Chromatography Forum

We do size-exclusion analysis of a carbohydrate polymer with a MW of about 1.5 million Daltons, so it almost totally excluded from this column. We usually see a pretty high peak at the void which tails off. However, there may have been some experimental samples that introduced some contamination to a the column, and now it seems that our peak is shifting to being more retained, with more of a guassian distribution. My question to those who have experience with size-exlcusion chromatography is how common is it to have contamination that causes secondary retention effects? There are some small-molecules present in the carbohydrate sample and those peaks appear unchanged; only the high-MW carbohydrate peak is affected. Any thoughts would be appreciated...

It is possible to get secondary effects, but specifically for your type of sample (a neutral very hydrophilic polyer) such a thing is difficult to imagine. Of course, if your polymer contains ionic sites, this would change things, but you would know if this is possible.

Another issue with high MW samples is their stability. Breakdown may occur with age, or by shear in the instrument (blocked frits etc.) Your MW is at the borderline where such effects become relevant.

Those who have been with this forum for a long time might recall that I have a "standing" question (foremost to Toso) about why their TSK Gel SuperSW 3000 changed permanently after contacting TFA (0.1%, mobile phase). The resolution of antibodies went way down (peaks broadened so changing rt), this could only partially be recovered by very long flushing/running on buffers like phosphate buffered saline (PBS).
Incidentally, in this system, other mobile phases (especially those modified by organics) can change the shape, probably via adsorption phenomena, but they do it reversibly.

Sorry that I still don´t have an answer.

The TSK SW series sorbents are silica based with a hydrophilic coating to prevent adsorption of proteins. Used with low pH buffer may affect this coating and expose the silanols. Check the column instructions manual for details on eluent compatibility.

Thanks guys. The only mobile phase we run is 0.05% sodium azide, but some samples were injected from an experimental process where they varied the acidity, so I wonder if that could be the culprit. For now I think we'll try the recommended cleaning procedures and see if that works.

Note that the comments from Hans and ivanwins were on the silica-based packing (SW). You are using the polymer-based packing, so I am not that much concerned about injecting some acid.

Is it possible that Toso has a "proprietary" surface on this polymer column as well?
For those using the SW: It is also not that sensitive to acid. As noted the change was partially reversible, so the change was only partially irreversible after being hours on ~0.1% aqu. TFA.

Just a theorethical remark...

Analysing and quantifying anything at void volume means we are quantifying each and every stuff unretained by the column - which is even more "serious" at SEC / GPC separations, since most large size molecules will exit the column at void volume.

Also, if the "shape" of the molecule changes when interacting with the eluent - that might happens with carbohydrates and proteins, you will experience retention changes.

I hope this helps to confuse all of us even more!!!