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1、Given that highly lipophilic LFP were the target molecules of the analysis, our approach concerning the finding the optimal separation conditions was to use more non-polar mobile phase than it is the case with the majority reverse phase HPLC separations. Previous experiments in our laboratory with LFP showed an optimal isocratic elution with an acetonitrile-methanol-water mixture having different ratios of these solvents, depending on a tissue from which analyzed LFP originated. This was adopted for the separation of LFP mixtures from human erythrocytes. The optimal isocratic separation was therefore with the mobile phase consisting of 60% organic solvents and 40% water. 2、The excitation/emission wavelengths of 335/360 nm set on the detector were in accordance with a maximum difference in fluorescence intensity between the two groups of samples. The flow rate of 0.4 mL min-1 was accepted for this separation after a few flow rates between 0.2 and 0.6 mL min-1 had been tried out. Higher flow rates tended to produce high backpressure. 3、Method Development for a Qualitative Analysis of LFP from Beef Heart Mitochondria The chromatographic separation was carried out with an injection of chloroform LFP extracts into the system without prior solvent evaporation, for chloroform by no means worsened the separation. When choosing the optimal mobile phase, we started with different mixtures of acetonitrile, methanol and water that proved to be good choices as running phases in our previous experiments. However, LFP mixtures from mitochondria were not resolved well when using various combinations of these solvents. |
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麦兜Magic(金币+158): ★★★★★最佳答案 2012-02-15 20:34:00
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1，鉴于高亲油性的类脂褐素是分析的目标分子，在涉及到寻找最佳的分离条件时，我们的方法是使用比与大多数反相高效液相色谱分离方法用更多的非极性流动相。 我们实验室之前对类脂褐素的研究表明，最佳的用不同配比的乙腈-甲醇-水混合溶剂的无梯度洗脱取决于源自于产生类脂褐素的组织。 这个方法同样适用于从人类的红细胞中分离类脂褐素混合物。 因此最佳的分离条件是流动相中有60%的有机溶剂与40%的水。 2.检测器的激发/发射波长设定在335/360nm，这个与样品的2个基团之间的荧光强度最大值一样。 我们在做了流动速率在0.2-0.6mL min-1一些实验数据表明0.4 mL min-1的流速是比较适合的。因为高流速倾向于产生高的 反压力。 3.定性分析牛心线粒体中类脂褐素的方法进展 用色谱法分离的操作是在没有蒸发氯仿的情况下在仪器中打入一针管有氯仿的LFP，因为氯仿对分离没有影响。 当选择最佳的流动相时，我们用不同配比的乙腈、甲醇和水的混合溶剂开始我们的实验，因为这些混合溶剂在我们之前的实验中已经证实了能够作为良好的流动相。 然而，当我们使用上面的不同配比的混合溶剂来分离从线粒体中提取出来的LFP混合物时，它并没有得到好的分离。 |
3楼2012-02-15 16:22:34
