Chromatography Forum

Hi there,
I want an opinion and any possible advise about some results i have extracted a few days ago.
I run a gradient elution program for the separation of two compounds(tetracyclines) and i observed the following situation: The late eluting compound(12,3min) and its main metabolite(12min) had narrower peaks than the earlier eluting peak(5min).From theory i know that as retention time increases, the width of a peak also increases and the plate number decreases.
Is there any mistake for this elution?I mean, is everything ok with the peak width of the late eluting peaks? I shall note that the first peak has As=1,59(not good enough)
(I use a mobile phase ACN: KH2PO4 50mM pH8 30:70 v/v as a starting point).
The gradient has as follow:
1-5min 30% ACN-70% buffer
5-8min 65% ACN-35% buffer
8-15min 65% ACN-35% buffer
15-17min 30% ACN-70% buffer
17-30 min 30% ACN-70% buffer
The column's dimension are: 250mmx4,6mm 5μm and the flow rate is 1mL/min.
Thanks for any suggestion and advise!

The peak at 5 min is eluting under isocratic conditions whilst the peak/peaks at 12 min under gradient conditions.

Best Regards

Hi Danko, thanks for the help...but is it acceptable anyway?
Thank again...

You bet it is. No lawyer will dream of suing you for that

Best Regards

Thanks, I TRUST U!

The only other comment that I would add is that the rule of thumb that later peaks are wider applies only to isocratic separations. In gradients (to a first approximation) all peaks have about the same width.

As danko pointed out, your 5 minute peak is eluting isocratically before the gradient part of your run.

Thanks a lot for your help!
Regards