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dandanxiong1983金虫 (小有名气)
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[求助]
高手帮我看下这个峰该怎么分开
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这两天在做一味中药的同时分析，9个物质一起分析的实验。其中RUTIN,QUERCETIN-3-GLUCOSIDE合并为一个峰怎么也分不开。图见附件 条件如下： A:MeOH/Water/Formic acid(20/80/0.25) B：MeOH 0-5min: A:B=100:0-90:10, 5-10min: A:B=90:10-80:20, 10-30min:80:20-80:20,30-45min:80:20-60:40 这个条件很适合其他物质的分离分析，其他7个物质都分离度较好，而且连ISOMERS 都分开了。请高手是否能在不改变原基本条件，在溶剂的梯度上给点意见。 |
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2012-07-02 14:13:23, 25.68 M
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lzjessie_wlf
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【答案】应助回帖
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感谢参与，应助指数 +1
soundhorizo: 金币+5, 感谢您的应助，欢迎常来分析版交流指导 ^^ 2012-07-02 23:33:22
dandanxiong1983: 金币+10, ★★★很有帮助 2012-07-17 19:48:11
感谢参与，应助指数 +1
soundhorizo: 金币+5, 感谢您的应助，欢迎常来分析版交流指导 ^^ 2012-07-02 23:33:22
dandanxiong1983: 金币+10, ★★★很有帮助 2012-07-17 19:48:11
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如果楼主倾向于使用条件1，那么可以对条件1加以修改。A 相改为18ACN+82H2O+0.25%HCOOH, B 相为MeOH. 这样就可以在使用到下列分离 条件的同时, 15 18 82 rutin, q3 separated 又最大限度的保持后续使用甲醇，水分离其他七个化合物的条件。 请教楼主，为什么不是水相和有机相中同时加酸？ 一般来说，两相同时加等量酸，可以降低在梯度洗脱时酸度对洗脱的影响。 如果楼主可以列出其他七个化合物在这个两个流动相条件下的分别出峰时间(retention time)，那么就更有利于理论上推段流动相的梯度分布。 当然，最后的流动相条件还需要实践来证明。 如果知道梯度的延迟时间，楼主还可以使用等梯度洗脱，大致推断出每个化合物大致洗脱出来的梯度。 粘贴一个网址给楼主，希望有所帮助。 如果网址不能打开，那么后面粘贴的一段希望对楼主有帮助。 http://www.ionsource.com/tutorial/chromatography/rphplc.htm Optimizing the Separation Once you have a separation you may want to optimize it. You may wish to shallow out the gradient to improve the separation, or you may wish to shorten the run time. Taking the illustration above one can see that all of the peptides are out by 40 minutes. This does not mean that we can change this 80 min run into a 40 min run, but there is room for improvement. The first step in the optimization is to determine the %B at which the last peak elutes. If you look at the blue gradient line you might guess that the last peak elutes near 40%B but this would be incorrect. All HPLC systems have a gradient delay. The gradient delay is the time between when the software tells the pumps to start pumping at a certain mobile phase composition and the time it takes for that solvent composition to reach the column and have an effect. A good guess for a gradient delay is 10 minutes. This would mean that our guess for the final mobile phase composition for the 40 min peak would be approximately 30%B. To observe the gradient delay time look at the illustration above and observe that the baseline returns to the starting conditions at 70 minutes and not at 60.1 minutes when our pumps have gone back to 2% B. One must take care to avoid having the last peak elute on the "equilibration cliff", (at 70 min. in this example). This can be avoided by ending the gradient at a %B that is slightly higher than that required by the last component. Based on the separation shown at the top of this section one could rewrite the gradient to look like this: This would make the gradient shallower and possibly give a better peak separation. To shorten the run time one could rewrite the gradient to look like this: This last change would cut 30 min. from the analysis time. Shorter analysis times are always better for work efficiency. With every minute you can cut from the HPLC method without sacrificing your chromatographic goals you will be rewarded with better work efficiency. With this change the last peak would most likely still elute at 40 minutes and the peptide separation would essentially remain the same as in the initial 60/60 analysis. |
24楼2012-07-02 23:08:45
xbqewpq
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感谢参与，应助指数 +1
dandanxiong1983: 金币+5, ★有帮助 2012-07-17 19:46:57
感谢参与，应助指数 +1
dandanxiong1983: 金币+5, ★有帮助 2012-07-17 19:46:57
| 是不是可以把条件一的甲酸调低些，改为0.1 |
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2楼2012-07-02 14:46:34
soundhorizo
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3楼2012-07-02 14:50:04
dandanxiong1983
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4楼2012-07-02 14:58:32
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