One-step coating of silica capillaries for selective protein retention by Cu(II)-IDA IMAC.

A simple protocol to obtain Cu(II)-IDA (iminodiacetic acid)-modified capillaries was developed for immobilized metal ion affinity chromatography (IMAC). It consisted in the synthesis of IDA-silane used for a one-step coating of fused silica capillaries. The approach prevented the hydrolysis of silica potentially induced by two step coatings (γ-GPTMS, then IDA) employed in the conventional method of bonding iminodiacetic acid. The IDA content was quantified using a model relating the electroosmotic flow generated in IDA-modified capillaries to the charges induced by IDA species. The retention behavior of holotransferrin and bovine serum albumin on these IMAC columns was then investigated. Holotransferrin revealed a high affinity for Cu(II)-supports through a specific interaction with Cu(II) ions whereas albumin did not show any retention. The use of such columns for sample pretreatment before an HPLC analysis was proved to be successful.