(Luận văn) determination of ammonia in water samples using nda fluorescence derivatization with different nucleophiles

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY an lu n va to p ie gh tn PHẠM THỊ HẢI VÂN w TOPIC TITLE: DETERMINATION OF AMMONIA IN WATER SAMPLES d oa nl USING NDA FLUORESCENCE DERIVATIZATION WITH DIFFERENT NUCLEOPHILES v an lu m ll fu an BACHELOR THESIS : Full-time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2011-2015 oi Study Mode nh at z z @ om l.c gm an Lu Thai Nguyen, 9/2015 n va a th i c si Thai Nguyen University of Agriculture and Forestry Degree Program : Bachelor of Environmental Science and Management Student name : Phạm Thị Hải Vân Student ID : DTN 1153070072 Determination of Ammonia in Water Samples Using NDA Thesis Title : Fluorescence Derivatization with Different Nucleophiles - Assoc.Prof WU ,Chien-Hou Supervisor (s): an lu - Assoc.Prof Dr Do Thi Lan n va Abstract: Fluorescence has been in use by physical chemists for many years gh tn to Based on emission capacity of substance that has absorbed light or other electromagnetic radiation, fluorescence seems as one method with highest p ie applicability in chemical analysis An attraction of fluorescence is its sensitivity, helps scientists save w which time in research Based on that d oa nl advantage, fluorescence has widely application for determining ammonia and other possible nitrogen-containing photoproduct compounds (amine, amino acid ) v an lu with derivatizing reagent, OPA (o-phthaldialdehyde) On the other hand, fu an naphthalene-2,3-Dicarboxaldehyde (NDA) has been estimated as another popular oi OPA derivatives(Carlson, 1986) m ll derivatizing reagent, that can form much more stable derivatives with amines than nh This study was to optimize the derivatization conditions including concentration at z of NDA, maximum excitation and emission wavelengths, reaction pH, reaction z @ temperature and time, and choice of nucleophiles for analysis of ammonia in aqueous gm Fluorescence, Naphthalene-2,3-dicarboxaldehyde (NDA), Ammonia, Cyanide, Sulfite 53 n a th ii va September, 30, 2015 an Date of Submission: Lu Number of Pages: om l.c solution Keywords: c si ACKNOWLEDGEMENT To have completed this thesis, in addition to the ongoing efforts of myself, I would like to thank for teachers in faculty of International Training and Development as well as teachers in Thai Nguyen University of Agriculture and Forestry, who have dedicated teaching to me the valuable knowledge during study time in university and gave me a chance to my thesis oversea It is with immense gratitude that I acknowledge the support and help of Biomedical Engineering & Environmental Science Department, an lu National Tsing Hua University for accepting me to working in this wonderful place va n Furthermore, express my sincere deepest gratitude to Assoc Prof Wu Chien Hou, to gh tn from Biomedical Engineering & Environmental Science Department, National Tsing Hua p ie University and Assoc.Prof Dr Do Thi Lan from Thai Nguyen University of Agriculture d oa nl w and Forestry, who directly guided and created favorable conditions for me during the implementation of this thesis v an lu Next, i would like spend special thanks to Mr Bill (Wang Chin Yi) PhD student and my friends in the laboratory facilitated and provided the information and data fu an necessary for my implementation process and helped me finish this thesis m ll Finally, I would like to sincerely thank my family, all of my friends who always oi nh beside me all the time, giving spiritual help for me complete the tasks assigned during at z learning and research doing this thesis experiment z @ In the process of implementing the project, my thesis might have inevitable gm l.c shortcomings Therefore, would like to receive the attention and feedback from teachers om and friends to this thesis is more complete an Lu Sincerely, a th iii n va Pham Thi Hai Van c si TABLE OF CONTENT LIST OF FIGURES LIST OF TABLE PART I INTRODUCTION 1.1.Research rationale 1.2.Objectives of the research 1.3.Research questions and hypothesis