SEC eluent modifiers

[SEC-Man]

I have problems with SEC analysis of polymeric anionic high mass material only soluble in organic solvents such as THF.
Waters HR columns exhibited non-SEC type elutions (non-Gaussian, multimodal distributions), confirmed by static light scattering detection.
Thus a modification of eluent seemed to be helpful to solve the problem. I tried THF + 5g/l TFA but results were even worse. Narrow distributed Polystyrene elution looked distorted for high mass calibrants (7.5 Mio Da).

Any good ideas for modifiers or recommendations with columns that withstand TFA?

Just contacted Waters to comment but still waiting on their answers.

[Uwe Neue]

What is the nature of your polymer?

[SEC-Man]

The polymer is an acrylic copolymer consisting of styrene (15 %),various non functional acrylates and methacrylates and acrylic acid (2-5 %). The material is neutralized with ammonia and dispersed in water. Analysis in acqueous SEC is not possible unfortunately due to insolubility in water (material is only dispersed in water or in an emulsion form).

It is supposed to have a broad distributed but monomodal (random polymerization) MWD.
Expected Mw is appr. 1 Mio Da (data gained by multi-angle-light scattering detection (Brookhaven).

We encountered good experiences with TFA modification for mid-size columns such as Mixed-C from PL but these do not apply for high mass, of course. Exclusion limit for Mixed-C = appr. 1.5 Mio Da

Anyway, we can get the "correct"Mw from MALS but what we need is a good and robust column set-up for QC running conventional SEC with RI.

[Uwe Neue]

My first reaction is that you may have a problem with the high MW standards. They are very sensitive to the procedure which is used to make the standards. Any shaking of the vials or similar things can break down the polymer. Also, they take some time to dissolve quietly and in the dark, and afterwards they do not last forever either. They also can break down when you suck them up into the injector loop. This needs to be done sloooowly...

So let us see that you got the PS standards under control, and then we worry about the polymer itself.

If you have done all these things and are familiar with the problems described above, let me know and I try to think of something else.

[SEC-Man]

Well, I had the same feeling. But when switching back to pure THF (as eluent) and reinjecting the same PS standards (from same vial) it looked much better again. And after a second inject the high mass standard (7.5 Mio) recovered from a broad flat top to Gaussian shape again.

Thus I suppose that trifluoro actec acid modifier is doing some "harm" to the PS-DVB pores.

I have read about similar findings with PS-DVB columns published by Pasch (Darmstadt). I will post the article tommorrow when I am back in my office.
He reported best results with a GRAM column from PSS and DMAC/LiBr/AcAcid. Unfortunately these column material doesn`t cover the high mass range I require (at least to my knowledge there is none).
And I don´t want to run an extra GPC with DMAC since routine SEC with plain THF should be possible as well.
Ideal would be a column material that could stand THF/TFA and fits to the mass range from 500 - 10 Mio Da.

I have various PL, Waters and PSS column material available but either the mass range won´t fit(PL, PSS) or the column doesnot stand TFA (Waters).

[Uwe Neue]

OK, so the first idea was wrong...

Now, you mention that you are injecting the same PS standard onto the column with and without TFA. If your PS standard was not dissolved in the mobile phase WITH the TFA, maybe it could shrink during the run and that this is the reason for the the weird peak shape (rather than the column).

If this is a possibility, you should dissolve your standard in THF with TFA, and bring your column back to the same mobile phase.

I have never tried anything with TFA in the mobile phase, but the reasoning is that if you are concerned about TFA doing something to the styrene-divinylbenzene column, I think there should be at least equal concern about what TFA does to your standard.

My proposal is a lot of work/time. If I come up with a better idea, I'll let you know.

[SEC-Man]

Oh, your suggestions sound very reasonable!

I do indeed dissolve all in pure THF and NOT in the mobile phase due to simple practical reasons.
I completely forgot about different swelling status of both samples and calibrants!!

It seems that columns with small pores (and smaller PS propably too) do not shrink that much to give indication as distorted peak shapes. Or: since these samples travel for a much longer time than high mass ones, their molecule sizes will reach their final shrinked status before leaving the column. With pores enough to undergo separation..

That would explain why the 7.5 Mio and 2.5 Mio narrow standard appeared distorted while the smaller ones were not.
Do you agree or any other explanations?

However, your advice was very helpful. We will check asap and report the findings. It will take some time, of course.
Thanks!

[bhuvfe]

DMAC/LiBr or DMF/LiBr is quite a common solvent system for organic GPC for polymers that give non-SEC interactions.

Does TFA solve you interaction problem for your sample?

If yes, can you use other standards that are,maybe, more soluble in a THF/TFA mobile phase (maybe PMMA)?

[SEC-Man]

I agree. DMAC or DMF are common solvents for SEC but as said previously I wanted to get rid off of these toxic material for QC labs.

My favorite solvent is thus always THF with any suitable modifiers added when necessary.

And we truly noticed good results with improved reproducibility for mid sized (< 1mio Da) polymers when adding modifiers to THF. Mixed-C from PL works pretty well but the mass range is not suitable for my actual problem.

TFA addition to THF suppresses adsorptive interaction of polyanionic material with the column material. It is of course not the PSDVB itself but the residues from the manufacturing process that cause the unwanted enthalpic phenomena.
TFA eluent modification comes from the well known practise in eluent tuning in HPLC.

I assume that solubility of standards are not the problem but different swelling and gel sizes of the column particles. Eventually also for the PS standards.
PMMA standards could be considered but I have nothing available actually.

[Uwe Neue]

I am less concerned about the columns swelling and shrinking. They survived your treatment and you got good results when you went back to the straight THF. Also, the higher pore-size columns are less sensitive to swelling/shrinking than the smaller pore-size columns. However, your standards are subject to the same "swelling and shrinking" as the columns, and much more so, since the columns are highly crosslinked, and your standards are not.

[SEC-Man]

Yes. I am really curious about the results when diluting in the eluent.
If all will work as proposed the solution is rather simple.
I will post the results but not before the next 2- 3 weeks.


Off-topic: How could I append a document to my postings?
sorry, if the question is stupid, but I am new here and I have no idea how to get this done. Wrong profile settings?