| Environmental problems have been posing a great challenge to separation technology. Microflow or nanoflow chromatographic methods have attracted strong interest. While there is a lot of problems when comes to the particulate-packed capillary columns which are the traditional columns used in HPLC. The appearance of capillary monolithic columns put forward a completely significantly alternative for traditional packed capillary column. But its development is still in the very beginning step. Therefore, novel monolithic columns were developed and their applications have been studied. The main contents include the following four parts:1 . A new poly (styrene-octadecene-divinylbenzene) (PS-OD-DVB) monolithic column was simply prepared by in-situ polymerization of styrene, DVB and octadecene with DMF and decanol as porogens in one step. It was found that this kind of monolithic column had a total porosity of 85%, perfect mechanical intensity and adequate loading capacity. Compared with PS-DVB monolithic column, PS-OD-DVB monolith showed similar ability for the fast separation of six proteins in 2.5 min, and both the PS-DVB and PS-OD-DVB monolithic columns have good durability for high pressure and have good reproducibility after repeated injections. Batch-to-batch reproducibility of column fabrication was good with RSD below 9.5%. Moreover, the experimental results indicated that the PS-OD-DVB monolith showed higher loading capacity and provided better resolution for the separation ofαandβchains of hemoglobin than the PS-DVB monoliths because of the importing of C18 chain. These results make it reasonable to believe that this kind of column is promising in fast and highly effective separation of protein and has great potential for separation of complicated bio-macromolecule samples.2. Eeffect of inner diameter of capillary monolithic column on separation of macromolecules inμ-HPLC. PS-DVB monolithic columns with different ID from 100μm to 320μm were prepared and used for separation of standard proteins. The effects of the ID on various column parameters were studied. It was found that the smaller the column diameter is, the higher the permeability. As to the separation efficiency, it was evident that more theoretical plates can be achieved in less time using the smaller diameter columns. It is interpreted that the interstitial structure of the pores formed during the polymerizing of the polymer matrix depends a lot on the ID of the monolithic column, and the monolithic matrix in-suit polymerized in column with smaller diameter consist of larger clusters and through pores. From the Van Deemter equation simulated for each column, it was found with the increase of the column ID, there is an overall increasing trend for the parameters for eddy dispersion, longitudinal infusion and mass transfer. The monolithic column with samller ID which has larger clusters and through pores gives better performance for protein sparation. These results and discussion will be helpful in practical use of monolithic column for separation of biomacromolecules and development of the theory of the monolithic column.3. Two kinds of polymethacrylate-based monolithic columns, prepared with or without 2-acrylamido-2-methyl-1-propanesulfoni acid (AMPS) in fused silica capillary, were used for rapid analysis of three cyanobacterial toxins (MC-LR, MC-YR and MC-RR) inμ-HPLC and pCEC systems. Under the optimal gradient elution conditions, polybutylmethacrylate monolithic column inμ-HPLC allows the separation of these toxins in less than 9 min with good reproducibility, recovery and satisfied LODs. For the column prepared with AMPS, introduction of sulphonic bands led to a mixed retention mechanism inμ-HPLC for separation of MCs. After optimizing the separation conditions, this method could separate three MCs in 5 min. In pCEC system, the supplementary high pressure could effectively suppress the bubble formation and shorten analysis time. Under the optimized conditions, three MCs could be baseline separated in less than 6 min in an isocratic elution mode. Compared with MCs separation inμ-HPLC, pCEC collude improve the efficiency obviously and gradient elution could also be avoided. pCEC is a flexible and versatile solution to method optimization studying, and there is a great potential that this system could be used as a fast separation tool for routine use in microcystin monitoring.4. A new monolithic column was prepared by a home-made monomer 11-acrylamidoundodecanoic acid (AAUA) and EDMA. The optimization of the preparation was carried out with the assistant of experimental design software. A D-optimal design was performed to evaluate the effect of preparation mixture (concentration of crosslinker, monomer and progens) and optimize the preparation solutions. This method allows one to obtain appropriate data that can be analyzed to study the individual effects and the interaction effects of several factors as well as to determine the optimum conditions through a relatively smaller number of experiments. It was found that the experimental results were in a good aggrement with the predicted value from the software, which proved that this optimization method is successful. The optimized column could provid satisfactory separation of alkylbenzene and alkyl phenyl ketone homologous with good reproducibility. Separation of seven N-methylcarbamates (NMCs) pesticides was also preliminarily studied and a satisfied result was achieved.
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