Preparation And Chromatographic Characteristics Of The Novel Aminocarboxy Multidentate Metal Chelate Cu(II) Columns

According to the effects of stability constants between chelating agents and Cu2+ as well as dentate numbers of ligands on chromatographic characteristics, we selected a series of aminocarboxy chelating agents. Such ligands which are the chelating adsorbents with the versatile and multipurpose can ensure the lower leakage of Cu2+ and effective elution and separation of proteins on the metal chelate Cu(II) columns. The aminocarboxy chelating agents can be used as cation exchangers to separate positively charged proteins, remove metal ion and as metal chelate columns to isolate proteins with the affinity for metal ions. Therefore, the chromatographic characteristics of proteins on these stationary phases, especially for metal chelating properties, were studied in details. These studies may provide the great significance for expanding the range and development prospects of the application and market efficiency of immobilized metal affinity chromatography (IMAC).Based on the above ideas, glutamic acid-bonded silica (Glu-silica), glutamic acid-bromoacetic acid-bonded silica (Glu-BAA-silica) and glutamic acid-bromosuccinic acid-bonded silica (Glu-BSUA-silica) columns with cation-exchange properties have been synthesized using L-glutamic acid, glutamic acid-bromoacetic acid, acid-bromosuccinic acid of the aminocarboxyl agents as ligands and silica gel as matrix. The standard protein mixture was analyzed and separated with the series of synthesized multidentate chromatographic columns, and compared to iminodiacetic acid (IDA) column which is one of the most commonly coloumns in the current market. According to the physical and chemical properties of stationary phases and the effects of solution pH and salt concentration on the retention of proteins, the electrostatic interaction characteristics with cation-exchange characteristic of the three kinds of multidentate ligand bonded-silica columns were demonstrated. The number of multidentate ligands had a little effect for the cation-exchange properties of the synthesized columns. In addition, separation performance of the novel Glu-silica coloumn for proteins was better than the traditional IDA column. The experiment shows that the stationary phases exhibited the metal chelate property after fixing copper ion (â…¡) on the three kinds of columns. As fixing metal ion on the Glu-Cu(II) column, the influence of the different fixation methods and different pH values on binding capacity of Cu2+ and stability and the bonded density of immobilized metal ion for the coordination of metal chelate column were examined. The relationship between the number of multidentate ligands and binding capacity of Cu2+ was studied. According to the immobilized metal ion, pH of solution, salt concentration and the type and concentration of competitive agents, the chromatographic behavior of proteins and the leakage rate of immobilized metal copper ion (â…¡) were investigated. The optimum conditions with effective separation for proteins and the lower leakage of Cu2+ were discussed. The influence of separation condition and the leakage rates of Cu2+ for the stability constant of ligands were studied, and compared with that of the traditional IDA-Cu(II) column. The results indicate that the method of dynamic method for fixing metal Cu2+ in the pH 5.0 NaAc-HAc was optium. The binding capacity of Cu2+ increased with increasing the dentate number of ligands. According to the established chromatographic conditions, protein mixtures were effectively separated on the three kinds of multidentate chelate Cu(II) columns. Comparatively speaking, the ligand of quinquedentate Glu-BSUA which is the biggest dentate number was the best choice of chelating agent. The experiment proved that the elution and separation of proteins were related with the dentate number of ligands. The dentate number of ligands had greater affects on the chromatography characteristic of the metal chelated columns. In addition, the ligand with higher chelate stability constant is not always the best choice. Separation performance of Glu-Cu(II) column for proteins was superior to that of the IDA-Cu(II) column. Under a proper eluting condition, less amount of copper ion was leaked from Glu-Cu(II) column as compared to IDA-Cu(II) column.These studies may provide reference values for solving the leakage of immobilized metal copper ion (II), reducing heavy metal pollution in the process of protein separations and purifications, and expanding the application of IMAC.