Lee Ju Yong, Kim Ki Hun, Moon Myeong Hee
Department of Chemistry, Yonsei University, 134 Shinchon-dong, Seoul 120-749, South Korea.
J Chromatogr A. 2009 Sep 11;1216(37):6539-42. doi: 10.1016/j.chroma.2009.07.044. Epub 2009 Aug 6.
A multiplexed hollow fiber flow field-flow fractionation (MxHF5) is introduced to increase throughput of an HF5 channel system for semi-preparative purposes. HF5, a cylindrical version of the flow field-flow fractionation (FlFFF) operated with a porous, hollow fiber membrane by controlling the ratio of radial and axial flow rates, is capable of fractionating proteins, cells, and macromolecules by size. An advantage of HF5 is its inexpensive channel construction, allowing for disposability that can reduce run-to-run carryover problems. MxHF5 constructed in this study was made with six parallel HF5 modules connected to seven-port manifolds for the semi-preparative scale separation of proteins or biological particles. For the evaluation of MxHF5 separation efficiency, protein standards were utilized to test peak recoveries, band broadening, and throughput. The assembly showed the possibility of handling up to 50 microg of proteins without incurring overloading. The developed channel was applied to demonstrate size sorting of lipoproteins for the future study of size dependent lipidomic and proteomic analysis. The current trial offers a unique advantage of scaling up HF5 separation without using wide-bore, hollow fibers which sacrifice separation speed.
为了提高用于半制备目的的中空纤维5（HF5）通道系统的通量，引入了一种多路复用中空纤维流场-流分级分离技术（MxHF5）。HF5是流场-流分级分离技术（FlFFF）的圆柱形式，通过控制径向和轴向流速比，利用多孔中空纤维膜进行操作，能够按大小对蛋白质、细胞和大分子进行分级分离。HF5的一个优点是其通道构建成本低廉，可一次性使用，这能够减少批次间的残留问题。本研究构建的MxHF5由六个平行的HF5模块组成，这些模块连接到七端口歧管，用于蛋白质或生物颗粒的半制备规模分离。为了评估MxHF5的分离效率，使用蛋白质标准品来测试峰回收率、谱带展宽和通量。该组件显示出能够处理高达50微克蛋白质而不会出现过载的可能性。所开发的通道被应用于证明脂蛋白的大小分选，以便未来进行与大小相关的脂质组学和蛋白质组学分析研究。当前的试验提供了一个独特的优势，即无需使用牺牲分离速度的大口径中空纤维就能扩大HF5的分离规模。
