pH gradients generated by polyprotic buffers. II. Experimental validation

The experimental validation refers to the computer program reported in the companion paper, able to simulate the course of pH, buffering power (beta) and ionic strength (I) of polyprotic buffers (either singly or in a mixture) titrated over any pH range. With simple oligoamines (up to five nitrogens) it is shown that it is impossible to generate linear pH gradients in the pH 4-10 interval, unless they are mixed in appropriate ratios. With pentaethylene hexamine, when used alone, it is possible to create a linear pH 4-10 interval, provided the molarity ratios are altered in the two chambers of the gradient mixer. The general rule operating for generation of linear pH intervals is constancy of buffering power throughout the titration. Local minima of beta produce steeper gradients, while local beta maxima flatten it. The ideal delta pK to arrange for linear pH gradients during titration is centred around 1 pH unit; thus polyprotic buffers with very large delta pK values (e.g., EDTA) appear to be totally useless for this purpose. The present computing algorithms should be quite efficient for optimizing existing buffer recipes for chromatofocusing or ampholyte displacement chromatography or for creating new, properly tailored, buffer mixtures.