Chromatography Forum

Hi

I have a peculiar problem in my RP-HPLC, I am using a RP-HPLC method for quantification purpose. I am using C18,4.6 X 50mm, 5µ, 300 Å (discovery column)…every time I run a fresh sequence in my first 3-4 injection the sample doesn’t show any retention (my sample elutes with void volume peak..) after 5th or 6th injection the sample recovers its retention (around 5.0min)..this happens every time I run a fresh sequence..(I am doing proper storing of my column ..60% ACN)…

Could some one tell me the reason for such a phenomena?

Thanks a lot

You should give some more details about the method but my first guess would be inapropriate equilibration of the column (i.e. you need to equilibrate your column longer before you start your sequence). We can be more precise if you give some more info about the method (i.e. mobile phase, equilibration time, mobile phase, isocratic or gradient etc...).