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- Posts: 44
- Joined: Mon Dec 18, 2017 9:09 pm
I am working on my LC-MS method development for a mixture of standards and I want to separate them in a reverse phase chromatography.
The main challenge for me is that I could not use acidic modifiers since one of my analytes that is indole-3-carbinol (I3C) will be converted to another compound inside the column under the acidic conditions. In addition, I am restricted to column temperature of 20 centigrade since even in the absence of acidic modifier at higher temperature than 20, my analyte (I3C) will be transformed into another compound.
Therefore, basically for getting a good peak resolution among 4 different reference standards, I am limited to the solvent strength and gradient factors. My main question is what strategy do you recommend I take? At the moment, I know methanol is working better for my method than ACN. I tried many different gradient program but the peaks can not be separated well. I could see only the changes in the retention time but not the separation. May I ask what do you recommend? Should I mainly work on my gradient or there are other parameters I can manipulate to get a good separation?
P.s: Most of my analytes are eluting out from the column when the gradient goes from 60 % MeOH to 100 % MeOH without any modifier.
Thank you in advance