Improvement of the spiral disk assembly for separation of proteins and other biopolymers. 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. Using a polymer phase system composed of 12.5%(w/w) PEG1000 and 12.5%(w/w) dibasic potassium phosphate and a mixture of myoglobin (horse skeletal muscle) and lysozyme (egg white) as test samples, the experiments were continued to test the following various parameters : dimensions of the locule of the spiral disk; distribution of the glass beads in the channel, opening of the barricade on both sides, effects of revolution speed, flow rate, elution modes, etc. 1. Dimensions of the locule: two units were made and tested: 2.8 mm x 2.8 mm with 1460 pits (barricaded bead chain spiral disk) and 2.8 mm x 6.0 mm 730 pits (barricaded locular spiral disk) both connected with 0.4 mm ducts above and below the barricades. The preliminary test showed that the locular spiral disk yields much higher efficiency. 2. Distribution of the glass beads: Using the locular spiral disk of 1.2 mm opening, several different distributions of the beads were tested. The best results were obtained with placing the beads in every other pit or every third pit. 3. Opening on both sides of the barricaded in the locular disk: three different openings were tested, i.e., 1.2 mm, 0.8 mm and 0.4 mm. It was found that the smaller the opening the higher the efficiency. The barricaded disk with 0.2 mm opening is now under fabrication in the NIH Machine shop. 4. Effect of revolution speeds of the locular disk: Following three rpm ranges were tested: 600, 800 and 1000. The results clearly showed that the efficiency expressed by Rs (peak resolution) is almost linearly improved with an increased rpm up to 1000. For example, using the lower phase as a mobile phase at a flow rate of 0.25 ml/min, the Rs value of is 0.91-0.97 at 600 rpm; 1.29-1.31 at 800 rpm; and 1.51-1.56 at 1000 rpm. 5. Elution mode: The following 4 elution modes were tested: the lower phase pumped from the inner terminal which is either the head or the tail; and the upper phase from the outer terminal which is again either the head or the tail. All these elution modes use the centrifugal force gradient generated through the spiral channel to facilitate the retention of the stationary phase (the reversed elution modes resulted in a great loss of the stationary phase and poor peak resolution). It was found that the elution of the lower phase showed a high peak resolution with satisfactory levels of the stationary phase retention around 50% of the total column capacity, whereas the upper mobile phase showed much lower peak resolution with an extremely high retention of the stationary phase some over 80%. In short the results of the above studies promise that the efficient separation of proteins would be achieved using a set of barricaded spiral disks connected in series.
| Ballinger, Megan N; Newstead, Michael W; Zeng, Xianying et al. (2012) TLR signaling prevents hyperoxia-induced lung injury by protecting the alveolar epithelium from oxidant-mediated death. J Immunol 189:356-64 |
| Ito, Yoichiro; Yu, Henry (2009) Triangular Helical Column for Centrifugal Countercurrent Chromatography. J Liq Chromatogr Relat Technol 32:560-566 |
| Ito, Yoichiro (2005) Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A 1065:145-68 |
| Ma, Ying; Lang, Lixin; Kiesewetter, Dale O et al. (2004) Purification of the precursor for the automated radiosynthesis of [18F]FCWAY by counter-current chromatography. J Chromatogr A 1034:149-53 |
| Yu, Henry; Ito, Yoichiro (2004) Preparative separation of proteins using centrifugal precipitation chromatography based on solubility in ammonium sulfate solution. Prep Biochem Biotechnol 34:1-12 |
