Novel column design for centrifugal countercurrent chromatography

过去报道的流体静力逆流色谱(CCC)使用安装在离心机碗周围的盘状柱进行分析分离。在这种分离中，离心力在线圈的每一圈中保持静止相，而流动相则完全占据另一半。由于流动相通过部分置换而稳定地渗过固定相，因此保留在分离柱中的固定相的体积基本上小于总柱空间的50%或通常约为30%。固定相的低保留率限制了系统的分配效率。在逆流色谱柱中，固定相留量是决定峰分辨率的重要参数之一。固定相保留率越高，峰分离度越好。 该项目引入了环形柱的新结构，如等边三角形线圈和分布在旋转板上的各种其他形状的管子，以提高固定相的保留率和峰的分辨率。 本研究使用了由美国马里兰州巴尔的摩的Pharma-Tech Research Corporation制造的无旋转密封离心机。它容纳一个直径约34厘米的铝制旋转板，以容纳环形分离柱。已经检查了全部由0.46 mm ID氟化塑料管(Zeus Industrial Products，Orangeburg，SC，USA)制成的三种柱结构如下：1.三角螺旋柱：柱是通过将管缠绕到2m长的等长三角形聚四氟乙烯(聚四氟乙烯)管(每侧6 mm)上而制成的，该管以6.7毫升的容量旋转约2000圈。通过使三角柱的一侧面向旋转板的方式进行两个半螺旋旋转，将盘绕柱布置在旋转板的外围。2：Z字形环形柱：这种柱的设计是通过将油管钩在旋转板上形成16个单元之字形图案的直立螺丝上完成的。每个塔单元由5个约3 cm长的锯齿形环组成，每个环与径向传递管串联，每层由16个锯齿形单元组成，整个塔由多层锯齿形管组成，使塔容量达到所需的几毫升。3.锯齿形环柱：将油管钩在绕着旋转板外围的直立螺丝上，形成类似于由短径向段(6 Mm)和长螺旋环(约3 cm)组成的圆形锯齿形的油管布置图案。该柱由多层管子组成，以产生几毫升的柱容量。所有这些柱的每个端子都使用一组管道连接器(美国加利福尼亚州棕榈泉的厄普丘奇科学公司)连接到0.47 mm直径的聚四氟乙烯流量管。将这些流管放在一起，向下穿过中心轴的中心，斜齿轮的中空水平轴，然后向上引导进入垂直中空管支架，最后从上板的中心退出离心机，在那里它们用一对夹子紧紧地固定在一起。 用正丁醇-冰醋酸-水(4：1：5，v/v)组成的两相溶剂系统，对二肽、色氨酸和三羟基酪酮进行了分离试验。用正己烷-乙酸乙酯-甲醇-0.1M盐酸(1：1：1；1，v/v)组成的两相溶剂系统对DNP-氨基酸样品进行了分离。 总体结果表明，改变原用于环形线圈逆流色谱的螺旋柱的构型，可以改善有效柱间距和固定相保留率。螺旋柱用等边三角形柱芯使有效柱间距加倍，在不增加柱压力的情况下，使固定相保持在总柱容量的40%以上。锯齿形和锯齿形两种管状结构进一步改善了流动相在低流速下对静止相的保留。

Hydrostatic countercurrent chromatography (CCC) reported in the past used a coiled column mounted around the periphery of the centrifuge bowl to carry out analytical separation. In this separation the centrifugal force retains the stationary phase in each turn of the coil, while the mobile phase entirely occupies the other half. Since the mobile phase steadily percolates through the stationary phase by partially displacing it, the volume of the stationary phase retained in the separation column becomes substantially less than 50% or typically about 30 % of the total column space. This low retention of the stationary phase limits the partition efficiency of the system. the amount of the stationary phase retained in the column is one of the important parameters to determine the peak resolution in countercurrent chromatography. Ther higher the retention of the stationary phase, the better peak resolution is obtained. This project introduces new configurations of the toroidal column such as an equilateral triangular coil and various other shapes of tubing distributed on the rotary plate to improve both retention of the stationary phase and peak resolution. The present study uses a rotary-seal-free centrifuge fabricated by Pharma-Tech Research Corporation, Baltimore, Maryland, USA. It holds an aluminum rotary plate about 34 cm in diameter to hold a toroidal separation column. Three column configurations all made of 0.46 mm ID fluorinated plastic tubing (Zeus Industrial Products, Orangeburg, SC, USA) have been examined as follows: 1. triangluar helical column: The column was made by winding the tubing onto a 2 m length of equilateral triangular PTFE (polytetrafluoroethylene) tubing (6 mm each side) making about 2000 turns with a 6.7 ml capacity. The coiled column was arranged at the periphery of the rotary plate by making two and a half spiral turns in such a way that one side of the triangular column is facing to the plate. 2: Zigzag toroidal column: This column design was made by hooking the tubing onto upstanding screws forming sixteen units of zigzag patterns over the rotary plate. Each column unit consisted of 5 zigzag loops each about 3 cm long which was serially connected with a radial transfer tubing, and 16 zigzag units were made in each layer, and the whole column consisted of several layers of zigzag tubing to make a desired column capacity of several milliliteres. 3. Saw-tooth toroidal column: The column is made by hooking the tubing onto upstanding screws around the periphery of the rotary plate to form tubing layout pattern similar to the circular saw-tooth consisting of short radial segments (6 mm) and long spiral loops (ca 3 cm). The column consisted of multiple layers of tubing to make several milliliters of column capacity. Each terminal of all these columns is connected to 0.47 mm ID PTFE flow tube using a set of tubing connectors (Upchurch Scientific, Palm Spring, CA, USA). These flow tubes are put together and passed through the center of the central shaft downward, the hollow horizontal shaft of a miter gear, then led upward into the vertical hollow tube support, and finally exit the centrifuge from the center of the upper plate where they are tightly with a pair of clamps. Each column configuration is tested in separation of dipeptides, tryptophyl-tyrosine and valyl-tyrosone with a two-phase solvent system composed of 1-butanol-acetic acid-water (4:1: 5, v/v). Zigzag and saw-tooth columns were also testfed with an additional two-phase solvent system composed of hexane-ethyl acetate-methanol-0.1M HCl (1:1:1;1, v/v) for separation of DNP-amino acid samples. The overall results indicated that effective column space and stationary phase retention are improved by changing the configuration of the helical column originally used for toroidal coil countercurrent chromatography. The use of an equilateral triangular core for the helix column doubles effective column space and retains the stationary phase over 40% of the total column capacity without increasing the column pressure. Both zigzag and saw-tooth tubing configurations further improved the retention of the staitionary phase at a low flow rate of the mobile phase.