Chromatography Forum

How can I manually calculate the molar mass distribution numbers (Mw, Mn, Mz) from my SEC/GPC chromatogram? Edit

For some unknown reasons, the software (Empower 2) doesn't provide mass distribution values for my samples. It integrates the chromatogram and detects all the peaks/retention times very well but my results table is just empty on these parameters (Mn, Mw, Mp, Mz, etc.). It gives me the raw measurements of peak height, area, and %area as well. Is there any way I can use these measurements to do my calculations? 

The methodology was as below:
Molecular weight characterization of the polymers were determined using a GPC unit (Waters 2695) equipped with a RI detector (Waters 2414), and a PDA (Waters 2998). The samples were characterized by passage through a guard column, followed by two Styragel 5 μm, HR 4E 7.8 × 300 mm column in series. THF was used as the eluent at a flow rate of 1.2 mL·min−1. Narrow poly(styrene) standards (580-156000 Da) were used to calibrate the GPC.

The chromatogram was obtained at 230nm wavelength and some of my peaks are within the range of standards.

Any comments, suggestions?

Sounds simple, but first step is to verify that you have the GPC option for Empower and that it has been activated.

Second step is to verify the project you are using also has the GPC option enabled.

Third step is to make sure all calibration standards have been run as narrow standards and that the appropriate moment and concentration information has been entered into the amounts window for those standards.

Fourth step is to ensure that samples have been run as broad samples.

Fifth step is to make sure the processing method you are using is a GPC processing method and that you have entered a compound on the slice tab of the PM and that the peak in your samples is being identified as this slice compound.

I am guessing something went wrong for you between steps 1-5. Let me know specifically where and I can provide more detailed help.

Thanks Shaun for the step-by-step instruction..

I'm not sure about the first two steps, since I used the profile from another user in polymer characterization lab where GPC is used on a daily basis. Users out there had no idea about the problem source, but I should definitely make sure that GPC is activated for the profile I used.

Also, for the peak names I didn't add any name assuming that the peak numbers would be enough for the software to do the calculations. So, I would go back to my results and enter the names and see if it helps.

Regarding the calibration standards and my samples, they were of the same type as you mentioned, narrow standard and broad sample respectively.

There should be a "slices" tab in your GPC processing method. This sort of replaces the components tab in regular processing methods.

You need to name a slice in accordance with the analyte name you entered for the amounts section of the sample set method. In your actual samples, you need to ensure that the peak being integrated is being named in accordance with your slice.

If you need further instructions or help, just let me know and I can grab screenshots and give better directions.

Thanks Shaun for the follow up..

I was walking through the step-by-step protocol you provided that I found out a very silly mistake. People in polymer characterization lab run the calibration standards once in a while and everyone else does the quantitations based on their calibration curves. However, none of their methods worked for my particular polymer and therefore I had to modify the acquisition method to cover the wavelength that my polymer has absorbance (PDA set to 220-800 instead of 254-800). As a result, when I tried to check the calibration curve that is applicable to my results, I didn't find anything.

So, I just prepared the polystyrene standards to make a new calibration curve. It's currently running with the same method that I ran my actual samples. Hopefully, it will do the trick. I'll keep you posted.

Thanks Again

Sounds like you have everything under control then

Please post back if there are any problems.

Just figured it out.
As I said, the problem source was the different detector settings I was using to acquire the data than the one that calibration curve was prepared with. So, at the wavelength I ran my samples, there was no applicable calibration curve and as a result the interface could not quantitate the molar mass distribution. 

Simply ran a set of standards by the same method and it worked like a charm.

Thanks again Shaun for helping out.