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高手请指点反相硅胶柱
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我要走反相硅胶柱,但是以前从没有走过,现在对这方面的知识了解的很少,在这里 请各位高手多多指点!请问走反相柱需要注意些什么?!请各位高手不吝赐教!!!!!多 谢! [search]反相[/search] |
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klivis
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bhz_2001
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MCI是反相柱的一种，其具体方法供你参考： 1. Preparation of the column Prepare the MCI gel in a beaker, using a 1:1 mixture of acetonitrile and water. Then pour the mixture onto the column and wash the MCI gel with CH3CN/H2O 1:1 (colored impurities, usually yellowish, can be washed out). After what the MCI gel must be conditioned with the eluent of the chromatography, e.g. H2O/CH3CN 9:1. Pass through a quite big volume of this solution to be sure that the mixture inside MCI is 9:1 and not with a lower ratio of H2O. The solutions used for the washing or for the conditioning must not be used again on the column. Notice : MCI gel might float on solvent with a high ratio of water; it is also very light and particles can be suspended in the solvent when you add it. Then pressure can make it go down. 2. Addition of the crude product and elution The technique is the same as for chromatography on silica gel. The only difference is that MCI gel is lighter than SiO2 gel. So the solution of crude substance (in the eluent) as well as the eluent should be added with special care to avoid destruction of the surface of MCI. 3. Recycling of the MCI gel Once the column chromatography is finished, you can regenerate MCI by the following washings : - CH3CN, to get rid off unpolar impurities which sticked to MCI at the top of the column (usually colored substances), - CH3CN/H2O 1:1, to go step by step to water phase washings, - H2O, to wash out remaining polar materials, - HCl 25%, to destroy remaining impurities and wash them out, - H2O, to go back to neutral pH, - H2O/CH3CN 1:1, to prepare the column for another chromatography. MCI gel can be washed directly on the column where it was used. It can also be combined to other used MCI gel to be washed together. 4. Choice of the column size and of the amount of MCI - Relationship between the crude amount m and the diameter D of the column : m=D2/10 example : a column with a diameter of 3 cm can be used to purify 32/10= 0.9 g of crude substance. - Height of MCI gel in the column MCI gel can be added into the column as to reach a height between 15 and 20 cm (maximum), according to the difficulty of the separation. 15 cm are enough if there are not too much impurities, and 20 cm can be used to separate mixtures. |
2楼2008-11-19 08:43:06
