Vr Vsf Ksmvc Vsf

where VR is the retention volume of the solute, VSF the retention volume of the solvent front (amount of the mobile phase in the column), and VC the total column capacity.

Retention of stationary phase The retention of the stationary phase in the separation column is an important factor in determining the resolution of solute peaks in CCC. Generally, the greater the retention of the stationary phase, the better the separation. In hydrostatic systems, in which the phase mixing is not violent, the retention of the stationary phase is conveniently adjusted by varying the flow rate of the mobile phase and/or the rotation speed in the centrifugal CCC system. In hydrodynamic systems, which provide efficient mixing of the two phases, stationary phase retention requires more careful selection of the two-solvent system as well as the choice of the mobile phase and its elution mode. In high speed CCC using the Type J planetary motion, the settling time of the two solvent phases under gravity provides a useful measure for the stationary phase retention and the elution mode. The test is performed as follows: the two phases are preequilibrated in a separatory funnel and 2 mL of each phase is delivered into a 5 mL capacity graduated cylinder equipped with a stopper (an ordinary glass test tube, 13 mm o.d. and 10 cm long with a polyethylene cap can also be used). The contents are gently mixed by inverting the container five times and the time required to form two clear layers is measured. If this settling time is within 30 s, the solvent system can be used for separation by eluting the lower phase from the head toward the tail or the upper phase in the reversed mode. If the settling time exceeds 30 s, the above elution mode should be reversed while the retention of the stationary phase is usually considerably lower than an optimum range. However, this settling time test is not applied to the cross-axis coil planet centrifuge systems based on the planetary motions of Type X, Type L and their hybrid systems (see Figure 3, left column). These centrifuge systems provide excellent retention of the stationary phase for almost all two-phase solvent systems including viscous polymer phase systems used for partition of macromolecules and cell particles.

The retention of the stationary phase in the hydro-dynamic CCC systems has been extensively studied using various two-phase solvent systems. These results are summarized in a set of phase distribution diagrams that will provide a valuable guide for users of high speed CCC systems (see Further Reading).

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