Development And Evaluation Of Spiral Tube High-speed Counter-current Chromatography

High-speed counter-current chromatography is a liquid-liquid partition chromatography which is based on the coil planet centrifugal force to retain stationary phase. The commonly used high-speed counter-current chromatography (type-J HSCCC) instrument usually uses a multilayer coil as a separation column, which is made simply by winding a single piece of PTFE (polytetrafluoroethylene) tubing directly around the hold hub. The HSCCC centrifuge based on multilayer coil can produce a high efficient separation with good retention of stationary phase for a variety of two-phase solvent systems, especially for those organic solvent-composed systems widely used for the separation of small molecular compounds in natural products. However, it often fails to retain a satisfactory amount of the stationary phase for polar solvent system and highly viscous, low interfacial tension aqueous two-phase systems (ATPS) solvent systems, that are useful for the separation of extremely polar compounds and biological macromolecules. In the conventional type J HSCCC centrifuge, the retention of stationary phase almost entirely depends upon the Archimedean screw force produced by the planetary motion of coil, which can be improved by forming a spiral tube configuration to increase the radially acting centrifugal force gradient. However in the traditional spiral column design, the spiral pitch is limited by the outer diameter of the PTFE tubing. During recent years, a spiral disk column design was promoted by increasing the pitch of the spiral.In this paper, a spiral tube HSCCC was designed and manufactured in our laboratory, and the retention of a series of solvent systems including both typical organic-water system and ATPS was investigated. The retention ability of spiral tube HSCCC was compared with the ability of traditional multilayer coil HSCCC. In addition, its preliminary application in the separation of small molecular TFs, anthocyanins, peptides, and macromolecular proteins were demonstrated.A type-J coil planet centrifuge with a 7.0 cm revolution radius was designed. Two spiral tube supports was tandem mounted on the CPC machine. The spiral tube support was designed using UG, and manufactured by FDM. Each support had a four-spiral groove in which a PTFE tube was embedded in order to increase the centrifugal effect by the increase of the pitch. Theβvalues range from 0.3 to 0.7. The two column tubes were connected in seriers, and the total capacity of the column is 30 mL.A series of solvent systems including moderately polar and polar organic-aqueous solvent systems, and aqueous two-phase systems were investigated on this spiral tube column to evaluate its retention ability of different solvent systems in different rotation directions, different mobile phases, different flow-rates, different flow ways and different g-levels. The overall results suggested that the spiral tube column can produce excellent retention of stationary phase for moderately polar organic solvent composed systems, such as heptane-methanol and hexane-ethyl acetate-methanol- water by eluting lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a flow rate of 2mL/min, and this makes it possible for fast separation of some small molecular compounds. The spiral tube column can also provide satisfactory retention for polar to aqueous two-phase systems such as 1-butanol-acetic acid-water and PEG 1000-K2HPO4-water at lower flow rate by eluting lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I), except for U-O-T mode.The stationary phase retention of all the solvent systems was gradually increased and approached at a constant value with the g-levels increase (speed increase), but different solvent system changed at different rates. For the hexane - ethyl acetate - methanol - water system, the stationary phase retention was gradually increased with the g-levels increase, and reached a constant value when the g-levels was 80g. For polar to aqueous two-phase systems such as 1-butanol-acetic acid-water and PEG 1000-K2HPO4-water system, the retention of stationary phase increased more obviously as the increase of g-level. The stationary phase retention of 1-butanol-acetic acid-water system tends to a constant when the g-levels approached more than 100g. Therefore, all the other solvent systems require a higher g-levels (110g) to achieve a better stationary phase retention except hexane - ethyl acetate - methanol - water system, for which a better stationary phase retention can be achieved at a lower g-level ( 80 g). Comparison of retention capacity of the spiral tube HSCCC with the traditional multilayer coil HSCCC at the same mode, same flow-rates, and different g-levels, a significant retention increase was clearly obtained with the spiral tube HSCCC, especially for polar to aqueous two-phase systems such as 1-butanol-acetic acid-water and PEG 1000-K2HPO4-water system (increased by 1.5 times).The preliminary separation of test samples proved that the spiral tube column can produce efficient separation for small molecular compounds such as theaflavins (TFs) and anthocyanins with less polar solvent systems composed of hexane-ethyl acetate-methanol- water and methyl tertbutyl ether-butanol- acrolein-water- trifluoroacetic acid. This is of great significance for fast analysis and preparation of small bio-active metabolites from plant and natural resources. Acceptable resolution was also achieved when it was applied for the separation of dipeptides including Leu-Tyr and Val-Tyr by using 1-butanol-acetic acid-water (4:1:5, V/V/V) solvent system. The proteins including cytochrome C and myoglobin, lysozyme and myoglobin were well separated by 12.5% PEG1000-12.5% K2HPO4-75% water (pH=9) system. This makes it possible for the separation of macromolecules, although it still needs to be modified to improve the retention and mass transfer in highly viscous ATPS.