Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 8N230, Bethesda, MD 20892, USA.
J Chromatogr A. 2010 Apr 2;1217(14):2117-22. doi: 10.1016/j.chroma.2010.02.003. Epub 2010 Feb 6.
The performance of the coiled column of centrifugal counter-current chromatography was investigated by changing the angle between column axis and centrifugal force in the separation of dipeptides or DNP-amino acids each with suitable two-phase solvent systems. In general, retention of the stationary phase (Sf) decreased, and peak resolution (Rs) increased as the column angle was increased. The first series of experiments was performed using a polar two-phase solvent system composed of 1-butanol-acetic acid-water (4:1:5, v/v/v) to separate two dipeptide samples, Trp-Tyr and Val-Tyr, at a flow rate of 1 ml/min at 1, 000 rpm. When the column angle was changed from 0 degrees to 90 degrees , Rs increased from 1.05 (Sf=60.1%) to 1.17 (Sf=38.7%) with the lower phase mobile and from 1.02 (Sf=67.8%) to 1.14 (Sf=47.4%) with the upper phase mobile, respectively. The second series of experiments was similarly performed with a more hydrophobic two-phase solvent system composed of hexane-ethyl acetate-methanol-0.1M hydrochloric acid (1:1:1:1, v/v/v/v) to separate three DNP-amino acids, DNP-glu, DNP-beta-ala and DNP-ala, at a flow rate of 1 ml/min at 1, 000 rpm. When the column angle was changed from 0 degrees to 90 degrees , Rs increased from 1.38 (1st peak/2nd peak) and 1.20 (2nd peak/3rd peak) (Sf=61.1%) to 1.66 and 1.45 (Sf=34.4%) with the lower phase mobile and from 1.14 and 0.63 (Sf=72.2%) to 1.53 and 0.87 (Sf=51.1%) with the upper phase mobile, respectively. The overall results of our studies indicate that increasing the column angle against the radially acting centrifugal force enhances the mixing of two phases in the column to improve the peak while decreasing the stationary phase retention by interrupting the laminar flow of the mobile phase.
本文研究了在离心逆流色谱中改变柱轴与离心力之间的角度对不同流动相系统中二肽或DNP-氨基酸的分离效果。结果表明，随着柱角的增大，固定相（Sf）的保留值减小，峰分辨率（Rs）增大。第一组实验使用由 1-丁醇-乙酸-水（4:1:5，v/v/v）组成的极性两相溶剂系统，在 1,000rpm 转速下以 1ml/min 的流速分离两种二肽样品 Trp-Tyr 和 Val-Tyr。当柱角从 0 度变为 90 度时，下相流动相的 Rs 从 1.05（Sf=60.1%）增加到 1.17（Sf=38.7%），上相流动相的 Rs 从 1.02（Sf=67.8%）增加到 1.14（Sf=47.4%）。第二组实验也使用更疏水的两相溶剂系统（正己烷-乙酸乙酯-甲醇-0.1M 盐酸（1:1:1:1，v/v/v/v）），在 1,000rpm 转速下以 1ml/min 的流速分离三种 DNP-氨基酸 DNP-glu、DNP-β-ala 和 DNP-ala。当柱角从 0 度变为 90 度时，下相流动相的 Rs 从 1.38（第一峰/第二峰）和 1.20（第二峰/第三峰）（Sf=61.1%）增加到 1.66 和 1.45（Sf=34.4%），上相流动相的 Rs 从 1.14 和 0.63（Sf=72.2%）增加到 1.53 和 0.87（Sf=51.1%）。我们的研究结果表明，增加柱角以抵抗径向离心力可以增强两相在柱中的混合，从而改善峰形，同时通过中断流动相的层流来减少固定相的保留。
