Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16845
DC FieldValueLanguage
dc.contributor李茂榮zh_TW
dc.contributorMaw-Rong Leeen_US
dc.contributor楊慶成zh_TW
dc.contributorChing-Cherng Yangen_US
dc.contributor.advisor鄭政峯zh_TW
dc.contributor.advisorJen-Fon Jenen_US
dc.contributor.author杜怡真zh_TW
dc.contributor.authorDu, Yi-Chenen_US
dc.contributor.other中興大學zh_TW
dc.date2013zh_TW
dc.date.accessioned2014-06-06T06:56:30Z-
dc.date.available2014-06-06T06:56:30Z-
dc.identifierU0005-0607201217253500zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/16845-
dc.description.abstract本研究開發出以鹽輔助均相液液微萃取之樣品前處理技術,並以HPLC-UV分離偵測bacillius subtilis CWS1微生物發酵培養液中的表面素(surfactin)。表面素為一種生物界面活性劑,具有高表面活性、易被生物降解暨對環境友善等優點,因此在未來有極大潛力取代合成的化學界面活性劑成為產品化商品。在產品量化生產過程中,產品的穩定性影響其有效性,為了能監測微生物培養液中表面素的濃度,快速地分析表面素技術是十分重要的。本研究使用實驗室自行設計的玻璃萃取裝置,將與水互溶的有機溶劑及稀釋過的培養液放入萃取裝置之後,加入鹽類並攪拌使溶液達飽和,之後將裝置至於離心機離心,最後取上層液打入HPLC進行分析。在鹽輔助均相液液微萃取技術中,針對萃取溶劑、萃取溶劑體積、添加的鹽類、硫酸銨鹽添加量、及水樣的pH值等參數進行探討。實驗結果顯示,5毫升經pH2的緩衝溶液稀釋的培養液加入300μL的Acetonitrile及4克硫酸銨鹽,攪拌之後離心1分鐘會有最好的萃取效果。本方法測得培養液中表面素的線性範圍為2-250 mg/L ,線性相關係數介於0.9970-0.9994之間,應用培養液在真實樣品中的回收率為77.7%~106.7%。此方法對於偵測微生物發酵培養液中的表面素是一個簡單、快速、省錢、對環境友善、及少基質干擾的分析方法。zh_TW
dc.description.abstractIn this study, a simple, rapid and efficient sample pretreatment method has been developed, termed new in-tube salt-assisted homogeneous liquid-liquid microextraction (SHLLME) followed by high performance liquid chromatography/ultra-violet detection (HPLC/UV) for the determination of surfactin in microbial fermentation broths of bacterium bacillius subtilis CWS1. Surfactin is one of a biosurfactant and it has the potential to replace synthetic chemical surfactant in the near future because of its high surface activity, biodegradability and environmental-friendly nature. In order to characterize the amount of surfactin for the microbial monitoring of fermentation production process, we have demonstrated a new method to analyze surfactin using a home-made extraction device. In this method, an extractant (water-miscible organic solvents) and salt were added to broth sample in an extraction tube. Then the device was sealed with plunger cork and taken for stirring and centrifugation processes. After that, the amount of separated extractant was measured using self-scaled glass capillary tube which was in-printed in the extraction device. Then the collected extractant was injected directly into HPLC-UV for analysis. Parameters influencing the extraction efficiency of SHLLE-HPLC/UV, such as the extraction solvent, solvent volume, pH, and salt-addition were thoroughly investigated and optimized. Under the selected experimental conditions, surfactin was extracted from 5 mL broth sample (at pH 2) by the addition of 300 μL acetonitrile (extractant) and 4 g ammonium sulfate, and followed by stirring for 5 min, and then centrifugation for 1 min at 3200 rpm, yields the maximum extraction efficiency. The proposed method provides a simple, rapid, inexpensive, eco-friendly and less matrix interferences process for the determination surfactin in microbial fermentation broth samples.en_US
dc.description.tableofcontents摘要 I Abstract II List of Tables XI 1. Introduction 1 1.1 Research motive 1 1.2 Literatures of surfactin 1 1.2.1 Biosurfactants 1 1.2.2 Surfactin 9 1.3 Traditional methods for surfactin analysis 12 1.4 Homogeneous liquid-liquid extraction (HLLE) 16 1.4.1 HLLE separation by temperature 17 1.4.2 HLLE separation by adding auxiliary solvent 17 1.4.3 HLLE separation by adjusting pH of surfactants 18 1.4.4 HLLE separation by ion-pair formation 19 1.4.5 Salt-assisted homogeneous liquid-liquid microextraction (SHLLME) 20 1.5 Sterilization with microwave 29 1.5.1 Microwave theory 29 1.5.2 Compared with traditional heating 31 1.6 Aim of the study 31 2. Experimental 32 2.1 Apparatus and Instruments 32 2.1.1 Apparatus 32 2.1.2 Instrument 32 2.1.3 SHLLME device 33 2.2 Reagents and Chemicals 33 2.2.1 Reagents 33 2.2.2 Media and microorganism 35 2.2.3 Preparation of solutions 36 2.2.4 The fermentation broths of microorganism 38 2.3 Instrumental set-up and experimental procedures 39 2.3.1 Instrumentation: HPLC 39 2.3.2 SHLLME set-up 39 2.3.3 SHLLME procedure 39 2.3.4 Silanization of glass tubes 40 2.3.5 Sterilization using microwave 40 2.4 Experiment conditions 44 2.4.1 Optimizations of SHLLME 44 2.4.2 Calibration procedure 46 2.4.3 Real samples analyses 47 2.4.4 MS and NMR characterizations 47 2.4.5 Microwave assisted sterilization 48 3.1 Optimizations of SHLLME 49 3.1.1 Selection of extraction solvents 49 3.1.2 Optimization of the extraction solvent volume 51 3.1.3 Selection of salt 51 3.1.4 Optimization of the amount of ammonium sulphate 51 3.1.5 Optimization of sample pH 54 3.2 Method validation 57 3.2.1 Instrument calibration parameters 57 3.2.2 Method calibration parameters 57 3.3 Analysis of surfactin in fermentation broths 58 3.3.1 Non-spiked real samples 58 3.3.2 Spiked real samples 58 3.4 MS and NMR characterization 62 3.4.1 MS for surfactin 62 3.4.2 NMR spectrograms for surfactin 62 3.5 Microwave assisted sterilization 78 3.5.1 Effect of microwave irradiation time 78 3.5.2 Effect of broth volumes for microwave sterilization 78 4. Conclusions 82 5. References 83zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0607201217253500en_US
dc.subjectSurfactinen_US
dc.subject表面素zh_TW
dc.subjectMicrobial fermentation broth samplesen_US
dc.subjectSalt-assisted homogeneous liquid-liquid microextractionen_US
dc.subjectHPLC-UVen_US
dc.subject鹽輔助均相液液微萃取zh_TW
dc.subject微生物發酵培養液樣品zh_TW
dc.subjectHPLC-UVzh_TW
dc.title以鹽輔助均相液液微萃取技術結合HPLC-UV快速偵測微生物發酵培養液中的表面素zh_TW
dc.titleRapid Determination of Surfactin in Microbial Fermentation Broths using Salt-Assisted Homogeneous Liquid-Liquid Microextraction Technique combined with HPLC-UVen_US
dc.typeThesis and Dissertationzh_TW
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