Dynamic cross-linking effect of Mg²⁺ to enhance sieving properties of low-viscosity poly(vinylpyrrolidone) solutions for microchip electrophoresis of proteins

We report a dynamic cross-linking effect of Mg²⁺ that enhances the sieving properties of low-viscosity poly-(vinylpyrrolidone) (PVP) solutions. A low-viscosity PVP solution was applied to nondenaturing microchip electrophoresis of protein samples using microchips made of poly(methyl methacrylate). The separation resolution of nondenatured protein markers in 1.8% PVP solution was improved by adding 1-20 mM MgCl₂. We studied the effect of the ratio of cross-linking agent on mobility of protein samples and showed that protein retardation (In μ/μ₀) is correlated with the ratio of cross-linking agent to PVP ([c^Mg2+/c_PVP]) as In μ/μ₀ = A′[c_Mg2+ /c_PVP] ^b′. A′ was related to the protein radius (R), and b′ was found to be 0.72 for proteins with R = 2.4 nm and 0.82 for proteins with R = 1.85 nm. A structural study of PVP in semidilute solutions using dynamic light scattering showed that incremental increases of Mg²⁺ ion concentration from 5 to 20 mM in 1.8% PVP solution increased the hydrodynamic radius of PVP polymers by 20%.