Yang Yi, Aisa Haji Akber, Ito Yoichiro
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 20892-1762, USA.
J Chromatogr A. 2009 Jul 3;1216(27):5265-71. doi: 10.1016/j.chroma.2009.05.024. Epub 2009 May 18.
The original spiral tube assembly for high-speed counter-current chromatography (HSCCC) is further improved by a new tube configuration called "flat-twisted tubing" which was made by extruding the tube (1.6 mm I.D.) through a narrow slot followed by twisting along its axis forming about 1cm twisted screw pitch. This modification interrupts the laminar flow of the mobile phase through the tube and continuously mixes the two phases through the column. The performance of this spiral tube assembly was tested by three types of two-phase solvent systems with different polarities each with a set of suitable test samples such as DNP-amino acids, dipeptides and proteins at the optimal elution modes. In general all these test samples yielded higher resolution with the lower mobile phase than the upper mobile phase. In the most hydrophobic two-phase solvent system composed of hexane-ethyl acetate-methanol-0.1 M hydrochloric acid (1:1:1:1, v/v/v/v), DNP-amino acids were separated with Rs-a (peak resolution based on the same column capacity adjusted for comparison) at 4.40 and 73% of stationary phase retention at a flow rate of 0.5 ml/min with the lower mobile phase. In the polar solvent system composed of 1-butanol-acetic acid-water (4:1:5, v/v/v), dipeptide samples were resolved with Rs-a at 4.06, compared to 2.79 with the cross-pressed tube assembly at 45% stationary phase retention, each at a flow rate of 1 ml/min. Finally in the aqueous-aqueous polymer phase systems composed of polyethylene glycol 1000 - dibasic potassium phosphate each 12.5% (w/w) in water, protein samples were resolved with Rs-a at 2.53 compared to 1.10 with the cross-pressed tube assembly at 52% of stationary phase retention, each at a flow rate of 1 ml/min. These results indicate that the present system substantially improves the partition efficiency with a satisfactory level of stationary phase retention by the lower mobile phase.
高速逆流色谱法（HSCCC）的原始螺旋管组件通过一种名为“扁平扭曲管”的新管配置得到进一步改进，该配置是通过将内径为1.6毫米的管挤压通过一个窄槽，然后沿其轴扭转形成约1厘米的扭曲螺距而制成的。这种改进中断了流动相在管中的层流，并通过柱连续混合两相。该螺旋管组件的性能通过三种不同极性的两相溶剂系统进行测试，每种系统都有一组合适的测试样品，如DNP-氨基酸、二肽和蛋白质，在最佳洗脱模式下进行测试。一般来说，所有这些测试样品在使用下层流动相时比上层流动相具有更高的分辨率。在由己烷-乙酸乙酯-甲醇-0.1M盐酸（1:1:1:1，v/v/v/v）组成的最疏水的两相溶剂系统中，DNP-氨基酸在流速为0.5毫升/分钟时，使用下层流动相，Rs-a（基于相同柱容量调整以进行比较的峰分辨率）为4.40，固定相保留率为73%。在由正丁醇-乙酸-水（4:1:5，v/v/v）组成的极性溶剂系统中，二肽样品的Rs-a为4.06，相比之下，在流速为1毫升/分钟时，交叉压制管组件的Rs-a为2.79，固定相保留率为45%。最后，在由聚乙二醇1000-磷酸氢二钾各12.5%（w/w）在水中组成的水-水聚合物相系统中，蛋白质样品的Rs-a为2.53，相比之下，在流速为1毫升/分钟时，交叉压制管组件的Rs-a为1.10，固定相保留率为52%。这些结果表明，本系统通过下层流动相在固定相保留水平令人满意的情况下，大大提高了分配效率。
