For maximal separation of impurities the best partition coefficient or K for a compound is around 1 where the compound will elute at a column volume. There is enough retention of the compound such that early eluting and late eluting impurities will be maximally separated. A dried sample is dissolved in equal volumes of each phase of a pre-equilibrated solvent system. Then the conc. is determined by absorbance or by HPLC analysis.
For samples to be purified the most accurate method is to separate by HPLC and the area or peak height of the major component is measured. The ratio of the value in the upper phase to the lower phase is the K (Cu/Cl). In the CCC run the K is the stationary to mobile phase (Cs/Cm) which can be calculated from the elution volumes. At a K = 1, the compound elutes at a column volume. The phase chosen as the mobile phase is that giving a partition coefficient close 1. The elution volumes from around 0.3 to 3 elution volumes comprise the maximal resolution zone. The Ks/m (stationary phase/mobile phase) calculated from the elution of a compound is the ratio of the elution volume of the chromatographic peak minus the excluded volume of the coil to the total volume of the coil minus the excluded volume of the coil. K = (Vp-Vm)/(Vc-Vm)
In the example shown, 3 peptides at once were added to the solvent system and by HPLC from the areas the K’s could be measured. The retention times of the peptides are 20 min, 24.5 min and 28.5 min, respectively. The peptides were separated by CCC using the lower phase mobile of sec-butanol/aq.1%Tfa. The order of elution followed the K, 6877 then 7020 followed by 507. From the known K, the mobile phase can be selected to give the K closest to 1. If the K or inverse K is outside of 0.3 to 3 then try another solvent system. |   |
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