Immobilized pH gradients: New pK values of acrylamido buffers in poly(N‐acryloylaminoethoxyethanol) matrices

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Abstract
A novel matrix consisting of N‐acryloylaminoethoxyethanol, a hydrophilic monomer extremely resistant to hydrolysis, was recently reported by Chiari et al. (Electrophoresis, 1994, 15, 177–186). When using it as a matrix for grafting immobilized pH gradients for isoelectric focusing, a shift in protein spot position was noticed. This was attributed to a shift in pK values of the Immobiline buffers when changing from the standard poly(acrylamide) to a poly(N‐acryloylaminoethoxyethanol) matrix. A series of 1 pH unit gradients was constructed, where a single buffering Immobiline was used and titrated with a counterion having a pK removed by at least 3 pH units from the nearest extreme of the generated pH interval. It was noted that all compounds became weaker acids and bases, with a ΔpK ranging from −0.02 to −0.06 for the acids (pK 3.6, 4.4, and 4.6) and a ΔpK ranging from −0.12 tp −0.20 for the bases. The new pK values for the seven commercially available buffers are thus pK 3.6 ‐> pK 3.58, pK 4.4 ‐> pK 4.36, pK 4.51 ‐> 4.45, pK 6.21 ‐> 6.09, pK 7.06 ‐> 6.94, pK 8.50 ‐> pK 8.37, and pK 9.59 ‐> pK 9.39. These values refer to 10°C in the gel phase, the first value in poly(acrylamide) and the second in poly(N‐acryloylaminoethoxyethanol).