Chromatography Forum

Hello

i get a strange problem with reverse phase liquid chromatography.

i inject a one hour-reaction-mixture.
eluant: acetonitrile 0,1 % TFA :water 0,1 % TFA with gradient mode. i get nice results (nice peaks).

Then i inject the same reaction-mixture at initial time (t=0 min): i don't get any peak , just a continous line. So i pass ethanol through the column C18 to make it clear. and then i re-inject the same reaction-mixture (t=0); no peak at all.
i pass again ethanol through column. and then i inject a molecule dissolved in eluant. i get one peak which is caracteristic of this molecule.

when i re-inject the reaction-mixture (t=0), i don't get any peak again.

i think that the the reaction-mixture (t=0) cause problems because the molecules are not very soluble in the eluant, may be because the reaction-mixture contains DMSO. So it blocks the column so that i don't get any peak.

but why it shows nice peaks after one hour of heating?? i don't understand.
does DMSO cause any problem in RPLC ??
Please may any one help me to understand.

Best regards.

and then i inject a molecule dissolved in eluant

Is this molecule contained in either of your reaction mixtures?

Is the reaction mixture being used to synthesize your molecule? If so, You shouldn't see anything at time zero.

If not:

Do you get a peak if you inject a sample molecule dissolved in eluant with added DMSO (same level as the reaction mix)?

I don't think the DMSO should affect and RP18 column (I've used STDs and samples in 100% DMSO in some analyses).

Column blockage is not an option as your system would show massive pressure fluctuations and probably shutdown.

How is the reaction mixture treated? Does the 1-hour period significantly alter the reaction mixture composition (e.g. removal of volatiles)?

I suspect the problem may be the solubility of the molecule in the reaction mix which is eliminated during the 1 hour reaction period.