lu an 1.4.Limitations of research n va PART II LITERATURE REVIEW gh tn to 2.1 Ammonia p ie 2.1.1 The properties of ammonia w 2.1.2 Origins of ammonia pollution in water d oa nl 2.2.Methods for determinate ammonia v an lu 2.2.1.Ion Chromatography m ll fu an 2.2.2.Voltammetry 10 2.2.3Fluorescence 11 oi nh 2.3.Overview of research and application of NDA in fluorescence 12 at z 2.3.1.o-phthaldialdehyde (OPA) 12 z @ 2.3.2.Naphthalene-2,3-dicarboxaldehyde (NDA) 14 gm om l.c PART III METHODS 16 3.1 Material 16 Lu an 3.1.1 Chemicals 16 n va a th iv c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 3.1.2 Equipment 16 3.2 Methods 18 3.2.1 Determine the optimum concentration 18 3.2.2 Fluorescence Spectrophotometer 21 3.3 HPLC system 24 PART IV RESULTS 27 4.1 NDA-ammonia-sulfite reaction 27 an lu 4.1.1 Optimization Excitation and Emission wavelength 27 n va -sulfite product 28 gh tn to 4.1.2 Effect of pH value on the fluorescence of NDA4.1.3 Effect of reaction time on the fluorescence of NDA- -sulfite product 29 p ie 4.1.4 Effect of reaction temperature on the fluorescence of NDA- -sulfite product 30 w -sulfite d oa nl 4.1.5 Effect of ratio between NDA and sulfite on the fluorescence of NDA- product 31 v an lu 4.1.6 Apply Pertinent Parameters on determine ammonia 33 fu an 4.2 NDA-ammonia-cyanide reaction 34 m ll oi 4.2.1 Optimization Excitation and Emission wavelength 34 nh -Cyanide product 35 4.2.2 Effect of reaction time on the fluorescence of NDA- at z -Cyanide product36 z 4.2.3 Effect of reaction temperature on the fluorescence of NDA- @ -sulfite gm 4.2.4 Effect of ratio between NDA and cyanide on the fluorescence of NDA- om l.c product 37 an Lu 4.2.5 Apply Pertinent Parameters on determine ammonia 38 4.3 Determine ammonia using HPLC-fluorescence system 40 n va a th v c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 4.3.1 NDA-ammonia-sulfite reaction 40 4.3.2 NDA-ammonia-cyanide reaction 42 PART V DISCUSSION AND CONCLUSION 45 5.1 Discussion 45 5.2 Conclusion 46 REFERENCES 47 an lu n va p ie gh tn to d oa nl w oi m ll fu an v an lu nh at z z @ om l.c gm an Lu n va a th vi c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 LIST OF FIGURES Figures Page an lu Figure 2.1 Synthesis NDA 15 Figure3.1 Fluorescence Spectrophotometer System 17 Figure 3.2 HPLC Fluorescence System Diagram 18 n va Figure 3.3 Warm Up Sample Solution gh tn to 20 Figure4.1 Excitation And Emission Of NDA-Ammonia-Sulfite Reaction p ie 27 w Figure Effect Of Reaction Ph On The Fluorescence Of NDA-NH 28 d oa nl Sulfite Product v an lu Figure Effect Of Reaction Time On The Fluorescence Of NDA-NH 29 m ll fu an Sulfite Product Figure 4 Effect Of Reaction Temperature On The Fluorescence Of NDA30 oi nh NH -Sulfite Product at z Figure Effect Of Ratio Between NDA And Sulfite At NDA=0.5mm z 31 @ gm Figure Effect Of Ratio Between NDA And Sulfite At NDA=1mm om l.c 32 Figure Effect Of Ratio Between NDA And Sulfite At NDA=2mm an Lu 32 n va a th c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 Figure Determine Ammonia By Fluorescence Using NDA-Sulfite 33 Figure Calibration Curve Of NH + From Standard Solution 34 Figure4.10 Excitation And Emission Of NDA-Ammonia-Cyanide Reaction 35 Figure 4.11 Effect Of Reaction Time On The Fluorescence Of NDA-NH 36 Cyanide Product an lu Figure 4.12 Effect Of Reaction Temperature On The Fluorescence Of NDA37 n va NH -Cyanide Product gh tn to Figure 4.13 Effect Of Ratio Between NDA And Cyanide On The 38 Fluorescence Of NDA-NH -Cyanide Product p ie w Figure 4.14 Determine Ammonia By Fluorescence Using NDA-Cyanide 39 d oa nl Figure 4.15 Calibration Curve Of NH + From Standard Solution 40 v an lu Figure 4.16 Determine Ammonia By HPLC-Fluorescence Using NDA- fu an 41 Sulfite With Solvent DIW And Methanol m ll oi Figure 4.17 Determine Ammonia By HPLC-Fluorescence Using NDA- nh 42 at Sulfite With Solvent Acetate Buffer And Methanol z z Figure 4.18 Determine Ammonia By HPLC-Fluorescence Using NDA- @ 43 l.c gm Cyanide With Solvent Acetate Buffer And Methanol 44 om Figure 4.19 Calibration Curve Of NH + From Standard Solution an Lu n