Capillary Electrophoresis And That Coupled With Chemiluminescence Detection In The Determination Of Polyphenols

Capillary electrophoresis (CE) coupled with chemiluminescence (CL) detection have certain advantages of high separation efficiency and sensitivity. In this dissertation, the instrumental development and application of CL detection in CE were reviewed. In the last 10 years, the extensive work of CE-CL was studied. However, one of the most formidable challenges in applying CL detection to CE is the on-line introduction of CL reagents into the separation system. Suitable interfaces are very important to establish a successful CE-CL system. In this paper, a novel interface for CE combined with CL was developed and utilized to separate polyphenols. A kind of micellar electrokinetic capillary chromatography (MEKC) using polyoxyethylene sorbitan monolaurate (Tween 20) was introduced for the simultaneous determination of five polyphenols also. At last, a microcolumn was prepared by filling with quartz sand of 120-150μm in a 2.2 mm i. d. quartz tube and adopted to separate amino acids. The research topics are discussed in detailed as follows:1.A novel on-line chemiluminescence detector was designed for capillary electrophoresis. A gap interface with height injection was made by two capillaries, which were placed in close proximity by careful micropositioning, and the distance between the capillaries was 50μm approximately. The preparation processes of the gap interface were easy with an oriented capillary, which was eliminated after the gap preparation. The gap interface was carefully inserted into a 1.0 mm i. d. quartz tube, as a detection window situated just in front of a photomultiplier tube (PMT), and a reagent tube surrounded by CL reagent solution.A high potential buffer reservoir was constructed from a running buffer cell and an electrode buffer one, which were jointed with a frit, in order to avoid the luminol electrolysis, the excursion of chemiluminescence baseline, and the increase of electric current in CE-CL.2.The CE-CL system was employed for the determination of four polyphenols in cigarette samples. The detection limits (S/N=3) of rutin, chlorogenic acid, quercetin and caffeic acid were 3.7×10^-9, 6.4×10^-8, 8.5×10^-8 and 1.3×10^-10mol/L, respectively, two order magnitude lower than those reported in the previous work.3.A simple and convenient method of MEKC using Tween 20 as single micelle and methanol as buffer additive was introduced for simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% (v/v) methanol was adopted in the separation. For rutin and esculetin were difficult to be separated by capillary zone electrophoresis (CZE) and SDS-based MEKC, Tween 20-based MEKC was used and two polyphenols were separated satisfactorily.The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily. The equilibrium constant K of Tween 20 and rutin (or esculetin) is calculated to be 4.53 ×10⁵ (2.23 ×105) L²/mol² by a differential spectrophotometry, respectively. And both of the binding numbers were 2. It shows that the satisfied separation of the polyphenols was achieved by the different interaction between Tween 20 and rutin (or esculetin) and the different mobility.The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The average recoveries of the polyphenols ranged from 95% to 112%. It means that the proposed method is suitable for the simultaneous determination of the polyphenols in the herbal samples.4.To overcome the disadvantages of CE due to its small inner diameter, such as poor detection sensitivity and low sample loadability, a microcolumn was prepared by filling with 100-125 μm quartz sand in a 2.2 mm i. d. quartz tube. The effect of electric field strength and quartz sand size on electroosmotic flow rate was studied. Nylon films were used firstly as frits. In this dissertation, the separation results of two amino acids were satisfying with the microcolumn and the detection limits (S/N=3) were improved, of which tryptophan and histidine were 0.30 and 0.27 mg/L, respectively. The electrophoretic separation can be developed as a complementary method for CE to improve the sample capacity and concentration sensitivity.