| Fundamental studies and application of a separation process called centrifugal precipitation chromatography in fractionation of host proteins, RNA and plasmid DNA, which were components in clarified lysate produced from bacterial culture, have been conducted in this dissertation.; First of all, the dispersion coefficient of the process was determined by monitoring and recording elution profile of tracer eluting out of the sample channel of centrifugal precipitation chromatography unit. After that, the suitable precipitant was selected. It was discovered here that 2-propanol, which is routinely used for DNA precipitation, could cause a transition from supercoiled to open-circular plasmid DNA, especially at high concentration, so it was not selected since supercoiled plasmid DNA is more desirable than the open-circular one. Another two alcohols: ethanol and methanol were also experimented but appeared not to be able to selectively precipitate different components in the clarified lysate.; Fractionation of proteins, RNA and plasmid DNA in centrifugal precipitation chromatography was achieved using cationic surfactant cetyltrimethylammonium bromide (CTAB) with an addition of inorganic salt such as sodium chloride or ammonium chloride. The successful separation of these components were obtained in both bench-scale and preparative-scale centrifugal precipitation chromatography, despite of difference in type of gradient and the surfactant concentration used in both processes.; The mathematical model previously developed for explaining gradient formation of ammonium sulfate in centrifugal precipitation chromatography was adopted for gradient formation of the cetyltrimethylammonium bromide in centrifugal precipitation chromatography. Due to the larger of the surfactant molecule when compared to ammonium sulfate, lower mass transfer of the surfactant across the membrane was observed. Then, the mathematical model for predicting elution profile of the solute in centrifugal precipitation chromatography was developed. Good agreement between experimental data and numerical result was achieved; however, more realistic correlation of certain parameter could have been helpful for improvement of the model accuracy.; Finally, it was found in this research that it might be possible to separate two conformations of plasmid DNA, supercoiled and open-circular, by using other cationic surfactant trimethyltetradecylammonium bromide (TTAB) instead of cetyltrimethyl-ammonium bromide. |
