Molecular Weight Determination by Counting Molecules

Molecular weight (MW) is one of the most important characteristics of macromolecules. Sometimes, MW cannot be measured correctly by conventional methods like gel permeation chromatography (GPC) due to, for example, aggregation. We propose using single-molecule spectroscopy to measure the average MW simply by counting individual fluorescent molecules embedded in a thin matrix film at known mass concentration. We tested the method on dye molecules, a labeled protein, and the conjugated polymer MEH-PPV. We showed that GPC with polystyrene calibration overestimates the MW of large MEH-PPV molecules by 40 times due to chain aggregation and stiffness. This is a crucial observation for understanding correlations between the conjugated polymer length, photophysics and performances of devices. The method can measure the MW of fluorescent molecules, biological objects, and nanoparticles at ultimately low concentrations and does not need any reference; it is conformation-independent and has no limitations regarding the detected MW range.