【作者】 陈立钢；

【导师】 丁兰； 于爱民；

【作者基本信息】 吉林大学 ， 分析化学， 2010， 博士

【摘要】 微波辅助萃取法由于其具有操作时间短、溶剂消耗量少、萃取产率高等优点,在药物分析、食品分析和环境分析等领域都得到了广泛应用。然而,目前微波辅助萃取的模式大多为静态方式。动态微波辅助萃取具有更为明显的优势,因为其可以加快目标化合物向萃取溶剂的传质速度。并且由于待测物及时从萃取体系中转移出来,因而可以减少不稳定化合物因长时间经受微波照射而引起分解。更为重要的是动态微波辅助萃取有利于与其它相关分析技术实现在线联用,进行在线分析,从而大大提高分析效率。TM010微波谐振腔是可将微波能高度集中于空间某一区域的微波器件,结构简单,易于加工。本论文采用TM010微波谐振腔为微波辅助萃取装置,建立了一种新型的动态微波辅助萃取法。并把这种方法与其它多种相关分析技术实现在线联用,对中药中的活性成分,食品中的药物残留等进行在线分析。具体内容如下:1.动态微波辅助萃取淫羊藿中的黄酮类化合物;2.动态微波辅助萃取在线结合强阳离子交换树脂净化测定动物饲料中的三聚氰胺;3.动态微波辅助萃取与紫外可见分光光度计在线联用测定红花中的黄色素;4.动态微波辅助萃取结合在线衍生以及光谱检测用于测定侧柏叶中的总黄酮;5.动态微波辅助萃取与高效液相色谱在线联用测定穿心莲中的穿心莲内酯和脱水穿心莲内酯;6.动态微波辅助萃取与固相萃取以及高效液相色谱在线联用测定粮食中的有机氯农药残留;7.动态微波辅助萃取在线结合2,4-二硝基苯肼衍生,限制性通过吸附剂净化和高效液相色谱测定水产品中的甲醛。在这些方法中,均考察并优化了动态微波辅助萃取条件,包括微波功率,萃取溶剂的组成、体积和流速,萃取时间以及样品质量等。对建立的每种方法进行了方法学评价,包括标准曲线、线性范围、检出限、回收率、日内和日间精密度,均得到了满意的结果。最终,这些方法在实际样品的检测中,也取得了理想的效果。更多还原

【Abstract】 Microwave-assisted extraction (MAE) has some advantages, such as short extraction time, small reagent consumption, high extraction yield and selectivity. It has been widely applied in pharmaceutical analysis, food analysis and environmental analysis. However, the mode used in most applications of MAE is static. Dynamic microwave-assisted extraction (DMAE) has more advantages and it can accelerate the transfer rate of analytes from sample matrix into extraction solvent. Moreover, the analytes would not decompose easily when increasing the microwave irradiation time in DMAE which can transfer the analytes with flowed solvent to the out of extraction vessel. The DMAE is also suitable for on-line coupling to related analytical techniques, and then greatly improves analysis efficiency.TM010 microwave resonance cavity is a microwave device which can highly concentrate the microwave energy in a certain regional. Its structure is simple and it is easy to be manufactured. The size of this cavity is small, and the utilization efficiency of microwave energy is high. Moreover, the cavity is suitable for on-line coupling to other analytical techniques. In this thesis, a novel DMAE technique was developed based on using the TM010 microwave resonance cavity as extraction device. This technique was also on-line coupled to related analytical techniques for analysis of active components in herbal medicines and drug residues in foods. The main contents and results of this thesis are as followed:1. The DMAE method developed in this thesis was used for the extraction of flavonoids from Herba Epimedii. The extraction conditions were optimized and the maximum extraction yield was achieved using 60% aqueous ethanol as extraction solvent, 0.7 mL min-1 of extraction solvent flow rate, 80 W of microwave power, 6 min of extraction time and 25 mg of sample amount. When compared with the proposed method, the time used in other extraction methods was long, such as 14 min, 1.0 h, 1.0 h and 4.0 h were used in pressurized microwave-assisted extraction (PMAE), ultrasonic extraction (UE), heat reflux extraction (RE) and Soxhlet extraction (SE), respectively. The extraction yields of flavonoids in Heaba Epimedii obtained by the MAE methods including DMAE and PMAE were higher than UE, RE and SE. However, in the PMAE, some flavonoids may be partly decomposed in the condition of long extraction time. This phenomenon was not easy to occur in DMAE.2. An analytical method was developed based on the on-line coupling of DMAE to strong cation exchange (SCX) resin clean-up, and was used for the determination of melamine in animal feed. The melamine was first extracted by 90% acidified methanol aqueous solution (v/v, pH=3) under the action of microwave energy, and then the extract was cooled and passed through the SCX resin. Thus the protonated melamine was retained on the resin through ion exchange interaction and the sample matrixes were washed out. The DMAE parameters were optimized by the Box-Behnken design and the maximum recovery of melamine was achieved using 7.6 mL of extraction solvent, 1.0 mL min-1 of extraction solvent flow rate, 34 W of microwave power. In all three parameters, the microwave power and extraction solvent volume have significant effect on the melamine recovery, and the extraction solvent flow rate has no significant effect. The optimal clean-up conditions were as followed: 90% acidified methanol aqueous solution (pH=3) as sample loading solvent, 1.0 mL min-1 of sample loading flow rate, 4.0 mL of 90% aqueous methanol as washing solvent, and 3 mL of methanol-ammonia (95:5, v/v) as elution solvent. The result indicated that when the clean-up