Among various spiral disks tested in the past, the mixer-settler spiral disk previously reported showed the best resolution of proteins with polymer phase systems. Based on the results of he previous experiment, a multiple disk assembly consisting of 8 units has been constructed and the performance tested with two standard protein samples, lysozyme (chicken egg white) and myoglobin (horse skeletal muscle), using a polymer phase system composed of 12.5%(ww) PEG1000 and 12.5%(ww) dibasic potassium phosphate. The experiments were continued to test the following various parameters : effects of revolution speed, flow rate, elution modes, etc. Using the best experimental condition, two sets of test samples consisting of 5 proteins and natural products such as daikon sprout extract and egg white were also separated using a suitable polymer phase systems. ? ? 1. Eight barricaded spiral disks (barricades: 2 mm wide leaving 0.4 space on both side and 0.5 mm long) were fabricated and assembled together after placing glass bead in every other barricaded section. It was tested with a dye eluted with water under various revolution speeds. When the disk is stationary, the dye was eluted with a broad peak. At a low speed of 200 rpm, the effect of mixing is already evident with substantial sharpening of the eluted peak. The peak sharpening is continued to be improved up to 600 rpm, and at 1000-12000 rpm, the peak height is slightly decreased apparently due to enhanced mixing. Each experiment produced a single peak indicating there is no inter-channel leakage of the water. ? 2. Using two standard proteins, myoglobin and lysozyme, performance of the multidisk assembly was tested in terms of theoretical plate (TP) and peak resolution (RS) as well as the retention of the stationary phase under various experiment conditions such as revolution speeds, flow rates and elution modes. The best Rs (4.5) with high stationary phase retention (68%) was obtained at a low flow rate of 0.25 mlmin under 800 rpm while the separation time is shortened by increasing the flow rate and rpm. Among all elution modes L-I-T (lower phase pumped from internal tail end) showed the highest peak resolution. ? 3. The results compared with the previous runs with a single disk, the Rs is substantially improved over the expected value apparently due to the reduced sample loading shock which imposes a higher impact on the single disk experiment. On the other hand, stationary phase retention is slightly deceased due to the prolonged elution time.? 4. The performance of the multiple barricaded disk assembly was also tested using two sets of 5 proteins samples, i.e., first set (cytochrome c, myoglobin, ovalbumin, lysozyme, and trypsinogen) and the second set (cytochrome c, human serum albumin, beta-lactoglobulin, alpha-chymotrypsin and trypsinogen) using appropriate polymer phase compositions. All peaks were reasonably well resolved. ? 5. Although symmetrical peaks of myoglobin and lysozyme strongly suggested that the intergrity of the protein molecule was well preserved, the effect of mixer-settler process with glass beads on the enzymatic activity should be further investigated. A very preliminary test on separation of myrosinase from Kaiware Daikon extract shows preserved enzymatic activity which will be reported in detail in the 2007-8 annual report. ? ? ? Report Bibliography ? Edit? 1. Y. Ito, R. Clary, F. Sharpnak, H. Metger, J. Powell, Mixer-settler counter-current chromatography with multiple-spiral-disk assembly, J. Chromatogr. A, in press? 2. Y. Ito, L. Qi., J. Powell, F. Sharpnak, H. Metger, J. Yost, , X.-Y. Cao, L.-S. Dong, L.-S. Huo, X.-P. Zhu, T. Li, J. Chromatogr. A 1151, 108-114, 2007.? 3. Y. Ito, F.-Q. Yang, P.E. Fitze, J. Powell, D. Ide, J. Chromatogr. A 1017, 71-81, 2003.? 4. Y. Ito, F.-Q. Yang, P.E. Fitze, J.S. Sullivan, J. Liq. Chromatogr. Rel. Technol. 26, 1355-1732, 2003.
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