Selection of Two Phase Solvent System

The first and most important step in CCC is to choose a proper solvent system that can satisfy the following requirements: the sample should be soluble and stable in the solvent system; the solvent system should provide a suitable partition coefficient for the target compounds; and it should produce a satisfactory retention of the stationary phase in the column. The partition coefficient and retention of the stationary phase are discussed below.

Partition coefficient CCC differs from other types of chromatography in that it uses two solvent phases and, therefore, the partition coefficient of the solute can be easily determined by a test tube experiment prior to the separation. In each measurement the sample is first partitioned between equilibrated two-solvent phases in a test tube, an aliquot of each phase is removed, and then the concentration of the solute in each aliquot is measured by ultraviolet or visual wavelength absorbance. Other methods of measurement such as radioactivity, enzymatic activity, etc., can be used. The partition coefficient, K, is the ratio between these two measurements and expressed in various ways such as K(U/L) = Cu/CL (solute concentration in the upper phase divided by that in the lower phase), K(S/M) = CS/CM (solute concentration in the stationary phase divided by that in the mobile phase), etc. When the sample is a mixture of multiple components, the partition coefficient of each component can be obtained by high performance liquid chromatogra-phy (HPLC), gas chromatography (GC) or thin-layer chromatography (TLC) analysis of each phase and by comparing the peak height or area of the corresponding peaks in the two chromatograms. In general the most suitable range of K value is 1 < K(S/M) < 2 for the hydrostatic CCC systems and 0.5 < K(S/M) < 1 for the hydrodynamic CCC systems. Once the K value has been determined, the retention volume of the solute can be computed from the following equation:

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