I've been attempting to use a Zetasizer Nano to measure the second virial coefficient of BSA, but my measured molecular weights do not change linearly with respect to concentration. Instead, they seem parabolic or exponential depending on the conditions and concentrations.
I haven't been able to find any references or even troubleshooting regarding this problem, and I'm at a loss as to what could be causing the nonlinearity. My dn/dc is set to 0.165 for BSA, I added a spherical shape correction, and I've tried different concentrations ranging from 24 to 0.1 mg/ml. I also measured as many concentrations as possible at 280nm, so I know my dilutions are reasonably accurate.
I've also taken the 2nd virial coefficient at various ~linear portions of my data (ex. the black dots, 5-11 mg/ml in my attached picture), but the resulting coefficient is still significantly different from literature values that use the same buffer/ionic strength.
Has anyone ever had reproducibly non-linear 2nd virial coefficient data?
Hello Avril,
static light scattering can be tricky. My first suggestion would be to always also do dynamic light scattering at each concentration. This will allow better identification of what exactly may be happening. Potential effects could be electrostatic interactions at low ionic strength, multiple scattering at higher concentration, inconsistent measurement position.
Here are some tips that might be of use: https://www.materials-talks.com/blog/2017/11/02/guide-to-molecular-weight-with-the-zetasizer/
Also, don't hesitate to send a data file https://www.materials-talks.com/blog/2014/08/19/faq-how-can-i-submit-a-data-file-to-the-help-desk/ which could help narrow the cause of the observed curvature.
Hi Avril,
As Ulf has suggested, I would perform some size measurements just to check that you are measuring what you expect to.
The curved nature of your data is surprising, as your scattering data is mostly linear.The measurement of Mw, requires the scattering values for a reference scatterer, toluene, and your continuous phase with no BSA. These can be measured every time you do a measurement or stored values could be used.
I suspect that one or both of these values that you are using may be off, which is giving you the curve in the data.
Ulf Nobbmann and Alexander Malm , thank you both very much - I'll try that out and see if I can get some answers. Much appreciated.
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