Study On The Preparation And Characteristic Of Polydentate Ligands IDS-silica Chromatographic Stationary Phase

As a chromatographic separation medium,immobilized metal affinity chromatography(IMAC)was often used for the analysis of biological macromolecular samples.But the problem of metal ion leakage in the use of IMAC column,that can lead to the contamination of the separated target material and reduce the efficiency of analysis.As a result,these restricted the application of IMAC.According to the constitution and action principle of IMAC,we found that the stability of IMAC column depends mainly on the complex stability of chelating ligand,metal ion and protein,and the chelating ligand is the key part of IMAC column.In recent years,some amino carboxylic chelating ligands have poor metal chelating ability.Based on these above problems,iminodisuccinic acid has attracted much attention,which is a new type of environmentally friendly aminopolycarboxylic acid chelating agent.Its metal chelating ability was similar to EDTA,which is often used in the adsorption removal of toxic metal.In this paper,IDS was fixed on Silica gel monomer as the chelating ligand for the first time.IDS-Silica stationary phase was successfully prepared by indirect synthesis method.We explored the optimal ratio synthesis of IDS-Silica stationary phase by using the method of p H potentiometric titration and HPLC experiments,which is IDS:Silica = 4.6:1.5.The stability order of IDS with divalent copper ion,ferri ion,divalent nickel ion,divalent zinc ion,bivalent cobalt ion,divalent calcium ion was evaluated by quantum chemical calculation and ICP-AES combined method.The results are all shown that IDS-Silica column had a dual characteristic of metal chelating and ion exchange properties when the IDS-Silica column fixed with these six kinds of metal ions.And we can predict the stability order of IDS with these six metal ions on IDS-Silica column,that is: Fe3+>Cu2+>Ni2+>Co2+>Zn2+>Ca2+.We can conclude that the metal chelating property of IDS is better than the other three kinds of ligands by comparing the binding amount of the metal Cu2+ ion on IDS,IDA,L-Asp and L-Glu this four chelating ligands.Finally,we study the stability constants of KML and the total bond number Λ0 on IDS-Cu(II)-Silica column with four proteins by using frontal chromatography(FC)method.Results showed that the stability of lysozyme(Lys)was the strongest in this four proteins on IDS-Cu(II)-Silica column,and the weakest adsorption was BSA.IDS is an aminocarboxy ligand,which has a strong metal chelating property.Fixing IDS on the silica matrix,we can not only prepare the IMAC with high stability,but also can widen the usig filed of IMAC.It is also proved that FC method is a new method which can be used to screen high stability IMAC column by measuring the adsorption stability parameters.