Size Exclusion Columns and Long tailed peak

[sturgman]

Hello everyone,

First Background:
We perform SEC measurements routinely and we ran into a problem with a recent sample. The polymer was Poly (N-phenylacrylamide) and the mobile phase was THF at 25 degrees C. We are running the following setup:

Waters 2695 at 0.3 ml/min
Injections of 100 uL
Sample concentration 1 mg/mL
Waters Styragel column bank (4 years old)
Waters 2996 PDA detector
Wyatt MiniDawn light scattering detector
Wyatt OptilabREX differential refractive index

Situation:
The problem is very erratic behavior of the detectors after the "solvent" peaks elutes. In addition the light scattering detector shows a very long tailed peak (even upon terminating data collection) and it took almost all night to return to baseline. Blank THF samples injected after this long tailed polymer show some strange behavior around the solvent peaks and the polymer peaks of standard injections are not as reproducible as they used to be. I think I have adsorbed polymer in my column and I want to know if I can save these columns or not.

Im currently letting mobile phase run through the columns for an extended period of time but if anyone has any suggestions on how to "clean" the columns it would be appreciated.

[Uwe Neue]

I do not yet understand the origin of the problem. Was this Poly (N-phenylacrylamide) some new polymer that you had never analyzed before? I assume that you dissolved it in THF, and did not see any gunk in the sample.
Has the quality of your THF changed?

[sturgman]

Yes Poly (N-phenylacrylamide) is a polymer we never injected before. If we just inject the monomer we get a single peak. Upon injection of what we believe to be polymer we get a long tailed peak that required a long time to return to baseline. This is why I think there might be some undesired interaction with the column.

Im going to continue purging this columns at elevated temperatures hoping to see clean chromatograms upon injection of clean blank samples and until I get reproducible behavior of my poly (styrene) standards. Its the only thing I can think off.

[Uwe Neue]

Do you have any literature on how this polymer should be run, i.e. solvents and temperature?

Otherwise, you appear to be on the correct path.

[sturgman]

Not much. One paper claims to run it in THF at 35 C using a similar setup to ours but with something called composite columns. They do not specify what that means. The paper is not very clear or specific in their procedures. Thanks for your time Uwe.

[bhuvfe]

Maybe reversing the flow into your column set (if it is possible with these kind of columns) or changing the mobile phase?

What do you see on your MALS? If interaction with the column is the cause of your problem shouldn't you see a funny Mw curve (i.e. non linear)?

[zilllski]

awesome work man! ^_^

[danko]

Hi Sturgman,

I think you’re dealing with a polymer that is not exactly soluble but stays suspended in the utilized solvent.
When this kind is injected onto a column, it typically sticks to the stationary phase and only elutes through dissociating slowly and thus becoming a monomer (i.e. soluble in the mobile phase).
I’ve seen that with some proteins.

So, the first action ought to be, as Uwe pointed out earlier, to dissolve (or try to) the polymer in the mobile phase (THF) and observe the solution/mixture very, very carefully to make sure that it is absolutely clear (no opalescence when direct light hits the container/test tube/vial or whatever.

As for the column regeneration, I think you’re doing the right think give it the necessary time with flow on.

Best Regards

[HW Mueller]

Sturgman, don´t your detectors allow to characterize the "polymer", at least regarding MW, so that you know more about the tail. etc.?

[sturgman]

HW,
The tail behavior is erratic and I cannot see it in all three detectors. The UV has a huge signal for these injections and the full PDA spectrum matches very closely that of the monomer. The light scattering detector does not provide much information regarding the molecular weight because I do not have an extinction coefficient for the polymer and the refractive index detector does not show a signal.

sturgman

[Jeffrey Bodycomb]

First, a comment on peak shape. High molecular weight polymers can break up in the column due to shear. Then, you will get to watch fragments elute for a few column volumes. If the light scattering detector shows a signal, this is probably what you are seeing. My columns survived this a couple of times.

Now a comment on the RI and light scattering signal. The light scattering signal is proportional to the square of the dn/dc of the polymer. The RI signal is proportional to the dn/dc of the polymer. If there is no RI signal, then you should not expect a light scattering signal. Of course the exception is a high molecular weight species injected at very low concentration.

And, that exception fits the idea that you have a high molecular weight material that is shearing on the column.

If you have enough material measure the light scattering and dn/dc in batch mode (without chromatography). Then, use that information to pick your columns. This is a variant on danko's suggestion....