Vortex Countercurrent Chromatography

在过去，流体动力学逆流色谱已经用盘绕柱进行，该盘绕柱在柱的行星运动下产生阿基米德螺旋效应。 分离是用各种流通式离心机系统进行的。 其中J型螺旋管行星离心机是目前应用最广泛的天然产物和合成产物的分离提纯设备。 本发明基于I型行星运动。在20世纪70年代，已经报道了使用盘管的这种螺旋行星离心机的CCC应用，其使用了大体积的离心机系统（参考文献1）。 I型螺旋行星离心机的行星运动与研究实验室中广泛使用的涡旋混合器中的行星运动相同，涡旋混合器具有几毫米的短旋转半径，并且可以在试管中形成液体对空气的涡旋。相比之下，I型螺旋行星离心机，围绕10 cm的大回转半径旋转，这可以在圆柱形保持器中产生两个不混溶的液相的涡旋运动，使得较重的相围绕较轻的相旋转。通过将一系列圆柱形单元与精细的传输管道连接，这样，圆柱体的下部外侧连接到相邻圆柱体的中心。在该洗脱系统中，较重相通过第一圆筒的中心引入，向下移动通过较轻相的柱，并从下部外侧入口离开，并在其余圆筒中重复该运动。 因此，足够量的较轻相保留在柱中，其与移动的较重相连续混合。 因此，引入移动的相中的样品溶液将在两相之间进行有效的分配过程，并从柱端洗脱。 当较轻的相是移动的相时，其通过第一圆筒的下部外侧入口引入，其中其流过较重的固定相的中心，并通过上部中心出口离开圆筒，并在其余圆筒中重复该过程。在分离柱的实际设计中，每隔一个柱体被倒置以减少连接流管的长度，连接流管构成无效的死空间。 分离柱的实际设计是由直径为17 cm、高为5 cm的高密度聚乙烯盘制成的。 在最外侧和最内侧的用于螺钉密封的孔旁边，6组圆柱形柱各布置成一个圆形：它们的尺寸从周边到中心为3 mm（120），4 mm（70），5 mm（60），7.5mm（40），1.0mm（20），直径为1.25 mm（10），其中圆柱体的数量在括号中表示。该柱夹有一对特氟隆片和金属凸缘，其用多个螺钉紧密压缩以形成密封的分离通道。 初步研究了以正己烷和乙腈组成的任意体积比的二元两相溶剂体系分离苏丹红染料（苏丹红I和苏丹红II）， 使用上部移动的相用3 mm直径圆柱体获得的结果以理论塔板数（TP）和峰分辨率（Rs）、相当于TP的体积（ml/TP）和相当于TP的高度（cm/TP）表示，其中ml/TP和cm/TP由第二个峰获得的TP计算。 数据表明，通过较高的rpm和较低的流速来提高分配效率。 将从3 mm涡流CCC圆柱体获得的这些结果与从常规HSCCC和基于Taylor Couette流动装置的涡流分离柱获得的数据进行比较，如该表所示。 由于每个系统的柱容量和长度不同，因此可以根据ml/TP和cm/TP对分配效率进行公平比较。 从表中可以清楚地看出，涡流CCC产生的高效率为2 cm/TP，接近其他两个系统的10倍。 这显然是由于水平涡混合的两相，防止纵向样品带扩展，发生在多层线圈中的类型J行星运动在HSCCC。 本系统存在固定相残留和固定相保留水平低的问题，特别是当下层相用作移动的相时。 在下一个塔设计中，这个问题可以通过将分离单元从圆柱体改为圆锥体来解决。 在这种配置中，作用在两相上的离心力引导上相向上和下相向下，以改善固定相的保留。 通过在单元的内壁上制造圆形起伏或回旋和/或向单元添加芯，可以进一步改进柱设计。 这些复杂的柱设计可以通过激光烧结制成，用于未来与美国马里兰州罗克维尔的CCBiotech合作进行快速原型制作。 最后，涡流CCC的独特特征总结如下： 1. 与现有的CCC系统相比，该系统产生高的分配效率（cm/TP）。 2. 该系统显示出低柱压，这允许使用较长的柱而没有溶剂泄漏的风险。 3. 该系统还将根据纳米颗粒的尺寸和密度，像旋风分离器一样，用于分离纳米颗粒，但效率要高得多。

In the past hydrodynamic countercurrent chromatography has been carried out with a coiled column which produces an Archimedean screw effect under a planetary motion of the column. The separation is performed with a variety of flow-through centrifuge systems. Among those, type-J coil planet centrifuge is now most widely used for separation and purification of natural and synthetic products. The present invention is based on the type-I planetary motion The CCC application of this coil planet centrifuge using a coiled tube has been reported in 1970s using a large bulky centrifuge system (ref. 1). The planetary motion of type I coil planet centrifuge is identical to that in the vortex mixers widely used in research laboratories which have a short revolution radius of several millimeters and can form a vortex of liquid against air in a test tube. The type I coil planet centrifuge, in contrast, rotates around the large revolution radius of 10 cm which can produce vortex motion of two immiscible liquid phases in a cylindrical holder in such a way that the heavier phase circles around the lighter phase This vortex motion of two liquid phases can be effectively utilized for performing countercurrent chromatography by connecting a series of cylindrical units with fine transfer ducts in such a way that the lower outer side of the cylinder is connected to the center of the neighboring cylinder. In this elution system, the heavier phase is introduced through the center of the first cylinder moves down through the column of the lighter phase and exits from the lower outer side inlet and repeats this motion in the rest of the cylinders. Thus a sufficient amount of the lighter phase is retained in the column which is continuously mixed with the mobile heavier phase. Consequently, a sample solution introduced in the mobile phase will be subjected to an efficient partition process between the two phases and eluted from the end of the column. When the lighter phase is