by
KC » Sat Apr 08, 2006 5:19 pm
Put solvent A (water) on channel A, and 100% methanol on channel B. Based on your 60% isocratic run I would:
1) Change mobile phase to 80% A and 20% B (now much weaker than what you were running)
2) Find the window in the method software that allows you to program the solvent composition over the course of the run. This should consist of a small table that has a column for Time, %A and %B (maybe even %C and %D but ignore them)
3) In the first line enter 0 for time and 80 for %A and 20 for percent %B.
In the second line enter 1.0 for time and the same 80 for %A and 20 for %B.
In the next line enter 10 min for time and 40 for %A and 60 for %B.
In the next line enter 15 min for time and the same 40 for %A and 60 for %B.
In the next line enter 15.01 for time and your original 80 for %A and 20 for %B.
In the last line enter 20 for time and the same 80 for %A and 20 for %B.
It should look something like this
TIME %A %B %C %D
0.0 80 20
1.0 80 20
10.0 40 60
15.0 40 60
15.01 80 20
20.0 80 20
Now this is just an example and it may work for your compound but also may need adjustment based on your compounds retention time. This also obviously depicts a 20 min run.
What will happen now is upon injection, your analyte will hopefully stop at the head of the column because the organic concentration in mobile phase is to low for it to move. The mobile phase will remain at this concentration for one minute. Starting at 1.0 min the methanol concentration in mobile phase will gradually increase until 10 min when mobile phase will be the same as you are running now 40% water and 60% methanol. It will remain at this concentration until 15 min where it will immediately switch back to your original time 0 mobile phase concentration for 5 min to re-equilibrate for the next injection.
What you are trying to do is separate two components with relatively similar polarity and with luck one will begin moving on the column before the other and provide you with a little separation between the two. You can play with these concentrations and make the ramp as drastic or gentle as you want to achieve the desired result. I usually like to see my analyte come off close to the end of the ramp period (ie about 9-11 min in this case). If you do this and don't see your analyte, make that 5 min hold between 10 and 15 min longer or change the time 10 min mobile phase to maybe 80% methanol rather than 60%.
How about it....you grab all that?
