Chromatography Forum

Hi all,

I am running water blanks on a polymeric SCX column with a steep NaCl gradient in 20mM MES, and I keep getting a double peak right at the end of my isocratic stage (0-1.5min). (gradient: 100mM to 750mM NaCl in 4.5 minutes, after 1.5 min isocratic 100mM). I don't understand why HPLC grade water would give a double peak...?
I am new to HPLC, so don't hesitate to state the obvious...

More information please:

what size column?
what flow rate?
what detector (and, if UV, what wavelength)?

Most likely a "system peak" (sometimes referred to as a "vacancy peak"). In principle, you can get one system peak for every component in the mobile phase if you inject something which does not contain that component. In practice, you may not see them at all (depends on your detector and on the retention of that component), and they can be either positive or negative (depending on your detector).

column is 4x250mm, SO3-linked polymer
1.25 ml/min
UV VWD at 280nm (analyzing protein)

Also, mobile phase is MES with 5mM CaCl2, 100 ppm TWEEN80 (Buffer B has 1M NaCl).

I am also seeing a broad, unidentified peak at the end of the run, during re-equilibration. I suppose if it's reproducible, it shouldn't be a problem for method validation...?

1.5 min is just about the "dead time" of that size column at that flow rate (the elution time of any unretained compounds). What you're seeing is indeed "system peaks" (aka "t0 noise"; aka "water dip"). Ignore it.

That said, you really should not be trying to quantitate anything that elutes at less than 2x t0 (at least 3x t0 would be better). Your gradient conditions are *very* steep for that size column and flow rate.

The broad peak at the end may simply be due to the RI shift (unfortunately, UV detectors do respond somewhat to RI) as the surfactant re-equilibrates. It may also be due to additional proteins or other junk coming off after your gradient is complete. You can check by running the gradient and putting in a long isocratic hold at the end.

Another concern is the dwell volume (gradient delay volume) of your system. Remember that if you have 1.25 mL of delay volume (which would not be unusual), it takes a full minute for your mobile phase composition change to get from the pump to your column.

Your response is always appreciated. Thank you for the valuable insight!