Micellar Electrokinetic Chromatographic Separation And Highly Sensitive Fluorescent Detection Of Jasmonates

Jasmonates (JAs), including jasmonic acid (JA), methyl jasmonate and jasmonic acid conjugates with amino acids (JA-AAs), are plant hormones that widely exsit in various plants. As JAs exhibit variety of biological activities, the development of analytical methods for JAs is of great significance for better studying of their physiological effects. However, the contents of JAs is very low in plants and they usually exist in different stereoisomers, so the development of new methods for high performance separation and highly sensitive determination of JAs is challenging work. Capillary electrophoresis (CE) has become an indispensable analytical method due to the advantages of high efficiency, fast analytical speed, wide application, little consumption of sample and solvent. With unique advantages such as high sensitivity, high selectivity and fast response, fluorescence detection (FD) is now coupled with high performance liquid chromatography (HPLC-FD) and applied to the analysis of plant hormones. This work focuses on the development of micellar electrokinetic chromatographic separation and fluorescent detection with fluorescent derivatization of jasmonates. The developed methods were well used in the analysis of real samples. The main contents of this thesis are described as follows:1. Simultaneous separation of JA-AAs by micellar electrokinetic chromatography (MEKC). JA-AAs are signaling molecules in the plant defense system and they act as signals in a stereospecific manner. The separation of stereoisomers of JA-AAs is a very challenging work since these stereoisomers have similar chromatographic and electrophoretic behaviors. Simultaneous separation of ten (±)-JA conjugates with five AAs including L-Tyr (tyrosine), L-leucine, L-Ile (isoleucine), L-valine, and L-phenylalanine and their stereoisomers has been achieved by MEKC with diode array detector. The main factors effecting on the separation were investigated, including concentration and pH of phosphate buffer, concentration of surfactant, applied voltage and capillary temperature. Under the optimal conditions, ten stereoisomers of JA conjugates with five AAs are completely separated. The proposed method showed good repeatability and stability. The RSDs of the migration times and peak areas of the ten stereoisomers were less than1.03%and2.07%, respectively. In the tested concentration range, good linear relationships (correlation coefficients above99%) between peak areas and concentrations of the analytes were observed. The proposed method has been successfully applied to the analysis of spiked rice floret sample and original reaction solution of (±)-JA-Ile conjugate and (±)-JA-Tyr conjugate. The recoveries ranged from91.7to107.6%for the rice floret sample and92.9to107.2%for the original reaction solution.2. Synthesis of fluorescent reagent and application in highly sensitive determination of JA by HPLC-FD. JA is one of the main representatives in JAs. Since JA lacks a suitable fluorophore, fluorescence derivatization of JA can improve the sensitivity for the fluorescent detection. Fluorescein was used as the basic structure, which conjugated with piperazine groups to form fluorescein-core piperazine (FCP).'H-NMR and MS were used to identify the structure of FCP. The fluorescent property of FCP was investigated and it was applied as a pre-column fluorescent labeling reagent for the detection of JA. Based on the enhanced fluorescence after derivatization, a simple and sensitive method was developed for determination of JA by HPLC-FD. The specificity of the proposed method was confirmed by HPLC-ion trap mass spectrometry (HPLC-IT-MS). The effects of several key factors such as amount of FCP and condensing reagent, time and temprature of derivatization reaction, the composition of mobile phase and wavelength of fluorescence detection were examined. Under the optimal conditions, FCP labled JA could be well separated from FCP. The linear concentration range for JA was6×10-9-1×10-6mol/L with the correlation coefficient of0.9971. The limit of detection was as low as4×10-10mol/L. The proposed method has been successfully applied to the assay of JA in plant samples.3. Separation of JAs by capillary electrochromatography (CEC). Possiblity of CEC for the separation of JA-AAs was explored. A graphene-?-cyclodextrin open-tube column for CEC was fabricated by a layer-by-layer method based on the mussel-inspire technology. The poly dopamine was firstly coated on the inner wall of the cappillary, followed by a graphene oxide film. Then ?-cyclodextrin was introduced to the graphene oxide film by physisorption effect. The column was applied to the separation of JA-AAs. The results indicate that the column has potential in the separation of stereoisomers of L-Tyr-(±) JA, L-Leu-(±) JA, L-Ile-(±) JA and L-Phe-(±) JA.