i) The reason I'm not using RP is that I already tried that, and couldn't
resolve the peaks. I thought IE would be easy in comparison!
ii) My peptide *is* N-capped, so cation IE would be out, even if I didn't
now have far too much fear of IE columns to consider ever buying another
one.
iii) The column I'm using is a brand-spanking-new Vydac VHP (DEAE) 5.00mm
ID column. I'm told these shouldn't really have any metal atoms lying
around, because of the type of matrix (spherical PS-DVB beads). I doubt
it'd be bad enough to chelate everything I threw at it, anyway(?)
iv) I'll give it a go with the 10% acetonitrile at pH2, like Andy Alpert
has suggested, but I still think there's something really odd here that
probably needs to be sorted out...
NEWSFLASH!
Since mailing the list, I've managed to get a peak! Sadly, it's not (I
believe) the peptide I want: I shoved a set of pI calibration peptides
down the column and eluted at pH8, 0-0.5M NaCl. This set included 6
proteins with pI more than one pH unit lower than pH8. I got *one* peak!
Is it just me, or this this not normal? In case anyone cares, they were:
Amyloglucosidase pI 3.50
Soybean trypsin inhibitor pI 3.75
Beta-lactoglobulin A pI 5.20
Bovine carbonic anhydrase B pI 5.85
Human carbonic anhydrase B pI 6.55
Horse myoglobin - acidic pI 6.85
There were also 5 others with higher pI's.
All the hardware I'm using is exactly the same as we use for RP (which
works fine, including with my peptide), except for the column itself and
two short sections of tubing.
Any suggestions as to what I could try next? The people who sold it to me
seem to be as confused as I am about what could be going on.
Jenny
PS: Is it normal to have to wait ages for these things to equilibrate? The
signal at 210 seems to just go down and down for ages.