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.

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2013
Total Cost
$194,746
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
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Zhang, Min; Khripin, Constantine Y; Fagan, Jeffrey A et al. (2014) Single-step total fractionation of single-wall carbon nanotubes by countercurrent chromatography. Anal Chem 86:3980-4
Zhang, Xinxin; Chen, Lin; Dang, Xuan et al. (2014) Neuroprotective effects of total steroid saponins on cerebral ischemia injuries in an animal model of focal ischemia/reperfusion. Planta Med 80:637-44
Ma, Ying; Ito, Yoichiro (2014) Chiral High-Speed Counter-Current Chromatography: Future Strategies for Chiral Selector Development. Curr Chromatogr 1:69-80
Shu, Yaping; Liang, Yong; Liang, Zhenjie et al. (2014) STUDIES ON A SIMPLE AND EFFICIENT METHOD FOR LARGE-SCALE PREPARATION OF GENKWANIN FROM DAPHNE GENKWA SIEB. ET ZUCC. USING NORMAL-PHASE FLASH CHROMATOGRAPHY. J Liq Chromatogr Relat Technol 37:773-785
Xie, Qianqian; Ding, Liping; Wei, Yun et al. (2014) Determination of major components and fingerprint analysis of Flaveria bidentis (L.) Kuntze. J Chromatogr Sci 52:252-7
Wang, Chun; Chao, Zhimao; Sun, Wen et al. (2014) Enrichment and purification of pedunculoside and syringin from the barks of Ilex rotunda with macroporous resins. J Liq Chromatogr Relat Technol 37:572-587
Wang, Chun; Chao, Zhimao; Sun, Wen et al. (2014) ISOLATION OF GLYCOSIDES FROM THE BARKS OF ILEX ROTUNDA BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY. J Liq Chromatogr Relat Technol 37:2363-2376
Li, Yang; Wang, Nan; Zhang, Min et al. (2014) Development of a method to extract and purify target compounds from medicinal plants in a single step: online hyphenation of expanded bed adsorption chromatography and countercurrent chromatography. Anal Chem 86:3373-9
Zhang, Xin-xin; Ito, Yoichiro; Liang, Jin-ru et al. (2014) Therapeutic effects of total steroid saponin extracts from the rhizome of Dioscorea zingiberensis C.H.Wright in Freund's complete adjuvant induced arthritis in rats. Int Immunopharmacol 23:407-16
Zhang, Xinxin; Liang, Jinru; Liu, Jianli et al. (2014) Quality control and identification of steroid saponins from Dioscorea zingiberensis C. H. Wright by fingerprint with HPLC-ELSD and HPLC-ESI-Quadrupole/Time-of-fight tandem mass spectrometry. J Pharm Biomed Anal 91:46-59

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