Bonded Phases - Choosing a Bonded Phase
Choosing a Bonded Phase
Many different organic moieties can be bonded to a wide range of different silica gel particles that includes the spherical variety. However, one of the most important features to consider when choosing a bonded phase for a particular application is the reproducibility of the product. The stationary phase must obviously accomplish the separation that is required, but, unfortunately. the different bonded phases will have a limited lifetime and this may range from a few hours (if operated at extremes of pH) to several months if operated under mild conditions. Consequently, all columns will eventually need replacing and if a specific analytical procedure has been developed using a particular stationary phase, then any replacement must have as near identical chromatographic properties to the original as possible. Column reproducibility is essential for forensic analysis or for those analyses where the results will be used to ensure adherence to regulatory standards as in environmental studies, pollution studies and forensic purposes.
The literature appears to indicate that the fluidized bed method of synthesis (particularly for the brush phases) provides the more reproducible products. Unfortunately, bonded phases synthesized by the fluidized bed method are not commonly available. As a result, all new replacement columns must be carefully evaluated to ensure they meet the required analytical specifications. Specification data confirming the reproducibility of any column purchased should be available from the manufacturer but in many cases, the tests carried out by the manufacturer may not be appropriate for the particular analysis involved. Thus, each replacement column should be carefully evaluated by the analyst concerned to ensure its performance is satisfactory. In past years, reverse phase column reproducibility has been markedly poor, and although the quality and reproducibility of contemporary products have improved considerably, quality control by the user concerned still must be considered essential. The following tests are recommended as minimal for a new or replacement column and the results should be compared with the data obtained from previous columns 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 determined by measuring the retention volume of an unretained solute
3/ The column efficiency should be measured employing a set of standard solutes. Where possible, the solutes should be selected 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 measured and their retention ratios calculated.
All the measurements should fall within 5% of the specified values. However, this 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.