State Key Laboratory of Biotherapy and Cancer Centre, West China Hospital, West China Medical School, Sichuan University, Chengdu, China.
J Chromatogr A. 2011 May 20;1218(20):3124-8. doi: 10.1016/j.chroma.2011.03.015. Epub 2011 Mar 16.
The elution-extrusion counter-current chromatography (EECCC) method was firstly developed by Berthod in 2003 and has been used in natural products separation in recent years. The advantages of this method have been well documented such as reducing the separation time and solvent consumption. In the EECCC method, the time point of the extrusion step is very important during the whole separation process as it directly affects the resolutions, separation time and solvent consumption. However, how to choose a suitable time point to perform the extrusion step without decreasing the resolution has not been studied yet. In the present study, a strategy for systematically calculating the time point for extrusion was developed in theory and five bioactive compounds from the extract of Dendrobium chrysototxum Lindl. were separated and compared using normal CCC and EECCC method. Our results demonstrated that the accurate time point to perform the extrusion could be calculated and reduced both separation time and solvent consumption without losing separation performance. Using this EECCC method, five bioactive compounds were separated and purified with high purity. The separation time and solvent consumption were decreased from 200 min to 100 min and 5-2.5L during the separation process while the resolutions were still acceptable. Finally, 63 mg, 48 mg, 97 mg, 162 mg and 43 mg of hydroxyl phenanthrenes and bibenzyls with the purity of 98.7%, 98.0%, 98.2%, 99.0% and 98.7%, respectively were isolated from 1.2 g crude extract of D. chrysototxum Lindl. initially purified by column chromatography in one step separation. The purities of compounds were determined by HPLC. Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and NMR.
洗脱-挤压逆流色谱法（EECCC）于 2003 年由 Berthod 首次提出，近年来已应用于天然产物分离。该方法具有减少分离时间和溶剂消耗等优点，已得到充分证明。在 EECCC 方法中，挤压步骤的时间点在整个分离过程中非常重要，因为它直接影响分辨率、分离时间和溶剂消耗。然而，如何选择合适的时间点进行挤压而不降低分辨率尚未得到研究。在本研究中，从理论上提出了一种系统计算挤压时间点的策略，并使用常规 CCC 和 EECCC 方法分离和比较了来自铁皮石斛提取物的五种生物活性化合物。我们的结果表明，可以计算并准确确定挤压的时间点，在不降低分离性能的情况下，同时减少分离时间和溶剂消耗。使用这种 EECCC 方法，可以分离和纯化五种具有高纯度的生物活性化合物。在分离过程中，分离时间和溶剂消耗从 200 分钟减少到 100 分钟和 5-2.5L，而分辨率仍然可以接受。最后，从最初通过柱层析一步分离纯化的 1.2g 铁皮石斛粗提物中，一步分离得到纯度为 98.7%、98.0%、98.2%、99.0%和 98.7%的 63mg、48mg、97mg、162mg 和 43mg 的羟基菲和二苄基。通过 HPLC 确定化合物的纯度。通过电喷雾电离质谱（ESI-MS）和 NMR 鉴定了它们的结构。
