Chromatography Forum

My ion exchange elution method increases my Buffer B (2M NaCl with 20 mM bis tris propane pH 6. concentration in a stepwise fashion, 5% at a time (0%, 5%, 10%, 15%, 20% --> 100%).

At 10% and 15% I see distinct absorption peaks at UV 280 and 265 nm, even though I've only injected a blank buffer (bis tris propane 20 mM).

Also, I see distinct absorption peaks any time I jump to 100% B or back down to 0% B. Again, these peaks appear even when the system contains no sample, only buffer. Is the sudden change in salinity causing my peaks?

[ Column: GE HiTrap Q HP 1 ml anion exhange)

Most UV detectors have*some* sensitivity to refractive index and can respond to abrupt changes in composition. The other possibility is that you have a low level of UV-absorbing contaminants in your "A" buffer which accumulate on the column al low ioic strength and the get bumped off when you step up. This is actually common in reversed phase, but I can imagine it happening in ion-exchange as well. As long as they don't interfere with your analytes, you can simply ignore the artifact peaks.