in environmental and pollution studies. The following tests are recommended as minimal for a new column and the results should be compared with the data obtained from the previous column as received.

1/ The column permeability should be measured i.e., the pressure required to produce a given flow rate e.g., a flow rate of 1 ml per minute.

2/ The column dead volume should be measured by determining the retention volume of an unretained solute

3/ The column efficiency should be measured for a set of standard solutes. If possible, the solutes should be chosen, from those likely to be present in the samples to be analyzed. Solutes eluting at (k') values of 2, 5 and 10 would be appropriate.

4/ The corrected retention volumes of a series of solutes spanning a (k') range of 1 to 20 should be determined and their retention ratios calculated.

All the measurements should fall within 5% of the specified values. However, these criteria may not be sufficiently stringent for some forensic purposes and, consequently, the tests given above should be considered as minimum requirements for litigation purposes..