SEC/MALS method for molecular weight

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

4 posts Page 1 of 1

I have a question about SEC/MALS (aka GPC with in-line light scattering detector) analysis for polymer molecular weight. An SEC/MALS system was turned over to me, and I am interested in optimizing the method to produce the most accurate data. The system is used to analyze acrylic solution polymers in approx. the 4K - 200K molecular weight range. The column in use is a single Agilent Resipore GPC column (mixed-pore column w/ 3um particle size and a stated MW separation capability of up to 500K). The original column was old and I was measuring low theoretical plates, so I replaced it with a new one of the same model. Peak shapes for some polymers changed after column replacement, and some also changed again when I tried running them at half the standard concentration (33 mg/mL instead of 66 mg/mL). I don't know why the 66 mg/mL concentration was originally chosen, but from the look of the chromatograms below, I think the column may be getting overloaded for higher MW polymers. (Recommended concentration of a monodisperse polymer standard for GPC/MALS per ISO 16014-5 is 5-10 mg/mL).

What seems strange to me about the chromatograms is

1) the way the right side of the lower 33mg/mL concentration (green traces) peaks shifts to the left and becomes less steep when compared to the higher concentrations (Overloading effect?)

2) The way the chromatograms do not appear to indicate overloading at the 66mg/mL concentration on the old and presumably occluded column (red traces) for polymer A and B, but do appear to indicate overloading on the new column at the same 66mg/mL concentration for polymer A and B. Each run's number average (Mn), weight average (Mw), Z average (Mz), polydispersity, and online-calculated dn/dc are included. Note that any peak after about 9.5 min in these chromatograms is non-polymeric low molecular weight material that is NOT included in the calculations.

I'm wondering about the most appropriate concentration to use for our samples, and if different concentrations should be used for different molecular weight polymers. Any thoughts on this would be appreciated, thanks!

System parameters:
Eluent: THF (BHT-inhibited)
flow rate: 1mL/min
injection size: 20uL loop
column: one Agilent Resipore column (300 x 7.5mm, 3um particle size, mixed porosity)
new column theo plates/meter: 47500
old column theo plates/meter: 33000
LS detector: Wyatt miniDAWN (3 scattering angles)
RI detector: Agilent G1362 RID
software: ASTRA

Do you realize that you are only looking at noise and minor changes in area due to poor retention? Your estimated column volume is ~ 8 mls and your flow rate is 1 ml/min. That means that it takes eight minutes for a compound to not interact (be excluded) from the column during the run. Look at your Peak Rt where you are making comparisons. None of it is relevant. Oh and yes, you are probably overloading the column by 2-5X. However, that seems like a minor problem compared to training.
This is a size exclusion chromatography column. The volume of mobile phase in this system (void volume) is about 4.5 mL, so 4.5 min is the earliest possible elution time, and the point where a large unretained polymer would begin to elute.
If anyone knows of another forum or resource for expertise on GPC/MALS, I would appreciate it. Thanks!
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