Natural Products Chemistry Group, Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands.
J Chromatogr A. 2009 Oct 23;1216(43):7268-74. doi: 10.1016/j.chroma.2009.08.043. Epub 2009 Aug 21.
An on-line method for the rapid pinpointing of radical scavengers in non-polar mixtures like vegetable oils was developed. To avoid problems with dissolving the sample, normal-phase chromatography on bare silica gel was used with mixtures of hexane and methyl tert-butyl ether as the eluent. The high performance liquid chromatography-separated analytes are mixed post-column with a solution of stable free radicals in hexane. Reduced levels of the radical as a result of a reaction with a radical scavenger are detected as negative peaks by an absorbance detector. After investigating a number of different reagents, solvents, concentrations and solution flow rates an optimized instrumental set-up incorporating a superloop for pulse-free delivery of the reagent solution is presented. Both 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,6-di-tert-butyl-alpha-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy (galvinoxyl) were used as stable free radicals. The method is suitable for both isocratic and gradient HPLC operation. The method has a simple experimental protocol, uses standard instruments and inexpensive and stable reagents, and accepts any hexane-soluble sample. It can also be used for semi-quantitative analysis. The method was applied to several pure, non-polar natural antioxidants, vegetable oils and lipid-soluble rosemary extract. The limits of detection varied from 0.2 to 176 microg/ml, depending on the compound tested.
开发了一种用于快速定位非极性混合物（如植物油）中自由基清除剂的在线方法。为避免溶解样品时出现问题，使用了裸硅胶的正相色谱法，并用正己烷和甲基叔丁基醚的混合物作为洗脱剂。高效液相色谱分离出的分析物在柱后与正己烷中稳定自由基的溶液混合。由于与自由基清除剂发生反应，自由基的水平降低，在吸收检测器中检测到为负峰。在研究了许多不同的试剂、溶剂、浓度和溶液流速后，提出了一种优化的仪器设置，其中包括一个超级环，用于无脉冲输送试剂溶液。使用 2,2'-二苯基-1-苦基肼（DPPH）和 2,6-二叔丁基-α-（3,5-二叔丁基-4-氧代-2,5-环己二烯-1-基）-对甲苯氧基（戊二酮）作为稳定自由基。该方法适用于等度和梯度 HPLC 操作。该方法具有简单的实验方案，使用标准仪器和廉价且稳定的试剂，可接受任何可溶于正己烷的样品。它也可用于半定量分析。该方法应用于几种纯的、非极性天然抗氧化剂、植物油和脂溶性迷迭香提取物。检测限因测试化合物而异，范围从 0.2 到 176μg/ml。
