Shinomiya Kazufusa, Kobayashi Hiroko, Inokuchi Norio, Kobayashi Koji, Oshima Hisashi, Kitanaka Susumu, Yanagidaira Kazuhiro, Sasaki Haruo, Muto Minoru, Okano Michiharu, Ito Yoichiro
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan.
J Chromatogr A. 2007 Jun 1;1151(1-2):91-8. doi: 10.1016/j.chroma.2007.02.116. Epub 2007 Mar 16.
The new small-scale cross-axis coil planet centrifuge (X-axis CPC) previously designed and fabricated in our laboratory has a distinctive feature such that four separation columns of similar weight are mounted symmetrically around the rotary frame to achieve stable balancing of the centrifuge under a high revolution speed. In this column layout, neighboring columns must be rotated in the opposite direction if viewed from the center of the centrifuge to avoid twisting the interconnecting flow tubes. The effect of rotational direction of the columns on the partition efficiency was evaluated with separation of a set of test samples such as cytochrome c, myoglobin, and lysozyme using an aqueous-aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate under 1000 rpm of column revolution. A series of experiments was performed using a set of two diagonally located columns (connected in series) each consisting of five coiled layers of 1 mm I.D. with a total capacity of 27.0 mL. Both right- and left-handed coils were tested each under the optimized conditions for choice of mobile phase and direction of the column rotation so that the satisfactory volume of the mobile phase was retained in the column by the aid of Archimedean screw effect. The results of these studies showed that one particular combination of handedness of the coil and direction of the rotation yielded the best peak resolution for each mobile phase. In order to demonstrate the capability of the apparatus, the purification of ribonuclease (RNase) from the extract of bullfrog egg, sialic acid binding lectin (cSBL), was carried out using both organic-aqueous and aqueous-aqueous polymer phase systems. When using the 16.0% (w/w) PEG 1000-6.3% (w/w) dibasic potassium phosphate-6.3% (w/w) monobasic potassium phosphate system, cSBL was successfully separated from other proteins present in the extract while commercial RNase A was eluted at near the solvent front by the lower phase mobile. The cSBL retained its native RNase activity. The overall results demonstrated that the present new small-scale X-axis CPC is useful for the purification of bioactive compounds without loss of their native activities.
我们实验室之前设计制造的新型小型交叉轴线圈行星离心机（X轴CPC）具有一个独特的特点，即四个重量相似的分离柱围绕旋转框架对称安装，以实现离心机在高转速下的稳定平衡。在这种柱布局中，如果从离心机中心观察，相邻的柱必须沿相反方向旋转，以避免扭曲互连的流动管。使用由12.5%（w/w）聚乙二醇（PEG）1000和12.5%（w/w）磷酸氢二钾组成的水-水聚合物相系统，在柱转速为1000 rpm的条件下，通过分离一组测试样品（如细胞色素c、肌红蛋白和溶菌酶）来评估柱旋转方向对分配效率的影响。使用一组两个对角定位的柱（串联连接）进行了一系列实验，每个柱由五个内径为1 mm的螺旋层组成，总容量为27.0 mL。在优化的流动相选择和柱旋转方向条件下，对右手螺旋和左手螺旋进行了测试，以便借助阿基米德螺旋效应在柱中保留令人满意体积的流动相。这些研究结果表明，对于每种流动相，螺旋的手性和旋转方向的一种特定组合产生了最佳的峰分辨率。为了证明该仪器的能力，使用有机-水和水-水聚合物相系统对牛蛙卵提取物中的核糖核酸酶（RNase）唾液酸结合凝集素（cSBL）进行了纯化。当使用16.0%（w/w）PEG 1000 - 6.3%（w/w）磷酸氢二钾 - 6.3%（w/w）磷酸二氢钾系统时，cSBL成功地与提取物中存在的其他蛋白质分离，而商业RNase A在下相流动时在溶剂前沿附近被洗脱。cSBL保留了其天然的RNase活性。总体结果表明，目前这种新型小型X轴CPC可用于纯化生物活性化合物而不损失其天然活性。
