LIQUID
CHROMATOGRAPHY
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. 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 (ie., 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 realised. 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 realised. A mixture of methanol and acetonitrile is employed, the acetonitrile being used to increase the dispersive interactions in the mobile phase. The reason for the particular solvent mixture is not clear and it would appear that the separation might be achieved equally well by using a stronger solution of methanol alone or a more dilute solution acetonitrile alone. There's no particular advantage to one solvent mixture over another except for the fact that 'waste' acetonitrile produces greater solvent disposal problems than methanol.
As the acetonitrile concentration became higher, however, the tetrabutyl ammonium salt would be desorbed from the reverse phase reducing the ionic interactions of the solutes with the stationary phase. At even higher concentrations of acetonitrile, the tetrabutyl ammonium salt would be completely desorbed and the interactions of the solutes with the stationary phase would become almost exclusively dispersive. This is an example where a complex phase system was necessary because there was limited column efficiency available. It is likely that a column with intrinsically more efficiency might achieve the separation with a much simpler solvent system and a more straightforward solvent programme.
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