School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU, 1, Rue Michel Servet, 1211 Geneva 4, Switzerland.
School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU, 1, Rue Michel Servet, 1211 Geneva 4, Switzerland.
J Chromatogr A. 2019 Aug 2;1598:85-91. doi: 10.1016/j.chroma.2019.03.042. Epub 2019 Mar 21.
Semi-preparative HPLC is one of the main techniques used for the purification of natural products (NPs). Generally, the sample has to be solubilized in organic solvent and injected on column through a loop valve. Since the solubility of crude natural extracts is often limited, a high solvent volume is needed for injection. This significantly compromises the resolution and increases the risk of overpressure in the system. To overcome this problem, a dry load injection procedure was developed to ensure optimum resolution even at high sample loading. The approach was first validated with a representative mixture of NPs standards, and successfully applied to two representative crude plant extracts: the dichloromethane extract of Annacardium occidentale and the methanolic extract of Hypericum perforatum. In all cases, the dry loading injection setup enabled an efficient introduction of the samples in the semi-preparative HPLC system. Different overload conditions of the columns were tested and the results demonstrated the robustness of the method and the possibility of applying it with a limited loss of resolution compared to liquid injection and without increasing pressure. The chromatographic resolutions were close to those obtained at the analytical level and separation were of much better quality when compared to liquid injection. This approach is especially relevant when purifying compounds isolated with high resolution from extracts that are poorly soluble in low volume of injection solvent due to the presence of lipophilic compounds and are thus not compatible for loop injection in typical reversed phase conditions. In addition, the dry load setup was also found to be useful when relatively polar components have to be separated in reversed phase conditions. In this case, loop injection with methanol generates strong peak distortion and broadening, while the dry load injection affords symmetrical peaks.
半制备高效液相色谱法是用于天然产物（NPs）纯化的主要技术之一。通常，样品必须溶解在有机溶剂中，并通过环阀注入柱中。由于粗提天然提取物的溶解度通常有限，因此需要注入大量溶剂。这会显著降低分辨率并增加系统超压的风险。为了克服这个问题，开发了干负载进样程序，以确保即使在高样品负载下也能获得最佳分辨率。该方法首先用 NPs 标准混合物进行了验证，并成功应用于两种典型的粗提植物提取物：辛那卡德木的二氯甲烷提取物和贯叶连翘的甲醇提取物。在所有情况下，干负载进样设置都能够有效地将样品引入半制备高效液相色谱系统中。测试了不同的柱过载条件，结果证明了该方法的稳健性，并且与液体进样相比，在分辨率略有损失的情况下，该方法具有应用的可能性，而不会增加压力。色谱分辨率接近在分析水平上获得的分辨率，并且与液体进样相比，分离质量要好得多。当由于存在亲脂性化合物而使提取物在低体积的注射溶剂中溶解度差，并且因此与典型的反相条件下的环阀注射不兼容时，从提取物中以高分辨率分离出的化合物进行纯化时，这种方法尤其相关。此外，当必须在反相条件下分离相对极性的成分时，干负载设置也被发现是有用的。在这种情况下，用甲醇进行环阀注射会产生强烈的峰变形和展宽，而干负载进样则会产生对称的峰。
