Spiral tube assembly for countercurrent chromatography

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Existing two-phase solvent systems for high-speed countercurrent chromatography cover the separation of hydrophobic to moderately polar solvent systems, but often fails to provides a suitable partition coefficient values for highly polar compounds such as sulfonic dyes, catecholamines and twitter ions. The present paper introduces a new solvent series which can be applied for separation of these polar compounds by spiral tube countercurrent chromatography. It is composed of 1-butanol, ethanol, saturated ammonium sulfate and water at various volume ratios. The system consists of 10 steps which are arranged according to the polarity of the solvent system so that the two-phase solvent system with suitable K values for the target compound(s)can be found in few steps of search. Each solvent system gives proper volume ratio and high density difference between the two phases, and provides a satisfactory level of retention of the stationary phase in the spiral column assembly. The method is validated by partition coefficient measurement of four typical polar compounds including methyl green (basic dye), tartazine (sulfonic dye), tyrosine (twitter ion) and epinephrine (catecholamine), all of which show low partition coefficient values in the polar 1-butanol-water system. Recently, the above ultra polar solvent systems (Organic/high ionic strength aqueous solvent system) has been improved by increasing the solvent composition in 20 steps (1 to 10 and 11 to 20). Using this new system, a graphic determination of two-phase systems can be done in two steps, i.e., first the sample is partitioned in system 10. If the K value is over 1, the second K determination is performed at system 1 whereas if the K value in system 1 is below unity, the second K determination is performed at system 20. By drawing the line between these two points, the optimum K value of near unity can be easily obtained from the crossing point with the K = 1 line in each case. The method was successfully applied to separation of tartrazine (K=0.77), tryptophan (K=1.00), methyl green (K= 0.93), tyrosine (0.81), metanephrine (K=0.89), tyramine (K=0.98), and normetanephrine (K=0.96). Three sulfonic acid components in D&C Green No. 8 were separated by HSCCC using the graphic selection of the two-phase solvent system This new method was also applied for separation of ultra polar samples polar compounds including nucleobases, nucleosides and nucleotides.