Page 2 of 2
Posted: Mon Jun 16, 2008 10:08 pm
by semivoadude
My column is a Varian omnisphere C18 150mm 4.6 5um. I tried using a diluted sample diluent (ACN:WATer 50/50), it didnt work. It also split one of the peaks.
The problem I have, is altering the gradient. Ive tried many variations in %ACN, but I dont understand. Does anyone have general advice when altering gradients? I only change one variable at a time and Im trying to devlelop correlations, so I can learn instead of just haphazardly changing the percentages.
thanks
Posted: Mon Jun 16, 2008 11:09 pm
by Kostas Petritis
The fact that you get a split peak is a good indication that your injection solvent is still too high. Before doing anything else, I would dissolve my analytes in 5:95 ACN:water, and then start a gradient from 5% ACN 50% of ACN in 30 min. That will give you a pretty good idea of what your next step should be... (if any).
Posted: Tue Jun 17, 2008 1:03 am
by semivoadude
http://tinypic.com/view.php?pic=izuc06&s=3
Here is a posting of my problem of seperating hydroquinone and 1 methyl imidazole from the ACN void volume.
Gradient:
Minutes % ACN
0 85
5 85
20 100
Column: C18 varian omnisphere 4.6mm 5um 150
MP's: MPA= water w/0.1%TFA
MPB= ACN w/0.1%TFA
I have run it more polar in the first two minutes but I cant get the 2 peaks to elute before or after the void. They are all over the place
Posted: Tue Jun 17, 2008 12:23 pm
by danko
Hi Semivoadude,
With this column I would expect the void volume to be between 1.5 and 2 mL.
Consequently the “retention timeâ€
Posted: Wed Jun 18, 2008 11:22 am
by shaun78
You claim in an earlier post:
that using 15% ACN the peaks eluted nicely before the void time.
The chromatogram you posted should peaks eluting in the void of the column.
Can you change the initial ACN to 2-5% and hold for 3 minutes? Does that make any difference in the chromatography?