Liquid Chromatography - Liquid Chromatography Applications 1


Subsequent treatment, would convert the epoxy group to diols that would insulate the analytes from the silica surface. Stalcup and Williams [42] analyzed a series of these type of materials and found that there was about 10 times as much spacer on the surface (ca. 2 mmol/m2) as there was derivatized cyclodextrin (ca. 0.2 mmol/m2). This type of chiral stationary proved to be very effective. The separation of the enantiomers of hexabarbital on this stationary phase by the direct injection of blood serum is shown in figure 56. Chromatogram A was obtained after 20 injections of serum and chromatogram B after 60 consecutive injections of blood serum. It is seen that here is very little column deterioration and that, although the tail of the major peak has become a little extended after 60 injections, the column could still be used very effectively for the analysis.

Liquid Chromatography Applications

Liquid chromatography has been used in an extremely wide range of analytical methods and it is impossible to give a comprehensive set of examples that would illustrate its wide applicability. The following are a few LC analyses that may indicate the scope of the technique and give the reader some idea of its importance and versatility.

An example of the use of reversed phase chromatography (employing a C8 column) for the separation of some benzodiazepines is shown in figure 57. The column used was 25 cm long, 4.6 mm in diameter packed with silica based, C8 reverse phase packing particle size 5 m. The mobile phase consisted of 26.5% v/v of methanol, 16.5%v/v acetonitrile and 57.05v/v of 0.1M ammonium acetate adjusted to a pH of 6.0 with glacial acetic acid and the flow-rate was 2 ml/min. The column efficiency available at the optimum velocity would be about 15,000 theoretical plates. The retention time of the last peak is about 12 minutes (i.e., a retention volume of 24 ml). At a flow rate of 2 ml/min., the mobile phase velocity will be well above the optimum and so the maximum efficiency has not been realized. The general technique used when there are more theoretical plates available than required is to increase the flow rate until the separation required is just realized. This procedure trades efficiency for time and allows the separation to be achieved in the minimum time given the column and phase system that has been chosen.