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.
Showing the most recent 10 out of 55 publications
