I am running a reverse phase method for estradiol using 55:45, ch3cn:water as the mp. My column is A YMC C18, 3.0 x 250 mm. particle size 5 ucron. column temp = 30c. I noticed the retention time and area count decreasing as the run continues. I am bracketing every six samples with standards. Does anyone know why the RT and area count are decreasing and should I be concerned about this?

Thanks-R

The two observations together indicate a problem with the flow, i.e. the flow seems to be increasing over time. Check if this is indeed the case, then we worry about the cause.

Check to make sure the frit (if any) on your water reservoir is clean. Too much ACN, assuming you are mixing on-line, will cause RT's to drop slowly.

Do your sample contain other compounds? eg. PEG. Do you have the same problem if you only inject your standard six times? I would think your sample ingredient play a role.

How old is your column? If you've checked your system and filters and they're fine, then I would try a new column. First thing I would do though is remove the mobile phase inlet filters and try the run with fresh ACN/H20. If that's the problem you can sonicate the filters in ethanol for a while.

Is your baseline on a downward slope? This can tend to shift retention time backward...you might want to check whether this is a occurring only at certain points in a run, i.e., what the conditions are surrounding this shift.