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 both heavier and lighter phases circle together forming a vertical interface between them. 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 outer side of the cylinder is connected to the center of the neighboring cylinder. In this elution system, the either heavier or lighter phase is introduced through the side of the first cylindrical unit filled with the other phase and exits through the central outlet to enter the second unit through its side inlet. This process is repeated in the rest of the units. This results in retention of the stationary phase in the column at near 50% of the total column space. Consequently, a sample solution introduced in the mobile phase is subjected to an efficient partition process between the two phases and eluted from the end of the column. 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 first separation column was 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 second separation column was made from the similar plastic disk by making several diameters of conical holes each arranged in a circle Their dimensions are from the periphery to the center 4-1 mm (4 mm diameter at the bottom and 1 mm diameter at the top), 5-1 mm, 7.5-1 mm, 10-1mm and 12.5-1 mm. The performance of these two vortex columns was compared in terms of theoretical plate, peak resolution (Rs) and retention of the stationary phase. Three sets of two phase solvents with a broad range of hydrophobicity were selected to separate each suitable set of test samples as follows: hexane-acetonitrile binary system applied for separation of Sudan I and II dyes: hexane-ethyl acetate-methanol-1M hydrochloric acid (1:1:1:1, v/v) for separation of DNP-amino acids (DNP-DL-glutamic acid and DNP-L-alanine);and polar 1-butanol-acetic acid-water (4:1:5, v/v) for separation of dipeptides (tryptophyl-tyrosine and valyl-tyrosine). 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. For example, in Sudan dye separation with hexane-acetonitrile binary system, the cylindrical column yielded HETP of less than 2 cm that is over 10 fold that obtained by the conventional multilayer coil mounted on the type-J coil planet centrifuge which requires over 20cm in HETP. This is apparently due to horizontal vortex mixing of the two phases within the plane perpendicular to the flow path that prevents longitudinal sample band spreading which occurs in the multilayer coil in type J planetary motion in HSCCC. These results have been presented at the 6th International Symposium on Countercurrent Chromatography which was held in Lyon, France in July 28-30, 2010. Also the provisional US patent on this device was filed through the Technology Transfer Department of NIH this year. The studies were continued to modify the inner wall of the cylindiral cavity by threading a special made tap (6-40) to increase the surface area for mass transfer. As expeacted, the threaded 3 mm ID column produced substantially higher partition efficiency interms of both theoretical plate number and compared to the untreaded counterpart. Based on these findings we have made a preparative column consisting of all 3 mm ID thread cylidrical holes to peform preparative-scale separations. As expected from the results obtained from the short column, the large column produced high partition efficiency over one throusand theoretical plates. Recently, a new vortex CCC column (non threaded) with 2 mm ID was built and tested. The results indicated that it shows partition efficiency comparable to 3 mm ID threaded column. One remarkable finding of the present system is that the column pressure during separation is only several psi which does not change by stopping the column rotation. This is clearly due to the lack of the Archimedean screw force used in the multilayer coil high-speed CCC and the hydrostatic pressure caused by the density difference between the two phases in the hydrostatic CCC systems. Therefore, the present system can be scaled-up without the risk of high pressure which might cause leakage of solvents and breakage of the separation column. Another advantage of the present system is that every point on the rotating column is subjected to the identical centrifugal force field which allows us to use the whole column space as the effective partition space. This further suggests that the column capacity of the present design can be enormously increased by eliminating the central shaft between the two rotary plates supporting the column and the counterweight to accommodate a large separation column. Finally the unique feature of the vortex CCC is summarized below: 1. The system yields high partition efficiency. 2. The system shows low column pressure. References 1. Y.Ito, Vortex counter-current chromatography system, US provisional patent N0. E-96-2010/0-US-01, filed on July 27, 2010. 2. Y.Ito;Z.-Y.Ma;R.Clary, J.Powell;M.Knight, T.M.Finn, Vortex counter-current chromatography presented at the 6th International Symposium on Counter-current Chromatography, Lyon, France on July 28-30, 2010. 3. Y.Ito, Z.-Y. Ma, R.Clary, J. Powell, M.Knight,T.M. Finn. vortex counter-current chromatography, J. Chromatogr. A. 1218, 6165, 2011. 4. Y.Ito, Z.-Y.Ma, R.Clary, J.Powell, M.Knight, T.M.Finn, Improved partition efficiency with threaded cylindrical column in vortex counter-current chromatography, J. Chromatogr. A, 1218, 4065 5. Y. Ito, R. Clary, J. Witten, Y. Zeng,Vortex Counter-Current Chromatography: Performance of a New Preparative Column, Chromatographia, in press.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2012
Total Cost
$254,847
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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Shibusawa, Yoichi; Yanagida, Akio; Ogihara, Atsushi et al. (2012) Separation of nucleobases and their derivatives with organic-high ionic strength aqueous phase systems by spiral high-speed counter-current chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 891-892:94-7
Ito, Yoichiro; Clary, Robert; Witten, Jacob J et al. (2012) Vortex Counter-Current Chromatography: Performance of a New Preparative Column. Chromatographia 75:1091-1097
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Wang, Xiaohong; Liang, Yong; Peng, Cuilin et al. (2011) PREPARATIVE ISOLATION AND PURIFICATION OF CHEMICAL CONSTITUENTS OF BELAMCANDA BY MPLC, HSCCC AND PREP-HPLC. J Liq Chromatogr Relat Technol 34:241-257
Cao, Xueli; Wang, Qiaoe; Li, Yan et al. (2011) Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 879:480-8

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