External gradient chromatofocusing
The invention provides novel methods for the separation of charged molecules such as proteins according to the isoelectric points (pI's) and includes the systems and buffering compositions employed for isolating charged molecules. The invention further provides for modifications to the above described chromatographic methods that enable the separation of charged molecules exhibiting virtually identical pI's by shifting both the buffer's pKa and the pI's of the eluted charged molecules while they are traversing the ion exchange column.
1. 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.
2. The composition of claim 1 , wherein each of the at least three buffering components contains at least one functionality selected from the group consisting of amino, amido, imino, imido, carboxylic, sulfonic, phosphoric and phosphonic.
3. The composition of claim 1 , wherein each of the at least three buffering components is present in a concentration of 0.001 mM to 1,000 mM.