the mobile phase it is introduced through the lower outer side inlet of the first cylinder where it flows through the center of the heavier stationary phase and exits the cylinder through the upper central outlet and repeats this process in the rest of the cylinders. In the actual design of the separation column, every other cylinder is inverted to reduce the length of the connecting flow tube which constitutes an inefficient dead space. The actual design of the separation column is made from a disk of high density polyethylene measuring 17 cm in diameter and 5 cm in height. Beside the outer most and inner most holes which are used for sealing with screws, 6 sets of cylindrical columns are each arranged in a circle: their dimensions are from the periphery to the center, 3 mm (120), 4 mm (70), 5 mm (60), 7.5 mm (40), 1.0 mm (20), and 1.25 mm (10) in diameter where the number of the cylinders is indicated in the parentheses. This column is sandwiched with a pair of Teflon sheets and metal flanges which are tightly compressed with a number of screws to form sealed separation channels. The preliminary studies were performed to separate Sudan dyes (Sudan I and II) with a binary two-phase solvent system composed of hexane and acetonitrile at an arbitrary volume ratio, and the results obtained with 3 mm diameter cylinder using the upper mobile phase are expressed in terms of theoretical plate number (TP) and peak resolution (Rs), volume equivalent to TP (ml/TP) and height equivalent to TP (cm/TP) where ml/TP and cm/TP are computed from TP obtained from the second peak. The data show that the partition efficiency is improved by higher rpm and lower flow rate. These results obtained from 3 mm cylinders of vortex CCC are compared with the data from the conventional HSCCC and vortex separation column based on Taylor Couette flow apparatus as shown in this table. Since the capacity and length of the column are different in each system, fair comparison in partition efficiency can be made from ml/TP and cm/TP. As clearly shown from the table, vortex CCC yields high efficiency of 2 cm/TP near 10 folds of those obtained from two other systems. This is apparently due to horizontal vortex mixing of the two phases which prevents longitudinal sample band spreading which occurs in the multilayer coil in type J planetary motion in HSCCC. The present system has a problem of carry over of the stationary phase and low level of the stationary phase retention especially when the lower phase was used as the mobile phase. In the next column design this problem can be solved by modifying the separation unit from cylinder to cone. In this configuration the centrifugal force acting on the two phases directs the upper phase upward and the lower phase downward to improve the retention of the stationary phase. The column design may be further improved by making circular undulation or convolution on the inner wall of the unit and/or adding a core to the unit. And these complex column designs may be made by laser sintering for rapid prototyping in collaboration with CCBiotech, Rockville, MD, USA in the future. Finally the unique feature of the vortex CCC is summarized below: 1. The system yields high partition efficiency in terms of cm/TP compared with the existing CCC systems. 2. The system shows low column pressure which permits use of a longer column without a risk of leakage of the solvent. 3. The system will also be applied for separation of nanoparticles according to their size and density like the cyclone separator but with much higher efficiency.