Analyses Of Several Bacteria And Their Metabolites By Capillary Electrophoresis

The research results are summarized as following: A new capillary electrophoresis method for determining Poly-β-hydroxybutyric acid (PHB) in Bacillus thuringiensis was established. Poly-β-hydroxybutyric acid in samples was hydrolyzed by sulphuric acid and neutralized by Ba(OH)2. The content of produced β-hydroxybutyrate was then determined by capillary zone electrophoresis (CZE) with indirect UV detection at 254 nm. With 5 mmol/L p-hydroxybenzate and 0.5 mmol/L tetradecyltrimethylammonium bromide (TTAB) at pH 8.0 as carrier electrolyte, β-hydroxybutyrate can be determined within 6 min. Standard regression equation was made by β-hydroxybutyrate, and the linear range was 2 to 1000 μg/mL. The relative standard deviations (RSDs) for migration time and peak area are both less than 1.0 %. The limit of detection for β-hydroxybutyrate was 0.2 μg/mL, which is 2 to 3 orders of magnitude lower than that of the gas chromatography (GC) method. The capillary electrophoresis method was successfully applied to determine Poly-β-hydroxybutyric acid in fermentation broth and single colony. The added standard recovery was 96 %. A new capillary zone electrophoresis method for the analysis of dipicolinic acid (DPA), a specific component found in spores but not in vegetative cells, was used to determine spore concentration in Bacillus thuringiensis according to the relationship between the spore concentration and the content of dipicolinate. The quantitative relationship was established by using purified spores. Electrolyte conditions that affected the separation efficiency of dipicolinate and the reproducibility were investigated. With 10 mmol/L phosphate, 10 mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.25 mmol/L tetradecyltrimethylammonium bromide (TTAB) at pH 6.2 as carrier electrolyte, dipicolinate can be determined within 8 min at an applied voltage of ?25 kV (anode at detector) and a capillary temperature of 25℃. The method has a high separation efficiency with which the number of theoretical plates is above 300 000 plates/m. The relative standard deviations for migration time and peak area are less than 0.5% and 2.0% respectively. The limit of detection for dipicolinate was 10 ng/mL, which corresponds to 7.2 ×105 spores/mL. The method was used to determine spores in fermentation broths, and the results obtained agreed well with the values obtained by plate counting. Validamycin A, a main component of the antibiotic validamycin complex which is widely used to control sheath blight disease of rice plants, can be determined by capillary zone electrophoresis with indirect UV detection. The influences of various separation conditions including background electrolyte and modifier concentration, pH and voltage were investigated. By using 10 mmol/L aminopyrine-2 mmol/L EDTA at pH 5.2 as carrier electrolyte, high efficiency separation of validamycin A was achieved with the number of theoretical plates up to 350 000 plates/m. Linear range was across three orders of magnitude. The relative standard deviations for migration times and peak areas are less than 0.5% and 3.0% respectively. The limit of detection for validamycin A was 1.0 μg/mL. The average recoveries were ranging from 103 to 104%. A method of capillary zone electrophoresis with direct UV detection for the determination of validamycin A was also established. With 100 mmol/L acetate at pH 4.7 as running buffer, separation voltage 15 kV, validamycin A in commercial products can be determined within 10 min. The method has high separation efficiency with which the number of theoretical plates are above 200 000 per meter. Linear range was across three orders of magnitude. The relative standard deviations for migration times and peak areas are less than 0.5 and 2.0% respectively. The limit of detection was 0.2 μg/mL. The mean percentage recoveries were ranging from 97 to 101%. These two methods have many advantages comparing with high performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MEKC). Linear polymer solutions with high concentration were employed as sieving buffer to separate DNA fragment. The concentrated polymer solutions usually have high viscosity, which was difficult to be pumped into or out of the capillary. In order to obtain satisfactory separation of smaller DNA fragment with low concentration polymer solutions, the additives such as glycerol,glucose and mannitol was tested. The addition of 10 g/L glucose to 5g/L hydroxyl-propylmethylcellulose solution in a Tis-Borate-EDTA background electrolyte can create entangled solutions and enhance the sieving ability. The smaller DNA fragment with length less than 1 000 base pairs can be separated satisfactorily. Like other colloidal particles, bacteria carry charged or chargeable groups on their outer surface creating an electric double layer upon contact with the aqueous phase. Therefore, bacteria undergo electrophoresis in free solution with their migration depending on the ionic strength and the pH of the buffer solution. Differential electromigration can thus be used to separate and characterise the intact cells. Bacillus thuringiensis and E. coli can be separated either in the zone electrophoretic or in the isoelectric focusing mode with high efficiency (up to 3 000 000 plantes/m in CZE). However, the relative standard devitions for peak area and peak high were not good enough.