Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Methods for Determining Protein Molecular Weight
Determination of Molecular Weight by Osmotic Pressure

The relationship between molecular weight (MM) and the Osmotic Pressure of a highly dilute solution is described by the van 't Hoff equation:

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where c_Б is the mass concentration of the solute in g/L, П is the osmotic pressure, and R and T are the universal gas constant and absolute Temperature, respectively.

The van 't Hoff equation holds true only for ideal solutions (solutions that are very dilute, approaching infinite dilution). Therefore, in experimental practice, the osmotic pressure is measured at various protein concentrations in the solution. The obtained data are plotted on a graph, and the molecular weight is determined by extrapolating to zero protein concentration.

In principle, the Procedure for determining molecular weight via osmotic pressure is straightforward and backed by a rigorous theoretical foundation. However, practical Structure/175.html">Implementation often presents significant challenges. First of all, it is essential to ensure that the protein solution is free of low-molecular-weight impurities capable of passing through the semipermeable membrane. The diffusion of such impurities through the membrane would lower the measured osmotic pressure due to the generation of an additional osmotic pressure directed in the opposite direction (from the solvent side). Furthermore, many complications in determining molecular weight by osmotic pressure can be avoided by conducting measurements at the isoelectric point.

To measure osmotic pressure, one can use the very simple Pfeffer apparatus shown in the figure. The osmotic pressure exerted by the solution inside the tube is generated by the solvent penetrating through the semipermeable membrane and is equal to the height of the liquid Column (h).