Zhou Tingting, Chen Bin, Fan Guorong, Chai Yifeng, Wu Yutian
Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China.
J Chromatogr A. 2006 May 26;1116(1-2):97-101. doi: 10.1016/j.chroma.2006.03.041. Epub 2006 Apr 18.
Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC-DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum (St. Jone's Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate-ethanol-water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).
通过高速逆流色谱法（HSCCC）进行制备性分离和纯化后，通常采用高效液相色谱法（HPLC）对收集的馏分进行分析，以确定各馏分的相对纯度。我们的论文首次报道了一种制备性分离-纯度检测联用系统：HSCCC与高效液相色谱-二极管阵列检测（HSCCC-HPLC-DAD）的在线联用。HSCCC中引入在线纯度分析，克服了HSCCC分离后分析的缺点，极大地提高了该技术的效率。HSCCC出口流出的液体被分成两部分：一部分被收集，另一部分通过切换阀直接引入HPLC-DAD系统进行纯度分析。因此，可以监测从HSCCC获得的馏分的纯度，并收集高纯度的馏分。该策略已通过从贯叶连翘（圣约翰草）中制备性分离和纯化金丝桃苷得到成功验证；使用TBE-300A HSCCC模型，采用体积比为5:1:5（v/v）的乙酸乙酯-乙醇-水两相溶剂系统，利用在线检测技术从贯叶连翘中分离和纯化金丝桃苷。分离过程在不到3.5小时内完成，从300毫克部分纯化的提取物中得到了83.0毫克纯度超过99.0%的金丝桃苷。这种新策略在从中药中制备生物活性化合物方面具有普遍实用性。
