By Anonymous on Friday, February 14, 2003 - 01:42 pm:
Dear all,
I have three compounds to separate by RP-HPLC. My first gradient is: starting with 50% ACN for 2 minutes, then ramp up to 55%ACN in 10 minutes. The three compounds are separated with retention time at 10.75m, 11.41m, and 12.03m. Trying to retain them on the column more and also to separate them better, I tried a second gradient as: starting with 30%ACN for 2 minutes, then ramp up to 60%ACN in 60 minutes. Strangely, this time, I only saw two peaks at 38.50m and 40.10m, LCMS run indicated that 40.10m peak has two compounds co-eluting. I am puzzled, I thought the 2nd gradient was supposed to at least give me similar separation to the 1st one. why the second gradient gave me a worse separation? Any advice? Thank you.
By Anonymous on Friday, February 14, 2003 - 01:58 pm:
Some components reacts different to a method change than others, that's why we sometimes that peaks walks trough others when we change %organic phase. It happens more then you think, sometimes you can adjust you method so that you decrease your runtime from 25 min to less then 10 min, without loose of resolution. At this point Drylab is a very good tool.
By Anonymous on Saturday, February 15, 2003 - 10:34 am:
Take a look into "A practical guide to HPLC method development" Snyder et al.