Chromatography Forum

i Am Using 0.1% H3PO4 in ACN And Also in Water on Gradient Method and Column is Waters Symmetry C18,250 X 4.6mm,5µm.
we Face a Problem When We Inject ACN or MeOH to Check Baseline for System Suitability it gives a small peak at R.T. where our a coumpound isomer is retained.
we tried it on five HPLC and also with new column. we used ACN , MeOH and Water From Merck Gradient Grade.
what was the main Problem.

1. Is this peak unretained?
2. Check if you get the peak without injecting sample!
3. If the latter is the case, use increasing equilibration times without injecting sample. If the peak increases, it is in the mobile phase.

Thanks
I also Increase Time up to 10 Minute. The Baseline Peak is Vary in Run to Run Injection.
If This is in the Mobile Phase then How we can remove this or what precautions Required for this

Degassing by Helium , Sonication , Filtration by 0.2µm Membrane is also done.

If This is in the Mobile Phase then How we can remove this or what precautions Required for this

1. Try to change one of the mobile phases components (some other mark - J.T Baker

2. Try to change a little bit your method (flow, temp... )

3. Change wave lenght ( for example +/- 5 nm)

Godspeed