Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion‐exchange adsorbents

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Abstract
Although it is commonly believed that a column packing used for chromatofocusing must have an “even” buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong‐acid cation‐exchange column packing or a strong‐base anion‐exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak‐acid or weak‐base functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using chromatofocusing and a strong‐acid cation‐exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ion‐exchange chromatography using a salt gradient for this purpose.