Immobilized metal-ion affinity chromatography: Imidazole proton pump and chromatographic sequelae. I. Proton pump

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Abstract
Complexation of imidazole (Im) with an iminodiacetate (IDA) metal chelate [IDA‐M(II)] ligand of a chelating gel results in an acidification of the mobile phase. The scope of the action of this IDA‐M(II)Im ‘proton pump’ in IMAC is determined by: (a) IDA‐M(II) density of the gel; (b) concentration of applied Im; and (c) the buffering capacity of the mobile phase. Application of Im onto a metal chelate column in a gradient rather than in a stepwise manner, mitigates the proton pump's action, as it does an increase of buffer concentration in the mobile phase. However, only an antecedent conversion of the metal chelate gel, IDA‐M(II), to its Im derivative, IDA‐M(II) Im, can effectively circumscribe the action of the proton pump. The same holds true, as anticipated, when another chelating ligand (nitrilotriacetate) is used.

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