Metal chelate-interaction chromatography of proteins with iminodiacetic acid-bonded stationary phases on silica support.
An iminodiacetic acid (IDA)-bonded stationary phase on a wide-pore microparticulate silica support was used for the chromatography of amino acids and proteins at pH 5.0 and 6.0. Without chelated metal the retention behavior of the stationary phase parallelled that of other silica-bound cation exchangers used in high-performance liquid chromatography of proteins. In metal chelate-interaction chromatography (MCIC) with IDA, chelated by Cu(II), Zn(II), Ni(II), Fe(II), or Fe(III), amino acids were most strongly retained on Cu(II)-IDA, whereas all metal chelates separated the proteins under investigation but with different selectivity. The effect of salt concentration in the eluent on protein retention was investigated and the pertinent electrostatic and hydrophobic interaction parameters were evaluated. The proteins were separated by MCIC with increasing salt gradient and, using the same column, by hydrophobic-interaction chromatography with decreasing salt gradient. In MCIC the addition of methanol to the mobile phase had disparate effect on protein retention, whereas addition of histidine or glycine, which acted as competing ligands, reduced the retention.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000032:analysis*; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D006736:Horses; D007098:Imino Acids; D000432:Methanol / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012822:Silicon Dioxide
D000596:Amino Acids; D002614:Chelating Agents; D007098:Imino Acids; D011506:Proteins; D012822:Silicon Dioxide; C008109:iminodiacetic acid; D000432:Methanol