Lu Yanbin, Berthod Alain, Hu Ruilin, Ma Wenyan, Pan Yuanjiang
Department of Chemistry, Zhejiang University, Hangzhou 310027, Zhejiang Province, China.
Anal Chem. 2009 May 15;81(10):4048-59. doi: 10.1021/ac9002547.
In countercurrent chromatography (CCC) the choice of the liquid system is the heart of any separation. It corresponds to the selection of the mobile phase and the stationary phase at the same time. Any change in one phase composition induces a change in the other phase composition which renders the choice of the appropriate liquid system difficult and lengthy. A scale of compositions of the heptane-ethyl acetate-methanol-water quaternary liquid system was referred to by letters from A to Z and called the Arizona (AZ) liquid system. Each composition of the AZ system has the same heptane/ethyl acetate and methanol/water volume ratios. It is shown that there is a continuous polarity change from the hydrophilic A composition (ethyl acetate-water) to the hydrophobic Z (heptane-methanol) mixture by measuring the distribution constant K(D) of a known test mixture. For all compounds, the log K(D) is linearly increasing with the water content of the lower aqueous phase of the composition used. The slopes of the log K(D) versus percent H(2)O have very different values which means that the chromatographic selectivity changes with liquid system compositions. The AZ system was associated to the elution-extrusion method to design a procedure to identify rapidly the appropriate solvent composition able to fractionate correctly a complex natural extract. With the use of an integrated three-coil CCC column (40 mL each coil) able to test three AZ compositions in parallel, it is shown that the optimum AZ composition is found in half a day using less than a liter total volume of solvents. Two natural extracts are rapidly screened using the proposed protocol. An extract of Piper longum L. of intermediate polarity was fractionated in five usable portions using the 3/2/3/2 (Q) composition of the AZ system. A polar extract of Polygonum cuspidatum was also separated in five fractions using the 1/6/1/6 (D) composition. In both cases, a 140 mL CCC column was used for a direct scale-up transfer with the same liquid system. Purified fractions were subjected to an antioxidant activity assay and liquid chromatography with UV and mass spectrometry detection (LC-UV/MS) analysis to determine the molecular weight, number, and quantity of compounds in the active fractions. Four fractions of P. cuspidatum showed excellent antioxidant activity. They were rapidly produced at the milligram level by the 140 mL CCC column and fractionated by semipreparative high-performance liquid chromatography (HPLC) in individual compounds that were each identified by NMR and MS and reevaluated for confirmation of bioactivity. The rapid screening CCC protocol associated to the preparative capability of CCC allows for a fast identification and characterization of active compounds in natural products.
在逆流色谱法（CCC）中，液体体系的选择是任何分离过程的核心。这同时对应着流动相和固定相的选择。一个相组成的任何变化都会引起另一个相组成的变化，这使得选择合适的液体体系既困难又耗时。庚烷 - 乙酸乙酯 - 甲醇 - 水四元液体体系的组成范围用字母A到Z表示，并称为亚利桑那（AZ）液体体系。AZ体系的每种组成都具有相同的庚烷/乙酸乙酯和甲醇/水体积比。通过测量已知测试混合物的分配常数K（D）表明，从亲水性的A组成（乙酸乙酯 - 水）到疏水性的Z组成（庚烷 - 甲醇）混合物存在连续的极性变化。对于所有化合物，log K（D）随着所用组成的下层水相的含水量线性增加。log K（D）对水百分比的斜率具有非常不同的值，这意味着色谱选择性随液体体系组成而变化。将AZ体系与洗脱 - 挤出方法相结合，设计了一种程序，以快速识别能够正确分离复杂天然提取物的合适溶剂组成。使用能够并行测试三种AZ组成的集成三线圈CCC柱（每个线圈40 mL），结果表明，使用总量不到一升的溶剂，半天内就能找到最佳的AZ组成。使用所提出的方案对两种天然提取物进行了快速筛选。使用AZ体系的3/2/3/2（Q）组成将中等极性的长蒌提取物分离成五个可用部分。使用1/6/1/6（D）组成将虎杖的极性提取物也分离成五个部分。在这两种情况下，使用140 mL CCC柱并采用相同的液体体系进行直接放大转移。对纯化的部分进行抗氧化活性测定以及液相色谱 - 紫外和质谱检测（LC - UV/MS）分析，以确定活性部分中化合物的分子量、数量和含量。虎杖的四个部分表现出优异的抗氧化活性。通过140 mL CCC柱以毫克水平快速制备这些部分，并通过半制备高效液相色谱（HPLC）分离成各个化合物，每个化合物通过核磁共振（NMR）和质谱（MS）鉴定，并重新评估以确认生物活性。与CCC的制备能力相关的快速筛选CCC方案能够快速鉴定和表征天然产物中的活性化合物。
