PVA-based buffering membranes with tunable pH values were prepared on a PVA substrate by reacting PVA, glycerol-1,3-diglycidyl ether, -NH2 group-containing buffers and -NH2 group-containing titrants in the presence of sodium hydroxide. The pH of the buffering membranes could be tuned in the 3 < pH < 10 range according to the Hendersson-Hasselbach equation while maintaining buffering capacities in excess of 100 mM. Detailed recipes for the preparation of six families of buffering membranes are tabulated and provided as Supporting Information. The buffering membranes were used to trap and desalt ampholyte solutions and separate proteins having a Delta pI as small as 0.1. The membranes were mechanically and hydrolytically stable and could be stored, even in 10 < pH sodium hydroxide solutions, in excess of 6 months.