For the count rate issue in your Zetasizer software, use high-quality Toluene, preferably optical-grade, for calibration. To calibrate Protein Molecular Weight, use a standard protein with a known molecular weight, determine its exact concentration, and perform serial dilutions to create a calibration curve correlating hydrodynamic radius to molecular weight. Search for relevant papers on "Zetasizer protein molecular weight calibration" for additional guidance. Remember to consult the instrument's user manual or seek expert assistance for accurate measurements. Good luck!

Hi Lassaad, thank you for the input. I'm trying to learn and understand this calibration at the moment. Any brand recommendation for standard protein reference I can use for?

Hello Amalia Qistina ,

the error message may be due to a bug in an earlier software version. Are you using the latest version 3.2.2.5 downloadable from https://www.malvernpanalytical.com/en/support/product-support/zetasizer-range/zetasizer-advance-range/zetasizer-ultra#software

Or your toluene may be contaminated and scatter too much light.

HPLC grade toluene would be good, there is also a sealed toluene cuvette available.

You can contact the global support for the Zetasizer at https://www.materials-talks.com/help-for-my-instrument-one-step-away/

There is no need to calibrate with any protein standard, only a one-time calibration with toluene is needed.

To make static light scattering measurements in the Zetasizer you typically would prepare a series of concentrations. There is some help in the software help and manual (both accessible from within your software on your computer). Additionally you may find this application note of some benefit as it describes an example https://www.malvernpanalytical.com/en/learn/knowledge-center/application-notes/an220627-protein-molecular-weight-dimer-zetasizer

PS: if you can share the toluene count rate in backscattering and attenuator 11 it might be useful.