Ion Exchange Chromatography Buffers
The goals of this exercise are to demonstrate how ion exchange chromatography works and at the same time to point up the rationale for choosing ion exchange buffers. For many of the buffers biochemists use, the acid or base form is electrically neutral and the other form is either anionic or cationic. The mode of chromatography used (more frequently anion exchange) determines which of these two possibilities is preferred. I am frequently surprised to see researchers who should know better indicate use of inappropriate buffers in published work.
I only did this once and it could use some touching up. The matrices I used were Dowex-1 (anion exchanger, chloride form) and Dowex-50 (cation exchanger, sodium form). I don't remember the mesh size, but the larger mesh number (smaller beads) the better. I made dilute (20 mM) buffer solutions (pH 7.5) using either triethanolamine-HCl ("Buffer A") or Na-HEPES ("Buffer C"), and identical buffers containing 2 M NaCl ("Buffer B" with triethanolamine and "Buffer D" with HEPES).
Students prepared minicolumns (1 mL bed volume) of Dowex-1 and Dowex-50. They washed the Dowex-1 column with 5 mL of Buffer B, then 5 mL of Buffer A to equilibrate it to pH 7.5, discarding the washes. Then they put over it, sequentially and collecting the run-through, 5 mL of Buffer A, Buffer B, Buffer A, Buffer C, Buffer B, and Buffer B. They finish by determining the pH of each fraction with a pH meter. The Dowex-50 column is treated the same way, except the washes are with Buffer D, then Buffer C, and the collected fractions are Buffer C, Buffer D, Buffer C, Buffer A, Buffer D, and Buffer D. The expected results are as follows:
Students are finally asked to explain any "unexpected" pH values. They should recognize that the pH of Buffer C will be decreased by passing through Dowex-1 because the anionic (basic) form of the buffer will be retained by the matrix and will subsequently be washed out by Buffer B, giving it a pH somewhat above 7.5. Similarly, the pH of Buffer A will be increased by passing through Dowex-50 because the cationic (acidic) form of the buffer will be retained by the matrix and will subsequently be washed out by Buffer D, giving it a pH somewhat below 7.5.
In fact, the pH changes the students observed were not as large as I had hoped, and the pH of untreated buffers didn't seem too stable. This could be corrected by adjustment of buffer concentrations, buffer/column volume ratios, finer Dowex mesh, or pH meters themselves. I still think this is a nice exercise, because it is simple to set up and to do, and kills two pedagogical birds with one stone.
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