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Preparation of immobilized pH gradients spanning 2–6 pH units with two‐chamber mixers: Evaluation of two experimental approaches

Alternative procedures for the preparation of wide‐range immobilized pH gradients with two‐chamber mixers are compared. In the first approach, both vessels contain the same mixture of buffering species, with the addition of either a strong acidic or a strong basic titrant; with the second protocol, the concentration of the buffers is different in the two chambers. The formulations for twenty 2–6 pH unit‐wide ranges were computed according to both methods. Relevant parameters of the resulting gradients are listed, including deviation from pH linearity, buffering power, ionic strength and efficiency. The effect of mistakes in the preparation of the gradients is also discussed. “Different concentration” gels allow for a better linearity of the pH course; “same concentration” gels offer a more even buffering power and ionic strength, and assure better reproducibility, in consequence of both the reduced number of manipulations and their higher resistance to mistakes. Reagent consumption and efficiency are very similar in both cases.