Patent ID: 7790025

Claim:
A buffering composition for chromatographically separating charged molecules having different isoelectric points which performs reliably on both strong and weak anionic and cationic columns wherein the at least one buffering component is comprising an aqueous solution of at least three buffering components with or without additional buffering components with widely spaced but overlapping pKas such that when the composition at a predetermined pH is titrated with the composition at a different predetermined pH, the change in the pH of the mixture of the two compositions is proportional to the fraction of the mixture represented by each of the two compositions as characterized by a polynomial derived from titration data; and this polynomial is used to execute a preprogrammed chromatographic method through the software of a liquid chromatography system to either: supply to an anion exchange adsorbent an eluent formed with a time dependent pH formed from the continuous mixing of a solution at the initial pH containing the at least three buffering components with or without additional buffering components pumped out from a first reservoir with a solution at pH different from the initial pH containing the at least three buffering components pumped out from a second reservoir, wherein the mixing proportions vary to maintain an unretained pH gradient with an externally defined slope; or supply to an anion exchange adsorbent an eluent formed with a time dependent pH formed from the continuous mixing of a solution at the initial pH containing the at least three buffering components with or without additional buffering components pumped out from a first reservoir with a solution at pH different from the initial pH containing the at least three buffering components pumped out from a second reservoir, wherein the mixing proportions vary to maintain an unretained pH gradient with an externally defined slope wherein the at least three buffering components comprise piperazine, 1-methyl piperazine, triethanolamine, bis-tris propane, and formic acid.