PHZone Refining CCC

This preparative CCC technique produces a train of highly concentrated rectangular peaks similar to those obtained in displacement chromatography. The method utilizes a retainer acid or base in the stationary phase and an eluter counterion in the mobile phase. Interaction of ionic analytes forms a series of solute zones with sharp boundaries that move together through the column at the same rate (isotachic movement). Each zone consists of a single species, has its own specific pH and is arranged in the order of its pKa and hydrophobicity. Charged minor components are concentrated at the boundaries of the major zone

Fraction number or time (min)

Figure 11 Separation of eight (Z)-(benzyloxycarbonyl) dipeptides by pH-zone-refining CCC.

Fraction number or time (min)

Figure 11 Separation of eight (Z)-(benzyloxycarbonyl) dipeptides by pH-zone-refining CCC.

Figure 12 Separation of ( + )-dinitrobenzoyl leucine (( $ ) DNB-leu) by pH-zone-refining CCC.

and are eluted as sharp peaks. Figure 11 shows the separation of eight (Z)-(benzyl-oxycarbonyl) dipep-tides by pH-zone-refining CCC. All components (100 mg of each) are well resolved with minimum mixing zones as evidenced by the sharp transitions of their partition coefficients (Kstd) measured with a standard two-phase solvent system.

The method can also be applied to preparative-scale chiral separation using a chiral selector in the stationary phase. Figure 12 illustrates separation of

Figure 13 Protein separation with an aqueous-aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) K2HPO4 in distilled water. The column was initially eluted with the lower phase. After point UP the column was eluted with the upper phase in the reverse direction to elute haemoglobin retained in the column SF, solvent front.

Figure 13 Protein separation with an aqueous-aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) K2HPO4 in distilled water. The column was initially eluted with the lower phase. After point UP the column was eluted with the upper phase in the reverse direction to elute haemoglobin retained in the column SF, solvent front.

2 g of (+ )-dinitrobenzoyl leucine by pH-zone-refin-ing CCC. pH-zone-refining CCC is described in detail in texts listed in Further Reading.

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