The conventional multilayer separation coil fails to retain polar two-phase solvent systems such as polymer phase systems and butanol/aqueous solvent systems which are useful for separation of bioactive compounds including peptides, protains, DNA, RNA, polysaccharides, etc. The retention of the liquid stationary phase in the conventional multilayer coil entirely relies on an Archimedean screw force generated by combination of coiled separation column and the special type of the planetary motion of the column holder (type J planetary motion). The new column design of the spiral tube assembly described below can utilize an additional physical force of centrifugal force gradient acting along the radial direction of the spiral, thus enhancing retention of the lower phase in the periphery and the upper phase in the proximal portion of the spiral column. The actual design of this column requires a special consideration on the tube support so that a long spiral column can be made from a single piece of tubing without junction. This can be achieved by two different ways: in the first design the connecting tubing between spiral layers is accommodated on the other side of the support. But it requires to pass a long tubing through the narrow central hole of the support and also the dead space in the tranfer tubing increases as the number of spiral layers is increased. These problems are solved by the second design that accommodates the transfer tubing between the spiral layers. Although this limits the number of spiral layers accommodated in a give column support, the problems is largely alleviated by a special process described later and we have chosen this design to develop the spiral tube assembly. The first model of the spiral tube support was made from an aluminum block in the NIH machine shop. It consists of 4 spiral grooves, each 5 cm deep and 1 m in length. It can accommodate 9 - 10 spiral layers of 1.6 mm ID, 40 m long PTFE (polytetrafluoroethylene) tubing with a total capacity of about 100 ml(ref.1). The performance of this spiral tube assembly was tested in separation of dipeptide samples, tryptophyl-tyrosine and valyl-tyrosine, with a two-phase solvent system composed of 1-butanol-acetic acid-water at a volume ratio of 4:1:5 at 800 rpm. The best results were obtained by LIT (lower mobile phase pumped from internal tail terminal of the spiral channel) and UOH (upper mobile phase pumped from the external head terminal of the spiral channel) elution modes where two dipeptides were resolved within 30 min at a high flow rate of 5 mL/min (re1. 1). However, the separation of proteins was inefficient as expected from the results obtained from the spiral disk assembly described in the previous report. The improvement in protein separation in the present system can be achieved by changing the shape of the tubing. When the tubing is pressed with a pair of pliers perpendicularly at 1 cm intervals, the peak resolution in protein separation was remarkably improved from 0.68 to 1.10. The separation of the dipeptides was also improved in spite of the reduced column capacity from 103 ml to 95 ml. This cross-pressed spiral tube assembly consisting of 9 spiral layers of 1.6 mm ID tubing with the total capacity of 95 ml could separate test protein samples at a relatively high flow rate of 2 ml/min (ref.2). As mentioned earlier, the column capacity of this spiral tube assembly was limited by the transfer tubing sandwiched between the spiral layers. This problem may be largely alleviated by compressing the 4 radial grooves using a specially-made tool with rectangular teeth which fit to the radial grooves. This process gives the following 3 major advantages for the separation First, the number of spiral layers is increased. second, a dead space in the transfer tubing is reduced, and third, laminar flow of the two phases is interrupted as in the cross-pressed spiiral tube. The performance of the tube assembly with these two tubing modifications of cross-pressing and radial groove compression was tested by protein separation with a polymer phase systems with a slightly smaller tubing of 1.35 mm ID with 15 spiral layers and with a total capacity of about 85 ml. In the LIT elution mode the separation of the two peaks wsa steadily improved with revolution speed, and at 1200 rpm two protein peaks were well resolved in 90 minutes at a high flow rate of 2 ml/min (ref.2). In order to examine the applicability of the present system, a series of experiments was performed using 5 typical two-phase solvent systems covering a broad spectrum of hydrophobicity including hexane/ethanol/water (5:4:1), Hexane/acetonitrile, Hexane/ethyl acetate/methanol/0.1M hydrochloriic acid (1:1:1:1), 1-butanol/acetic acid/water (4:5:1), and 12.5%(w/w)PEG1000/12.5%(w/w)dibasic potassium phosphate in water each with suitable test samples. The results obtained with the cross-pressed and radial-groove compressed tubing indicated that the present system can yield an excellent peak resolution in all solvent systems by choosing a suitable elution mode (ref.3). More recently the column design has been further improved by flattening tubing by extruding it through a narrow slot ollowed by twisting along its axis to form about 1 cm srew pitch. This flat-twisted tubing further improved the separation of dipeptides and proteins by eluting the column with lower mobile phase (ref.4). As described above, the spiral tube assembly can be successfully applied to the separation of dipeptides and proteins using highly polar solvent systems with sufficient stationary phase retention. Although a plain spiral tube assembly can be used for separation of peptides, the partition efficiency is remarkably increased by modifying the tube configuration in two steps, cross-pressing or flat-twisting in addition to radial groove compression especially for protein separation with a polymer phase system. Since the present system allows a high flow rate with high retention of the stationary phase, good peak resolution is obtained in a short elution time. This improved spiral tube assembly will be efficiently applied to all the two-phase solvent systems for HSCCC. References 1. Ito, Y., Clary, R., Powell, J., Knight, M., Finn, M.T. Spiral tube support for high-speed countercurrent chromatography, J. Liq. Chrom. &Rel. Technol. 31(9) (2008) 1346-1357. 2. Ito, Y., Clary, R., Powell, J., Knight, M., Finn, M.T. Improved spiral tube assembly for high-speed counter-current Chromatography, J. Chromatogr. A 1216 (2009) 4193-4200. 3. Ito, Y., Clary, R., Powell, J., Knight, M., Finn, M.T. Spiral tube assembly for high-speed countercurrent chromatography: Choice of the elution modes for four typical two-phase solvent systems, J. Liq. Chromatogr. &Rel. Technol. 32 (2009) 2013-2029. 4. Yang, Y., Aisa, H.A., Ito, Y. Flat-twisted tubing: Novel column design for spiral high-speed counter-current chromatography. J. Chromatogr. A 1216 (2009) 5265-5271.
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