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Column recondition
Posted: Tue Jan 22, 2008 10:49 am
by yzhen77
Hallo,
the detection of one mycotxoin patulin with our HPLC column Purospher (250-4, RP-18e, 5 µm) at isogratic flow give the best seperation (with 95% water as mobile phase, RT at 12 min), but we need to run each sample 90 min to recondition the column, thus no peak shift happend. Are there some methods to fulfil the reconditioning and to reduce the time at the same time. Is a gradient elution maybe better? thanks!
Best regards,
Zhen
Posted: Tue Jan 22, 2008 11:11 am
by zokitano
If you could, I'll recommend you to try that method on embedded (aqua) columns, which can give better reproducibility when working with high aqueous mobile phases.
Otherwise, if you have encountered other peaks that elute after your peak of interest you could use a gradient after 12th minute to fasten the "column cleaning" and then return to the initial isocratic conditions prior the next injection. When using this approach, do not forget to reequilibrate the column with the initial mobile phase composition prior injection.
Regards
Posted: Tue Jan 22, 2008 11:39 pm
by danko
It sounds strange to me, that re-conditioning of the column should be required in an isocratic elution.
The column is conditioned all the time when running isocratic. Unless gradient is applied somewhere in the run, there is no need for re- conditioning.
Another strange thing is, the shifting retention times. I have this feeling that your sample is dissolved in a stronger solvent (e.g. a lot of ACN or methanol) than your mobile phase and the conditions get disturbed every time a sample is injected.
So would you please share 1. The nature of the sample solvent and 2. What du you observe after 12 min (i. e. how does the baseline look like)?
Best Regards
Posted: Wed Mar 05, 2008 2:21 pm
by yzhen77
hallo Danko,
you are right. I have tried to run with gradient after 15 min, the result is quite good, the only problem is the baseline shift a little. I suppose I need a more time to recondition (right?) the column.
thank you for your suggestion and sorry for the delayed reply.
best regards,
Zhen
Posted: Wed Mar 05, 2008 3:39 pm
by danko
It was Zokitano that suggested the gradient option: So I’m not entitled of this honour .
Anyway, the column you’re using should be reconditioned for approx. 12 min (after the gradient). I’m assuming the flow rate is 1 mL/min.
Best Regards