step was not applied, the extract obtained by the DMAE was cloudy and had strong color, and it would not be suitable for subsequent liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. On the other hand, a transparent and colorless solution was obtained by using the SCX resin clean-up. The limit of detection (LOD) is 12.3 ng g-1. The intra- and inter-day precisions expressed as relative standard deviations (RSDs) are 5.1% and 7.3%, respectively, and the recoveries of melamine are in the range of 76.1%-93.5%. The proposed method was successfully applied to determine melamine in different animal feeds including two chicken feeds, two pig feeds and one cattle feed which obtained from the local market. Melamine was detectable with the contents of 279, 136 and 442 ng g-1 in three samples.3. An analytical method was developed based on the on-line coupling of DMAE to ultraviolet-visible (UV-Vis) spectrophotometer, and it was used for the determination of safflower yellow in Flos Carthami. In most extraction systems, the extraction process is not known, which results in a high level of uncertainty concerning the point at which the extraction process is complete. The practical solutions to this problem are usually to use large extraction time in order to ensure complete extraction. In this work, the extract obtained by the DMAE was on-line diluted, and then was directly introduced into the UV-Vis spectrophotometer for on-line monitoring. It is possible to visually observe the extraction process. The DMAE conditions were optimized and the maximum extraction yield was achieved using 60% aqueous methanol as extraction solvent, 1.0 mL min-1 of extraction solvent flow rate, 60 W of microwave power and 3.0 mg of sample amount. The LOD is 8.0μg mL-1. The intra- and inter-day precisions (RSDs) are in the range of 1.6%-3.2% and 2.8%-4.2%, respectively. The proposed method was compared with off-line DMAE, PMAE and maceration extraction (ME). The result indicated that the low extraction yield was obtained and long time was used by the ME. The extraction yield of safflower yellow obtained and the time used by DMAE and PMAE were similar. But the DMAE offered the capability of coupling with the spectrophotometer for on-line analysis, thus simplified analysis procedure and saved analysis time.4. An analytical method was developed based on the on-line coupling of DMAE to derivatization and UV-Vis detection, and was used for the determination of total flavonoids in Platycladus orientalis (L.) Franco. The DMAE conditions were optimized and the maximum extraction yield was achieved using 80% aqueous methanol as extraction solvent, 1.0 mL min-1 of extraction solvent flow rate, 80 W of microwave power and 5.0 min of extraction time. A derivatization reaction between aluminium chloride and flavonoid prior to UV-vis detection endows the method with high sensitivity and selectivity. The optimal derivatization conditions were as followed: 1.5 % aluminium chloride methanol solution as derivatization reagent, 1.5 mL min?1 of derivatization reagent flow rate and 100 cm of reaction coil. The LOD is 0.28 mg g-1. The intra-day and inter-day precisions (RSDs) are 1.5% and 4.6%, respectively. Mean recovery is 98.5%. This method was compared with RE. The results showed that the higher extraction yield of total flavonoids was obtained by DMAE with shorter extraction time and small quantity of extraction solvent. Moreover, this method realized on-line analysis and simplified analysis procedure.5. An analytical method was developed based on the on-line coupling of DMAE to high-performance liquid chromatography (HPLC), and was used for the determination of andrographolide and dehydroandrographolide in Andrographis paniculata Nees. The extraction was performed in a recirculating system. When a number of extraction cycles were completed, the fractional extract (20μL) was driven to the HPLC system for separation and detection. The DMAE conditions were optimized and the maximum extraction yield was achieved using 60% aqueous methanol as extraction solvent, 1.0 mL min-1 of extraction solvent flow rate, 80 W of microwave power and 6.0 min of extraction time. The LODs are 0.5 and 0.6μg mL-1 for andrographolide and dehydroandrographolide, respectively. The intra-day and inter-day precisions (RSDs) are 2.1% and 3.7 % for andrographolide and 1.7% and 4.1% for dehydroandrographolide, respectively. Mean recoveries for andrographolide and dehydroandrographolide are 97.7 % and 98.7 %, respectively. Compared