Chromatography Forum

I have heard the terms “solvent breakthrough peak” and “system peak” thrown around as I begin GPC work. I have seen it as positive and negative peaks near the end of injects. What is the cause for these peaks? What impacts whether they are negative or positive?

Difficult to answer without knowing how familiar you are with how GPC works, so forgive me if I sound like I'm either overwhelming you or patronizing you!

In GPC very large sample molecules will go around the packing particles and elute near the beginning of the chromatogram ("total exclusion volume"). Very small molecules will penetrate the packing particles and therefore elute at the end of the chromatogram ("total permeation volume"). The smallest molecules in your sample usually are those of the solvent used to dissolve it (I generally prefer the term "diluent", because that lessens confusion with the mobile phase solvent). If the diluent is identical to the mobile phase, the detector will not see a peak, because the composition remains the same. If the diluent is even slightly different in composition from the mobile phase, then the detector *will* see it. Whether the resulting peak is positive or negative depends on the detector and on the difference between diluent and mobile phase.

I'm glad this is a recent thread. I'd really appreciate any thoughts on the following situation / help rationalizing what I've observed.

I'm performing SEC, and thought I understood my total permeation volume which we've loosely called the salt peak.

While working through the cause of an early (~5min) negative peak I discovered its actually from the prior injection - I have an "over-lap-o-gram"
I confirmed this by extending my run time an additional 10 min (30 --> 40min), which resulted in that negative deflection showing up between 30 & 40min and it was absent in the next run.

Injecting a 16% soln of acetone (v/v in diluent), (10 µL) confirmed the total permeation volume was seen in under 30 min (UV 220nm). If I had a positive peak after my salt peak I would think retention was through a specific interaction (adsoption) in addition to size exclusion, but I'm at a loss to explain why a negative peak is seen with UV detection. I understand that a more transparent zone is in the detector, but can't figure why.

Diluent is pH 5 acetate, while MP is 20 mM MOPS, 200 mM NaCl, pH 7.5, 7% ACN in water.

One thought is that a component of the sample or its diluent (perhaps ACN?) is displacing a mobile phase component in equlibrium with the packing (adsoptive interaction). The displaced molecules then elute with the "salt peak". The more strongly retained molecules are isocratically eluted by a RP mechanism, which accounts for their retention beyond the total permeation volume. I'm guessing ACN, because the zone is detected as a decrease in UV absorbance.

Any thoughts / corrections / suggestions?