Research On Modification Of Hydroxyethylcellulose By Grafting And Its Application In Separation Of Protein And DNA

Capillary electrophoresis (CE) is one of the most significant techniques for the analysis of charged biomacromolecules such as proteins and DNA and so on. It offers high separation efficiency, rapid analysis time, and low consumption of samples and ease of automation. When the CE is applied to separate proteins, the electrostatic interaction between positively charged basic proteins and the negatively charged capillary wall induced by ionized silanol group on a silica surface (pH>3) leads the strong adsorption of protein onto the capillary surface. Furthermore, the peak tailing, band broadening, low separation efficiency, and irreproducible migration time and even their irreversible adsorption with loss of samples are also brought by this electrostatic interaction. Coating the capillary with polymer solution has been proved to be a very efficient and simply approach to control the wall-protein interactions. The capillary can be modified with polymers by covalent bond or physical adsorption. The procedures employed to prepare a covalent coating are laborious and time consuming. However, physically adsorbed coating becomes more attractive due to its fast coating procedure and reproducibility.High-sieving ability and self-coating ability is very important for the sieving matrices which are applied for dsDNA separation by CE. At present, among non-gel sieving matrices (i.e., noncross-linking polymer solutions), linear polyacrylamide (LPA) with high molecular weight (MW) possesses high sequencing ability and long read length. However, high-MW LPA solution is very viscous and has no self-coating ability. On the contrary, poly (N, N-dimethylacrylamide) (PDMA) shows excellent self-coating ability but offers relatively poor sieving performance. Therefore, searching for the sieving matrices with low viscosity, high-sieving ability and self-coating ability is still an important issue for high-throughput DNA analysis. A lot of copolymer solutions have been developed and tested as sieving matrices because the existing homopolymer solutions can hardly meet all the separation expectations for CE. Copolymers can be provided with satisfactory abilities resulted from different homopolymers.In this dissertation, a range of grafted copolymers based on hydroxyl- -ethylcellulose (HEC) were synthesized according to different separation purposes. We design work on the following aspects: 1. Preparation and characterization of grafted copolymers based on HECHydroxyethylcellulose-graft-poly (N, N-dimethylacryl- -amide) (HEC-g-PDMA) and hydroxyethylcellulose-graft-poly ((N, N-dimethylamino) ethyl methacrylate) (HEC-g-PDMAEMA), were synthesized by using ceric ammonium nitrate (CAN) initiator in aqueous nitric acid solution. This kind of polymer is very attractive because of the commercial availability of HEC, the ease of the copolymer synthesis route, and purification process. The copolymers were characterized by 1H NMR and FTIR and the results demonstrate the preparation of HEC-g-PDMA and HEC-g-PDMAEMA.2. HEC-g-PDMA for protein separation by CEBoth the backbone HEC and the grafted chain PDMA of HEC-g-PDMA are provided with self-coating ability and especially the rather hydrophobic PDMA makes the polymer coating have excellent stability. HEC-g-PDMA was shown to be capable of stabilizing and suppressing the EOF. Meanwhile, highly efficient (105 plate/m) and rapid separation of three basic proteins (i.e. Cytochrome C, Lysozyme, Ribonuclease A) has been obtained over the wide pH range of 3-6. The best separation performance was achieved when the highest grafted density HEC-g-PDMA-3 was applied to separate proteins. Furthermore, the Quartz Crystal Microbalance (QCM) was employed to quantificationally characterize the ability of HEC-g-PDMA for resisting protein adsorption.3. HEC-g-PDMA for dsDNA separation by CEHEC-g-PDMA solution can be potentially applied to separate dsDNA because both the backbone HEC and the grafted chain PDMA of HEC-g-PDMA are provided with sieving ability. Additionally, the grafted PDMA has excellent dynamic coating ability which is important to stabilize and suppress the EOF during the separation. The effects of HEC-g-PDMA solution concentration, the grafted density of the copolymer, and the applied electric field strength on dsDNA sample (Φ×174/HaeIII digest) separation performances were studied in detail. The best separation result was achieved when the lowest grafted density HEC-g-PDMA-1 was applied to separate dsDNA. However, high resolution polymer solution simultaneously possessed high viscosity and long migration time for HEC-g-PDMA-1.4. HEC-g-PDMAEMA for protein separation by CEHEC-g-PDMAEMA coating was employed to separate proteins over the pH range of 3-8. The grafted PDMAEMA chain of HEC-g-PDMAEMA is pH sensitive and protonates in low pH. The amount of charge of PDMAEMA can be varied by adjusting the pH of buffer for protein separation and the hydrophilicity of HEC-g-PDMAEMA can also be varied by adjusting the ratio of backbone (HEC) to grafted chain (PDMAEMA). In this way, a favorable EOF can be realized for a special separation purpose by adjusting the amount of charge of PDMAEMA. Therefore, high efficient protein separation can be achieved because of the application of the highly stable and hydrophilic HEC-g-PDMAEMA coating.