Application Of HPLC With Post-column Chemiluminescence Detection To Alcohol Acid And Flavonoids In Plant Samples

Chemiluminescence(CL) is defined as the emission of light from an electronically excited state species which is produced during the course of a chemical reaction, and is observed when the electronically excited product or intermediate formed decays to the ground state with a photon emitted. There are many merits on CL such as high sensitivity, wide linear range, rapidity, simplicity and so on. High performance liquid chromatography (HPLC) has mostly been used to enhance the selectivity of analytical methods, because of its high efficiency for separation of analytes from various interfering substances. One of the major research areas about HPLC involves the development of sensitive detection methods. Significantly more sensitive detection can be realized by HPLC when the analytes are subjected to the use of chemiluminescence (CL) detection. The chemiluminescence (CL) detection system combined with HPLC separation method can offer excellent analytical selectivity and sensitivity. No doubtably, chemiluminescence linked to HPLC will be widely applied in environmental science, biological science, pharmaceutical science and clinical science.The thesis consists of two parts. One is a review related to the application of HPLC with post-column chemiluminescence detection to plant and pharmaceutical samples. Emphasis is taken on the development in the recent ten years.The other is research reports which is composed of 3 components as follows. 1) Simultaneous determination of alcohol acid in certain Taxodiaceae plant roots byHPLC using post-column chemiluminescence detection;2) Determination of the Levels of Nevadensin in Lysionotus Pauciflorus Maxim by HPLC-CL;3) Simultaneous Determination of Rutin and Quercetin in Carthamus tinctorius by High-Performance Liquid Chromatography with Chemiluminescence Detection.1 .Simultaneous determination of alcohol acid in certain Taxodiaceae plant roots by HPLC using post-column chemiluminescence detectionA novel method for the simultaneous determination of alcohol acid compounds such as Tartaric acid, malic acid and shikimic acid in certain plants of Taxodiaceae family by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection was developed. The procedure was based on the chemiluminescent enhancement by alcohol acid compounds of the semicarbazide hydrochloride and KMnO4 system in a sulfuric acid medium. The separation was carried out on a Hypersil ODS column with a mobile phase of Perchloric acid (pH 2.25). For three alcohol acid compounds, The detection limit (3a) of 3.54X10"8 g/mL,2.24X10"9 g/mL and 2.02 X10'8 g/mL respectively, and the relative standard deviations (n=6) for the determination of 5.0 XI0"6 g/mL compounds were in the range 1.4%,0.9% and 2.8%. The CL reaction was found to be well compatible with the mobile phase of HPLC and no baseline drift occurred in HPLC-CL detection. The method has been successfully applied to the determination of Tartaric acid, malic acid and shikimic acid in Taxodium ascendems and Taxodium distichum.2. Determination of the Levels of Nevadensin in Lysionotus Pauciflorus Maxim by HPLC-CLAn HPLC-CL linkage method was developed for simultaneous determination of Nevadensin in Lysionotus Pauciflorus Maxim, based on the strong sensitive chemiluminescence of the CH3CN-H2O2 system in alkaline medium. The separation was carried out on a Hypersil ODS column with a mobile phase of CH3CN: HCON (CH3)2:0.01mol/LH2C2O4 (27:6:67,V/V/V). The linear ranges for Nevadensin determination was 0.02~50|xg/mL with a detection limit (3o) of 0.01u.g/mL. The relative standard deviation for tetracycline were from 1.46% to 1.87% (n = 5).The method can be useful for the determination of Nevadensin in Lysionotus PauciflorusMaxim samples.3. Simultaneous Determination of Rutin and Quercetin in Carthamus tinctorius by High-Performance Liquid Chromatography with Chemiluminescence DetectionA method using HPLC-CL linkage was developed for simultaneous determination of Rutin and Quercetin in Carthamus tinctorius, based on the strong sensitive chemiluminescence of the K^Fe (CN) 6 systems in alkaline medium. The separation was carried out on a Hypersil ODS column with a mobile phase of ethanol-0.01% triethylamine (2:1,V/V). The linear ranges for Rutin and Quercetin determinations were 2.0X10'7 g/mL~2.5X 10"5 g/mL and 5.1 X 10'7 g/mL~1.3X lOVmL with a detection limit (3a) of 1.20 X 10"8 g/mL and 2.5 X 10'8 g/mL. The relative standard deviation for 5.0X 10'6 g/mL Rutin and Quercetin was 1.4% and 2.3% (n = 6). The method can be useful for the determination of Rutin and Quercetin in Carthamus tinctorius samples.