Chromatography Forum

I've build a calibration curve for GPC analysis with 6 narrow standards ranging from 6 120 000 to 1050 as Mp value. My R2 coefficient is 0.999944 and my highest % residual is 4% obtained with Waters Breeze software on a 3rd order fit curve. So I think my curve is pretty good for analysis. To verify my curve, I've injected the 6 120 000 standard as a broad unknown. The results are good for Mw (about 5% deviation) and Mp (about 2% deviation) but are really off for Mn. The deviation is 40-50% below expected value. The supplier standard Mn value should be
5 585 000 but I obtained about 3 000 000 on each run I did. Does somebody have an idea on how to correct that and have Mn value near standard supplier value?

Hi,

I don't have a solution, but want to draw your attention to the fact that half of the peak is in "uncalibrated zone", so your calculation is based on extrapolation, not calibration - this might create a significant bias. Second consideration - what is your column's exclusion limit? Your peak might be distorted if some part of polymer is above exclusion limit.

Good luck.

The Mn values are known to be prone to high errors - they are extremely dependent where you place the peak ending point. In your case you are integrating probably too far after the end of the peak.

Rolandas, my exclusion limit is 10 000 000 on my Styragel HT6 so it should be OK with that standard. I've tried recently with Mp = 49 170 standard and got same results. Standard supplier's Mn should be 47 650 and I got about 30 000 so it' still a 36% deviation.

ivanvins, I tried to manually adjust peak ending point in order to correct error due to automatic integration but the Mn didn't vary so much and still far away from what I expected.

I have done before a calibration with my standards processed as broad standards (I wrote Mw, Mp and Mn from supplier's values) but calibration curve was bad even in 1st order fit, my % residual on some value was 30%. I reprocessed my standard as narrow standard with supplier's Mp value only (the curve I use now) so the software calculate for the curve Mn, Mp, Mw, Mz and Mz+1 and get good curve except for Mn value.

If you can send me your chromatograms exported as AIA files (uniform sampling) with the standard Mn, Mw and Mp details, I can try to evaluate them in our software Clarity. Also, a picture of the baseline used may help - is the peak end on the chromatogram line or does the baseline continue bellow the chromatogram?

vins@dataapex.com

Ivan Vins

Sorry Ivan, our GPC computer is not on network. All I can do is print chromatogram, scan them and send it to you in PDF format.

The picture of chromatogram including the baseline with peak start/end will help.

You do not need to be on network to send files - just copy the exported chromatograms in AIA format (*.cdf files) to floppy or flash memory and attach them to an e-mail send from another computer. (you may need to check Help for the AIA export, in Millennium this was not a straightforward task)


Ivan