制备型电泳法分离腺苷酸混合物

Separation of Adenosine Phosphates by Preparative Electrophoresis

考察了制备型电泳法分离小分子生物物质的可行性及影响因素。在填充排阻凝胶的色谱柱两端各连接一个电极室以放置电极,在色谱柱上外加电场,进行单磷酸腺苷酸(AMP)和二磷酸腺苷酸(ADP)混合物的分离。以pH值4.6的乙酸钠-乙酸缓冲溶液为流动相,考察分离介质、电场方向、电场大小和断电时间对分离度和样品回收率的影响。结果表明,具有很大孔径的排阻凝胶有利于样品的分离;电泳方向与液体流动方向一致时,电场越大,分离度越好,但收率越低;电泳方向与流体流动方向相反时,通电时间不能过长(否则样品不能出峰),分离度差,但收率高;在样品到达电极前停止通电,可以将AMP与ADP分离,而且减少了电解对样品的破坏。建立的制备电泳系统可用于离子型小分子生物物质的制备分离。

The applicability and major influencing factors of preparative electrophoresis on the separation of small-molecule biological compounds had been investigated. Two electrode chambers containing two electrodes were separately connected to two ends of a column packed with size-exclusion gel. By these electrodes, the electric field could be applied across the column. Electrophoresis of adenosine monophosphate（AMP） and adenosine diphosphate（ADP） with sodium acetate-acetic acid buffer solution with pH value of 4.6 as mobile phase was performed. Effects of separation media, electric field, electric direction and stop time of electric field on resolution and sample recovery were studied. The size-exclusion gels with large pore size were beneficial to the separation of samples. When electrophoresis and liquid flow were in the same direction, higher electric field resulted in higher resolution, but lower recovery due to electrolysis. When they were in opposite direction, resolution was poor due to the shorter electric field application time （otherwise sample solutes could not be eluted）, while higher recovery was obtained. To power off the electric field before sample reached the outlet electrode could well separate AMP and ADP and prevent sample from being destroyed seriously by electrolysis reaction. The preparative electrophoresis in this paper could be used to separate certain ionic small-molecule biological compounds.