Chromatography Forum

Hello -


The peak shape of my std injection is fine when the injection volume is 50 uL, but terrible fronting (almost as if another peak is emerging in front of the peak of interest) is observed when the injection volume is reduced to 25 uL. Can someone explain the theory behind this?


I appreciate your help.


Liz

Assuming that your injections are from the same sample you are overloading (regarding separation) the column at 50µL so that a bad separation of at least two compounds is obliterated.

Hi Liza,

Provided my assumption is right, that your standard consists of only one compound which should result in only one peak, please go back and check whether it is the case - you experience fronting when the injection volume is 25 uL and no fronting when the volume is 50 uL .Because if it is visa versa, as I suspect, then you are witnessing “the strong solvent effectâ€

Thanks for your prompt replies.


To clarify, there is only one compound that's injected. Only one peak should result. The fronting is experienced when the injection volume is doubled. Could you explain the "strong solvent effect"?

please clarify: 8/30 you said

The fronting is experienced when the injection volume is doubled.

8/28:

The peak shape of my std injection is fine when the injection volume is 50 uL, but terrible fronting (almost as if another peak is emerging in front of the peak of interest) is observed when the injection volume is reduced to 25 uL.

which is your case?
q: when you changed the volume, did you change the concentration?

if it was former, you overloaded.
if the latter, it does not make sense if the concentration was the same. so if the latter, can you go back repeat this like concecutively inject the same solution from the same vial 50ul, 25 ul, 50 ul and 25 ul to see repeatability .

Sorry Liza, you are apparently not presenting a clarification but a 180° about face? If 50µL of identical sample does the fronting you very likely have the classic case of strong solvent effect mentioned above: 50µL is apparently enough to "wash" the analyte into the column (no or low retention, initially, since the sample solvent displaces the analyte from the stat phase more strongly than the mobile phase) while 25µL is too small to see this (high retention from the beginning).

Hi Liza,

I thought that was the problem.

Here is the explanation of “The Strong Solvent Effectâ€

Sorry for the confusion...my original question is stated incorrectly. I did a little reading & confirmed that it is the strong solvent effect that caused the fronting. The solutions for this problem is in fact to reduce the injection volume or to change the composition of the sample solvent closer to that of the mobile phase. Reduction in the injection volume did the trick in my case. Sorry for the confusion & thanks for all your help - I appreciate it.