Polymer GPC

[jdlh199]

I'm trying to do some aqueous GPC using PEG calibration standards to produce a molecular weight estimation of a polyacrylamide sample which should have a Mw ~3,500. I'm using a GS-520 HQ column with a DIW eluant running at 0.5 ml/min with UV detection at 195nm. All samples and std's are just made up in DIW.

My PEG calibration STD's produce a perfect linear calibration curve using log(Mw) vs Rt. However, my unknown polyacrylamide sample is eluting after my lowest std (Mw=100) and gives a back-calculated Mw estimation of just 72.

It seems no coincidence to me that acrylic acid has a mass of 72. Did my sample really completely degrade to acrylamide in such a short space of time (24 hours), or am I doing something wrong with the analysis.

Also, I only have a UV detector and am monitoring 195nm. I presume this is okay, but I haven't been able to find UV absorbance spectra for PEG or for polyacrylamide.

[Uwe Neue]

I do not know the column that you are using. For multiple reasons, it is best to adopt the manufacturer's recommended mobile phase and calibration procedure for the polymer that you want to analyze.

That said, I would analyze polyacrylamide with some salt in the mobile phase. If you have (residual) charges on the surface of the packing and charges on your polymer (from partial hydrolysis or initiator, for example), you can get strange results that are due to ionic repulsion, which of course does not happen with PEG. Since you know your polymer, you may be able to make a judgement if ionic charges can be part of the polymer.

Another possibility is that you see something else, like an additive with a low molecular weight that outshines the response of your polymer at your selected wavelength.

[jdlh199]

It's a Shodex Asahipak column:

http://www.shodex.com/english/cg09.html

http://www.htslabs.com/HTML/HPLCColumns/Shodex/methods_and_purifications/english/da060101.htm

Column manufacturer recommends DIW for PEG samples, which is why I presumed it would be the same for polyacrylamide.

I'll try it again with a little sodium phosphate or NaCl and see what happens.

I did think that there may be another UV absorbing additive, but there are not even any minor peaks at the rough correct Rt, and the sample is supposed to be a pure polymer.

[Uwe Neue]

Not yet entirely sure what is going on. The Shodex site says that this column is a column with multiple interactions, including ionic interactions, and they do not give specifics for polyacrylamide.

The other thing is the question of where the polyacrylamide should elute. SEC works on size, and an estimate of the equivalent size of the PEG and PAA gives a ratio of about 2.4. Therefore a 3500 MW PAA would elute at a PEG MW of 1500. This is at the upper end of your column. You may not have the right column for your assay.

The recommended mobile phases in my book for polyacrylamides and related samples all specify some amount of salt, in the order of 0.5M, which is a fairly large amount. I don't know why this is the case, but the addition of salt is commonly done to suppress ionic interactions. If you decide that you do now have the right column for your purpose, I suggest to look at the Waters catalogue. In my opinion, the Ultrahydrogel 250 column seems to be the right column for what you want to do.

[jdlh199]

Hi Uwe,

Thanks for the advice. I'm still relatively new to chromatography and am kind of learning on the job, this forum has been a great help!

Can you explain (or point me to a good ref source) of what you mean when you mention equivalent size and say that a 3500 MW polyacrylamide will elute at around 1500 MW PEG? How do you arrive at the 2.4 ratio estimate?

Also, my PEG calibration line runs from 100, 200, 400, 500, 1000 etc up to 20,000 MW and produces a perfect linear line with an RSQ >0.99, so I presume that detecting the PAM at ~1500 Mw PEG should be possible with this column. It's the fact that its eluting after my 100 std that is confusing me.

I'd love to buy a new column every time I need to, but like many small dept's I have to work with what we have!!

I'll try running with a salt eluant and see if that makes a difference. Thanks for your advice!

[Uwe Neue]

Polymers of the same chain length have roughly the same size random coil in solution. This is the essential principle of SEC. The repeat unit of PEG is CH2-CH2-O, the repeat unit of the acrylamide is CH2-CH2. So three acrylamide units give the same chain length as two PEG units. The rest is the molecular weight of the repeat unit, which is 44 for the PEG and 71 for the acrylamide.

There is a reasonable chapter on SEC in my "HPLC Columns". It does not give you this particular trick of estimating equivalent sizes, but it gives you the background on the molecular weight equivalence to size.