Chromatography Forum

Help!! I'm running a clients samples by GPC for Mw determination. The samples are low Mw polyacrylamide. Our first run was an attempt at using PEG reference standards for calibration. Here's what we noted:

1. Calibration is perfectly linear from 200,000 to 100 Mw for PEG samples.
2. Clients samples elute after the lowest PEG standard, suggesting they are very low Mw (<100!!) - this is unlikely
3. A sample of acrylamide monomer elutes after the clients polyacrylamide samples suggesting he does have a polymer not a monomer.

So I suggested we buy and try using some broad Mw polyacrylamide reference standards instead of narrow PEG ones. The only ones available were 20,000 and 3,500 Mw. Along with the monomer, this should provide a very limited rough calibration curve!

But... the PAM broad standards both elute together very early in the chromatogram, earlier than the 200,000 Mw PEG standard.

HPLC conditions:

Column: Shodex OHPak SB803-HQ (polyhydroxymethacrylate) 8x300mm
Eluant: DIW
Flow: 1 ml/min
Detection: UV @ 195nm and refractive index.
Samples all dissolved in DIW on a rotator overnight.

Maybe there's ionic activity? But I've tried using a PO4 eluant and an acetate eluant instead, but this seems to severely affect the background level. I'm going to try a different PO4 pH and see what happens.

But does anybody have any suggestions? Why would the clients PAM samples and the PAM reference standards behave differently under the same conditions?

I haven’t done much GPC, so I hope this helps.
I think you should try to use a standard as similar as you can to your sample.
Why don’t you use polyacrylamide standard?

I did buy PAM standards, read my post again.

So I suggested we buy and try using some broad Mw polyacrylamide reference standards instead of narrow PEG ones. The only ones available were 20,000 and 3,500 Mw. Along with the monomer, this should provide a very limited rough calibration curve!

But... the PAM broad standards both elute together very early in the chromatogram, earlier than the 200,000 Mw PEG standard.

Why would the clients PAM samples and the PAM reference standards behave differently under the same conditions?

Thanks anyway!

My guess is that the PEG standards are linear and the acrylamide samples have multiple cross linking. SEC shows the apparent molecular weight – 3D structure has a major impact on how things separate. Often this will be buffer dependant and you can get very different profiles for samples using a different mobile phase (especially for proteins).

The textbook mobile phase for polyacrylamide is water with 0.5 M LiNO3 or even with a phosphate buffer at pH 7. A possible explanation for your observation could be ion-exclusion effects from a partial hydrolysis of the acrylamide together with anionic sites on the packing. The salt will make this effect disappear.

I agree with Uwe , after re-reading your original posting - what you discribe is too dramatic to be molecular structure and is probably ionic .

Be careful with phosphate salts for chromatography buffers, I have seen some with very high UV absorbance. Make sure to use "chromatography grade" . Also at the low wavelenght (195 nm) you will see absorbance from many factors, including disolved oxygen, Degas your buffers.

Also don't forget to concider the buffer components from the samples & standards. Include buffer blanks.

OK, so here's what I tried next, I disconnected the UV and went with refractive index only!

As per PAM suppliers suggestion, I tried using a 0.3M NaNO3 buffer. I also used 2 columns in sequence!

Here's a few chromatograms. Peaks are not great by any means, but not bad I guess for rough R&D!

Blank: http://i19.tinypic.com/301376a.jpg
Monomer: http://i18.tinypic.com/44rjaq9.jpg
PAM 3500 (Mw): http://i15.tinypic.com/4fudlaq.jpg
PAM 20,000 (Mw): http://i15.tinypic.com/4i4jplw.jpg
Calibration curve: http://i18.tinypic.com/33d8nrb.jpg

Hi, Djlh199,

So the polymer MW 3500 came at Rt 16.075, is this right?

It seems to me a Shodex Protein 802.5 can do better than that unless your polymer has wilder distribution than mine.

Please inform us if you did achieve better peak shape.

Acoording to Shodex website, your SB-800 HQ should better than the Protein 802.5.
Good luck,