-
- Posts: 5
- Joined: Tue Nov 14, 2017 11:16 pm
I've banging my head against the wall with this separation for a while. I've been bouncing between columns, eluents and gradients and ALWAYS get the same results--4 closley eluting peaks with no separation. The most I've managed to do is swap the positions of two of the peaks. Running on an agilent 1100.
https://imgur.com/a/sqjuk
My analyte of interest is at 2.195.
Flowrate: 1.2 ml/min
Column: YMC-Pack ODS-AQ; 250x4.6 S-5um 12nm
Temp: 40 C (Yes, I've tried at lower temps)
Mobile phase: A 50mM Phosphate buffer @ 2.5 ph; B ACN
Gradient of 82:18 to 70:30 over 5 min (0-5min, 6min hold).
UV/VIS @ 218 nm.
The sample is cornflour extracted with 50% 10mM TCEP in water 50% EtOH. I'm trying to pull out cysteine. If I use 100% EtOH I have a nice clean cysteine peak, but the introduction of water gives me the bonus coeluting peaks (which is expected). The problem is that I have a 30% loss of cysteins in 100% EtOH. Cleanup with SPE has been largely unsuccessful.
I'm sure I'm missing something obvious, though I'm starting to think a new column may be needed. From digging around the literature it looks as though the ODS-AQ column is pretty similar to whats being used (eg primsep 100), so I'm hesitant to dump $1k on something that likely won't even work. Any input would be great. Thanks!
PS Precolumn derivitization doesn't work for some reason and postcolumn is not an option.