with UE used in the Chinese pharmacopoeia, the proposed method was demonstrated to obtain higher extraction yield in a shorter time with small quantity of solvent.6. An analytical method was developed based on the on-line coupling of DMAE to solid-phase extraction (SPE) and HPLC, and was used for the determination of organochlorine pesticides (OCPs) in grains. The DMAE conditions were optimized and the maximum extraction recoveries of OCPs was achieved using 95% aqueous acetonitrile as extraction solvent, 1.0 mL min-1 of extraction solvent flow rate, 80 W of microwave power and 10.0 min of extraction time. The optimal clean-up conditions were as followed: C18 as sorbent, 20% aqueous acetonitrile as sample loading solvent, 2.0 mL min-1 of sample loading flow rate. Several applications of on-line SPE–HPLC have been reported in the literatures, and are often used for direct analysis of liquid samples including water, plasma and urine. In this work, the DMAE coupled with on-line SPE–HPLC was used for direct analysis of solid sample. Because 95% aqueous acetonitrile was used as extraction solvent, prior to SPE, the extract had to be on-line diluted for trapping analytes on the C18 sorbent. In the same time, the fractional extract obtained by DMAE was collected in a sample loop and subsequently introduced into the SPE column for avoiding the leakage of DMAE extraction vessel when the vessel was directly connected with the high pressure SPE column. This was preformed by two 6-port switching valves and a 10-port switching valve. The LODs of OCPs are in the range of 19–37 ng g?1. The recoveries are in the range of 86%–105%, and the RSDs are ranging from 1.2% to 8.7%. The risk of organic solvent volatilization is decreased, since the overall analysis procedure took place in a closed system. It can be considered that this method is promising and can be used for the analysis of other pesticide residues in solid samples.7. An analytical method was developed based on the on-line coupling of DMAE to 2,4-dinitrophenylhydrazine (DNPH) derivatization, restricted access material (RAM) clean-up and HPLC, and was used for the determination of formaldehyde in aquatic products. The DMAE conditions were optimized and the maximum extraction yield was achieved using 5.0 mL water as extraction solvent, 1.0 mL min-1 of extraction solvent flow rate, 50 W of microwave power. The optimal derivatization conditions were as followed: 80μg mL?1 of DNPH as derivatizing reagent (pH, 4.0), 60°C of derivatization temperature and 5.0 min of delay time after DMAE. RAM is designed specifically for the removal of macromolecules partially based on a size-exclusion mechanism. Only small molecules are able to penetrate into the pores of RAM and interact with the stationary phase bonded on their inner surface. The RAM was mainly used in the biological area, such as plasma and urine, for the removal of protein. It also can be used for analyzing environmental samples for the removal of humic substances. However, its applications in analyzing food samples were still scarce. A Capcell pak MF Ph-1 column packed with RAM was used as pre-column for the clean-up of the extract. The material in this column possesses long hydrophilic polyoxyethylene chains and hydrophobic phenyl groups on the surface of 80? silica in order to limit the access of large molecules such as proteins and retain small molecules. In this work, 4.0 mL 5% aqueous acetonitrile was selected as sample loading and washing solvent in order to efficiently retain the analyte and wash the sample matrix. The LOD is 0.27 mg kg-1. The intra-day and inter-day precisions (RSDs) are 3.5% and 5.0%, respectively. The recoveries are in the range of 70.0%–105.0%. In order to demonstrate the applicability of the proposed method, it was used for determination of formaldehyde in various aquatic products including hairtail, yellow croaker, cod, catfish, whitebait, quid, cuttlefish, sea cucumber, shrimp, npatunede cumingi and jellyfish. The contents of formaldehyde in these aquatic products were in the range of 3.2-29.6 mg kg-1. The results obtained with the proposed method are in agreement with those obtained by the state standard method (off-line steam distillation method) used in China. The overall analysis time is greatly shortened in the proposed method, and one sample can be analyzed in less than 30 min. The sample preparation and analysis took place in a closed system, which avoided the formaldehyde volatilization and exposure to the people.更多还原