Can I use the BEH125 SEC Protein Standard Mix (186006519) to create a calibration curve on the ACQUITY UPLC Protein BEH SEC 125Å, 1.7 µm, 4.6 mm x 150 mm column (186006505)? - WKB228431

ENVIRONMENT

• UltraPerformance Liquid Chromatography (UPLC)
• Bridged ethylene hybrid (BEH)
• Size exclusion chromatography (SEC)
• Protein separation
• BEH125 Protein Standard
• ACQUITY UPLC Protein BEH SEC

ANSWER

No

ADDITIONAL INFORMATION

You cannot determine the molecular weight of an unknown protein based on any type of calibration curve.
The reason for this is that two proteins can have the same molecular weight but different sizes (hydrodynamic radii) in solution.
Consequently, you cannot use a calibration curve for biomolecules like you can for polymers.

The calibration curves Waters publishes for SEC columns are not the same as calibration curves for GPC columns.
The published SEC calibration curves are made simply to show what the upper and lower limits are for that column based on pore size.
It is based not really on MW but on hydrodynamic radius of the protein. Because we know the MW of the proteins tested, we can state the MW range.

ACQUITY UPLC BEH SEC Columns and Standards Care & Use Manual

Guide to Size-Exclusion Chromatography (SEC) of mAb Aggregates, Monomers, and Fragments