Preparation And Investigation On Performance Of Functionalized Thermally Response Chromatographic Materials

In the research of life sciences, there are many important functional biomacromolecules, which have low abundance, but were hard to be separated and easy to be resolved. Chromatographic materials play an important role in the enrichment and separation of biological molecules. However it often require a high pH, a high salt concentration of mobile phase, even a competitive inhibitor added into the mobile phase, etc. All of these will make the biological samples inactivated. Thus it is the urgent for analysts to find a convenient, fast, and efficient method to obtain the target molecules. Fortunately, temperature responsive chromatographic materials choose only water as mobile phase, we could achieve separation and enrichment of the target molecules only need to change the temperature. A ligand of target molecule was introduced into the temperature responsive material, then the target molecules could be captured by it, when the temperature was changed, the target molecules could be released and acquired. In brief, the key is to make the research of the preparation method on functional thermo-responsive material.This research was based on the method of Atom Transfer Radical Polymerization, and the graft conformation of thermo-responsive chromatographic materials was optimized. Base on the above, two borate thermo-responsive chromatographic materials were prepared, which could easily seperate and enrich the cis-diol containing biomolecules. At the same time, in this research, we found one general preparation method of thermo-responsive chromatographic materials with amino ligands. Because ATRP was not apt for the preparation of some monomer, thus for purpose of finding a preparation with more broad application, we chose RAFT(Reversible Addition-Fragmentation chain Transfer) skill to assist this research. As following:1. In order to study the relationship between the structure of the grafted conformation and the performance of thermo-responsive chromatography, the thermo-responsive stationary phase with different grafting density under the same grafting rate were prepared. Through the study of the separation of steroids, both of 10% graft density and 14% grafting could make out a optimized stationary phase with a short retention time and a good performance of thermo-responsive and separation degree. It is noted that the grafted chain length was important in this preparation process, the same as the grafting rate.Four thermo-responsive stationary phase were prepared with a same chain lengthand but different graft density. According to the analysis of separation of steroids, we found that when the optimized graft density was 30%, the thermo-responsive performance and separation ability were the best.Thus, the graft conformation is also important for the thermo-responsive performance. At last, a stationary phase with better performance of thermo-responsive and separation was dependent on suitable graft density, adapt chain length and appropriate graft ratio.2. Nowadays, borate thermo-responsive chromatographic materials could seperate and enrich the cis-diol containing biomolecules under high pH conditions. However, this will disrupt the biomolecules, which were easy to be hydrolysed. The service life of stationary phase was shortened. Thus, to get the separation under moderate condition, we prepared AAPBA grafted thermo-responsive chromatographic materials. A separation and enrichment testing of adenosine and deoxyadenosine indicated that the best pH range was pH 5.0 to pH 9.0.We also prepared DMAEMA and VPBA ones. They could only selectively capture the adenosine or rapidly release it at pH 7.0. The enrichment efficiency of adenosine was up to 89.8% by four times. And successfully enriched the sugar peptide converted from the digestion by HRP. And researched the performance in detail of DMAEMA and VPBA with different molar ratio to find that adapt ratio could got best performance.3. In the synthesis part, different ligands were used to prepare various function of thermo-responsive affinity chromatographic materials, and the ligands should contain amino groups or post-synthesized ones with amino groups. To obtain a general thermo-responsive chromatographic materials, thermo-responsive chromatographic materials containing N-acryloyloxy succinimide(NAS) was synthesized. After that, through the replacement reaction of amino and succinimide, the amino could be introduced. In this research, the thermo-responsive affinity chromatography contatining APBA was prepared. Normal polymerization conditions were also investigated by a selective separation test of different temperatures of cis-diol containing structure analytes, found that the prepared stationary phasenot only could selectively capture and release adenosine, but also can be used to the extraction of cis-diol containing structure analytes from plants.4. ATRP reaction is important in the preparation of functional thermo-responsive materials, but the application of ATRP reaction was not wide, the preparation of thermo-responsive materials containing acid functional groups need novel methods. RAFT method was used to prepare the thermo-responsive material. The graft density and graft ratio of the polymer were controlled by the introduction of 2-acetyl amino acid, which could help control the density of chain transfer agent on the silicone surface. Through the study of the characteristics under different temperature, the grafting density and the graft ratio were optimized. This method has strong controllability and mild application condition. Additionally, it provides us a new preparation method of more functional thermo-responsive chromatographic materials.