Once it was shown that the eight-disk rotor could be successfully used to separate peptides, additional studies were conducted with mixtures of peptides with known partition coefficients.
Separation is conducted by first selecting a solvent system where the analyte has a suitable partition coefficient of around one. The solvent system is mixed, equilibrated, and separated. The rotor in the planetary centrifuge is filled with the stationary phase, the sample is dissolved in equal volume of both phases, and then a small volume is loaded through the sample loop.
Centrifugation is then started, usually at 800 rpm, and the flow of the mobile phase is run at 1 or 2 mL/min. Fractions are collected, and the absorbance is read to determine location of the peptide. For recovery, the fractions can be directly lyophilized, or the fractions can be evaporated down in a centrifugal evaporator. The fractions are analyzed by HPLC to determine purity and identity.
Figure 1 shows a chromatogram of a peptide mixture. HPLC analysis of the countercurrent chromatography fraction showed that the order of elution corresponded with partition coefficients and each peptide was completely separated. Since the stationary phase is the upper or organic phase, the more hydrophobic peptides were eluted later.
The spiral disk assembly has also been evaluated for preparative purification. Numerous sample loadings were made, from 10 to 85 mg, with purification resulting in all cases in the 153 mL rotor. The major component was separated from minor components. Peptides that are hydrophobic and difficult to recover from reverse-phase HPLC were recovered in high yields. For a crude peptide with dark color due to acid sensitivity of a tryptophan amino acid residue, the percent trifluoroacetic acid was reduced to 0.1%, and the lower phase was used as the mobile phase so the fractions collected could be directly lyophilized to prevent acid-catalyzed darkening of the peptide (Figure 2).