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Chemistry Forums for Students => Analytical Chemistry Forum => Topic started by: cyril on October 24, 2005, 05:26:54 PM

Title: HPSEC Polystyrene sulfonate standards chromatogram
Post by: cyril on October 24, 2005, 05:26:54 PM
I am currently setting up an HPSEC analysis to characterize humic substances molecular weight, following someone else previous analysis.

But I am a bit confuse about the calibration by polystyrene sulfonate (PSS) standards. The shape of the elution peak I have are not symmetric and possess some tailing. I supposed that it was due to the polydisperse nature of the standards. I found in the litterature an example of the PSS chromatogram under the same conditions as mine which are the same as my results (assymetric peak with tailing).

For the moment everything seems OK.....but ...The person that did some study on this project before me had symmetric peak, and my research director thinks it is the way it should be.

This is why I am confuse....Am I wrong and why?? ??? :P

Thanks for any help...

Title: Re:HPSEC Polystyrene sulfonate standards chromatogram
Post by: Dude on October 24, 2005, 06:17:13 PM
I also think that you should have symmetrical peaks.  Although I am not familiar with aqueous GPC, I suspect that your standards are sulfonated by reaction of the polystyrene standards (which typically have a polydispersity in the 1.01 - 1.06 range) with H2SO4.  The polydispersity should not increase appreciably after sulfonation.  Although looking at the Polymer Labs site (, there does appear to be some tailing in the standards.  I suppose it then becomes a question of how much is acceptable.  If these are the display samples, then tailing might be somewhat common.

I would approach your situation in the following manner.
1.  Make sure your pump is working correctly (ie check valves aren't stuck).  This can be done by simply collecting the fluid coming out into a small graduated cylinder (or by weighing it after a fixed interval knowing the density of the fluid) and confirming that it matched your set flow rate.
2.  Organic GPC columns are notorious for "drying out", which is sort of like the channeling effect observed if you have ever done preparative column chromatography in organic chemistry.  If your GPC hasn't been used in a while, call up Waters or Polymer Labs and ask their technical service department if there is a test you can do to confirm if your column is OK.  
3.  If the solution isn't in 1 or 2, then I would look at your standards (unless you have a set of home-made standards from a previous student).
Title: Re:HPSEC Polystyrene sulfonate standards chromatogram
Post by: cyril on October 25, 2005, 11:35:12 AM
Thanks for your reply,

The standards I am using are from Polysciences. the polydispersity are 1.13, 1.25 and less than 1.20 for the other ones. the eluant phase is an aqueous phosphate buffer with a ph of 6.8, with added sodium chloride to yield an ionic strength of 0.1M.

Could this value explain the tailing?

The GPC software that I am using can integrate narrow standards as well as broad standards.
Shouldn't I be using the broad standards calibration method with this standards?

The machine has been check by maintenance service at the beginning of this year, after the previous study as been perform. At first I thaught that the column was saturated and I tried with a smaller injection volume and also with a smaller flow rates, but I obtained the same shape for the peaks.

Still looking for some informations on this....thanks for any help

 ;D :P