Spiral tube assembly for countercurrent chromatography

用于逆流色谱的螺旋管组件

• 批准号: 8746593
• 负责人: Yoichiro Ito
• 金额: $ 19.47万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746593
• 关键词: 1-Butanol; Acids; Ammonium Sulfate; Application procedure; Biological Factors; Carboxylic Acids; Catecholamines; Chromatography; Code; Color; Development; Dilatation and Curettage; Dyes; Epinephrine; Ethanol; FD& C Yellow No. 5; Goals; High Pressure Liquid Chromatography; Image; Ionic Strengths; Ions; Mass Spectrum Analysis; Measurement; Metanephrine; Methods; Methyl Green; Minor; N-methylacetamide-oxotremorine M; Nuclear Magnetic Resonance; Nucleic Acids; Nucleosides; Nucleotides; Paper; Partition Coefficient; Peptides; Phase; Proteins; Pyrazoles; Regulation; Reporting; Resolution; Sampling; Series; Sodium Chloride; Solvents; Speed; Sulfonic Acids; System; Tartrazine; Techniques; Tryptophan; Tube; Tyramine; Tyrosine; United States Food and Drug Administration; Water; analytical method; aqueous; countercurrent chromatography; density; design; diphenyl; improved; indexing; novel; time use

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.

现有的用于高速逆流色谱的两相溶剂系统涵盖了疏水性向中度极性溶剂系统的分离，但通常无法为高极性化合物（如硫磺染料，儿茶酚胺和Twitter离子）提供合适的分区系数值。本文引入了一个新的溶剂系列，可以通过螺旋管逆电色谱法将这些极性化合物分离。它由1-丁醇，乙醇，硫酸铵和水组成，以各种体积比。该系统由10个步骤组成，这些步骤根据溶剂系统的极性排列，因此可以在几个搜索步骤中找到具有合适k值的两相溶剂系统。每个溶剂系统都给出了两个阶段之间适当的体积比和高密度差，并在螺旋柱组件中提供了令人满意的固定相的保留水平。该方法通过分区系数测量的四种典型极性化合物进行验证，包括甲基绿色（碱性染料），塔尔塔嗪（硫磺染料），酪氨酸（Twitter离子）和肾上腺素（Catecholamine），所有这些（catecholamine）在极性1-二硫代水系统中均显示出低分配系数。最近，通过以20个步骤（1至10和11至20）增加溶剂成分，可以改善上述超极性溶剂系统（有机/高离子强度水性系统）。 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在每种情况下。该方法成功地应用于tarrazine（K = 0.77），色氨酸（K = 1.00），甲基绿色（K = 0.93），酪氨酸（0.81），Metanephrine（K = 0.89），酪氨酰胺（K = 0.98）和Normetanephrine（K = 0.96）。 D＆C绿色8号中的三个磺酸成分通过HSCCC分离，使用两相溶剂系统的图形选择该新方法也用于分离超极性样品极性化合物，包括核苷酸酶，核苷和核苷酸。 在本报告中，我们应用了上述溶剂系统将FD＆C黄色第5号的新型小杂质分开，即PK5和PK7，如下所示：美国联邦联邦法规中的规格，《颜色添加剂FD＆C黄色》第5号（颜色指数号19140）限制了四量盐盐的水平4-（4'，5-二硫酸1-1--二苯基-2-基）hidyazono-4,5-二氢-5-5-oxo-1-（4-硫苯基）-1H-吡喃唑-3-吡唑-3-羧酸和三偶氮盐的三偶氮盐4,4'-4,5-二氢-5-oxo-4-（4-硫苯基）hidyazono-1H-吡唑-1,3-二甲基苯乙烯磺酸，分别是辅助物质，分别为PK5和PK7，分别为pk5和pk7。 美国食品药品监督管理局需要少量的PK5和PK7来进行确认分析和开发分析方法。本研究描述了螺旋高速反向色谱（HSCCC）的使用以及最近引入的高度极性有机/高离子强度水性溶剂系统将PK5和PK7与FD＆C黄色的5号样品分开，其中包含3.5％PK5和0.7％PK7。 使用溶剂系统的上相1-BuOH/Etohabs/饱和硫酸铵/水，1.7：0.3：1：1，V/V/V/V/V作为移动相。 在应用了一种特殊开发的方法来从HSCCC收集的馏分中去除硫酸铵后，这些分离导致了PK5和PK7的富集混合物（分别为46％和21％）。 富集混合物的分离，这次使用该溶剂系统的下层作为流动相，导致61 mg的PK5收集的馏分中，其纯度范围为88.0％至92.7％（在254 nm时通过HPLC）。 从色谱柱含量的上阶段回收了PK7（20.7 mg，纯度83％）。 此过程的应用还导致纯化FD＆C黄色5至> 99％纯度的主要组成部分。分离的化合物的特征是高分辨率质谱和几种1H和13C核磁共振光谱技术。