Spiral tube assembly for countercurrent chromatography

现有的用于高速逆流色谱的两相溶剂系统涵盖了从疏水到中等极性溶剂系统的分离，但往往不能为高极性化合物（如磺酸染料、儿茶酚胺和微离子）提供合适的分配系数值。本文介绍了一种适用于螺旋管逆流色谱分离极性化合物的新型溶剂系列。它由1-丁醇、乙醇、饱和硫酸铵和不同体积比的水组成。该系统由10个步骤组成，这些步骤根据溶剂体系的极性排列，以便在几步搜索中找到适合目标化合物的K值的两相溶剂体系。每个溶剂系统给出适当的体积比和两相之间的高密度差，并在螺旋柱组件中提供令人满意的固定相保留水平。通过对甲基绿（碱性染料）、酒石酸锌（磺酸染料）、酪氨酸（微离子）和肾上腺素（儿茶酚胺）4种典型极性化合物的分配系数测定，验证了该方法的有效性，这4种极性化合物在极性1-丁醇-水体系中的分配系数均较低。近年来，上述超极性溶剂体系（有机/高离子强度水溶液溶剂体系）通过20步（1 ~ 10和11 ~ 20）增加溶剂成分得到了改进。使用这种新系统，两相体系的图形测定可以分两步完成，即，首先在系统10中对样品进行分区。如果K值大于1，则在系统1进行第二次K测定；如果系统1中的K值低于1，则在系统20进行第二次K测定。通过在这两点之间画一条线，可以很容易地在每一种情况下K = 1线的交叉点处得到接近统一的最优K值。该方法可用于酒黄石（K=0.77）、色氨酸（K=1.00）、甲基绿（K= 0.93）、酪氨酸（0.81）、肾上腺素（K=0.89）、酪胺（K=0.98）、去甲肾上腺素（K=0.96）的分离。采用两相溶剂体系的图形选择，采用HSCCC分离了D&C绿8号中的3种磺酸组分，该方法也适用于超极性样品核碱基、核苷、核苷酸等极性化合物的分离。

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. In this report we have applied the above solvent system to separate novel minor impurities of FD&C Yellow No. 5, namely Pk5 and Pk7 as follows: Specifications in the U.S. Code of Federal Regulations for the color additive FD&C Yellow No. 5 (Colour Index No. 19140) limit the level of the tetrasodium salt of 4-(4',5-disulfo1,1'-biphenyl-2-yl)hydrazono-4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylic acid and that of the trisodium salt of 4,4'-4,5-dihydro-5-oxo-4-(4-sulfophenyl)hydrazono-1H-pyrazol-1,3-diylbisbenzenesulfonic acid, which are subsidiary colors abbreviated as Pk5 and Pk7, respectively. Small amounts of Pk5 and Pk7 are needed by the U.S. Food and Drug Administration for confirmatory analyses and for development of analytical methods. The present study describes the use of spiral high-speed counter-current chromatography (HSCCC) with the recently introduced highly polar organic/high-ionic strength aqueous solvent systems to separate Pk5 and Pk7 from a sample of FD&C Yellow No. 5 containing 3.5% Pk5 and 0.7% Pk7. Multiple 1.0 g portions of FD&C Yellow No. 5 (totaling 6.4 g dye) were separated, using the upper phase of the solvent system 1-BuOH/EtOHabs/saturated ammonium sulfate/water, 1.7:0.3:1:1, v/v/v/v, as the mobile phase. After applying a specially developed method for removing the ammonium sulfate from the HSCCC-collected fractions, these separations resulted in an enriched mixture (160 mg) of Pk5 and Pk7 (46% and 21%, respectively). Separation of the enriched mixture, this time using the lower phase of that solvent system as the mobile phase, resulted in 61 mg of Pk5 collected in fractions whose purity ranged from 88.0% to 92.7% (by HPLC at 254 nm). Pk7 (20.7 mg, 83% purity) was recovered from the upper phase of the column content. Application of this procedure also resulted in purifying the major component of FD&C Yellow No. 5 to >99% purity. The separated compounds were characterized by high-resolution mass spectrometry and several 1H and 13C nuclear magnetic resonance spectroscopic techniques.