va a th c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 LIST OF TABLE Table Page Table 1.1 Physical and chemical properties of ammonia Table 2.1 Physical and chemical properties of o-phthaldialdehyde 14 lu an Table 3.1 dilute NH working solution 20 n va to 25 p ie gh tn Table 3.2 HPLC-Fluorescence instrument setting condition d oa nl w oi m ll fu an v an lu nh at z z @ om l.c gm an Lu n va a th c si 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37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 LIST OF ABBREVIATIONS High Performance Liquid Chromatography IC Ion chromatography NDA Naphthalene-2,3-dicarboxaldehyde OPA o-phthaldialdehyde DIW Deionized water PPB Parts Per Billion an lu HPLC n va p ie gh tn to d oa nl w oi m ll fu an v an lu nh at z z @ om l.c gm an Lu n va a th c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 m=100 L and blank to solution of NDA= 2mM, borate buffer (pH 9) = 10mM and cyanide = 5mM at reaction temperature = 40 ℃ and reaction time = 25 minute 110 100 1uM 2uM 5uM B la n k 90 intensity (a.U 80 70 60 50 40 an lu 30 20 va 10 n 460 480 500 520 540 560 580 600 to W a v e le n g th (n m ) p ie gh tn Figure 4.14 determine ammonia by fluorescence using NDA-Cyanide w According to standard of method we get result of a typical calibration curve of + at d oa nl figure 4.15 was y = 8.5683x + 64.295 and a correlation coefficient = 0.9743 v an lu Base on the slope of the curve and correlation coefficient of this method, the result of fu an relative error = 1.19% Compare with intensity change at figure 4.14, cyanide are suitable nh prepare high intensity and stably oi m ll to determine ammonia by fluorescence Spectrophotometer with advantage is easy to at z z @ om l.c gm an Lu n va a th 39 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 120 Intensity (a.u.) 100 y = 8.5683x + 64.295 R² = 0.9881 80 60 40 20 0 an lu Concentration (mM) n va to Figure 4.15 Calibration curve of + from standard solution gh tn 4.3 Determine ammonia using HPLC-fluorescence system p ie Base on the result of experiment determine ammonia by fluorescence Spectrophotometer w using ammonia with different nucleophiles we apply for HPLC fluorescence system, on d oa nl method emerged as the most in chemical analysis with advantage over other techniques v an lu are their high selectivity, sensitivity, reliability, versatility and generally cost of fu an operation low oi m ll 4.3.1 NDA-ammonia-sulfite reaction nh The result of experiment determine ammonia by HPLC fluorescence system using NDA at z sulfite show in figure 4.16 follow experiment on section 3.2.3 is the collecting result of z for determine ammonia using NDA-sulfite @ concentration by fluorescence gm l.c Spectrophotometer include Buffer (pH9) 10mM, NDA 2mM, sulfite 2mM, ammonium om M, M, m and blank at temperature = 60 ℃ and reaction time = 20 minute Lu an However for HPLC fluorescence system we use the total volume of sample = 1mM n va a th 40 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 The graph 4.16 is the result of apply NDA-ammonia-sulfite solution to HPLC system with solvent is DIW and methanol Follow the graph we get signals but it not stable It mean that when the concentration of ammonia increase the intensity value of sample changing Especially, at some peak the intensity of blank sample are higher than other concentration u m u m u m 1um 2um b la n k an lu Relative fluorescnce intensity(mv) 1000 800 600 n va 400 gh tn to 200 0 10 15 20 p ie T im e ( m in u te ) w Figure 4.16 Determine ammonia by HPLC-fluorescence using NDA-sulfite with solvent d oa nl DIW and methanol v an lu To make sure application capacities of sulfite in determine ammonia by HPLC system fu an using NDA We use the condition similar as experiment at 4.16 However, at that m ll experiment we change solvent of HPLC system to acetate and methanol The result of that oi nh experiment was present in figure 4.17 at z According the graph, we also get signals and at signal (10.43 minute and 13.15 minute) z @ gm intensity of solution seem stable The intensity of blank sample is lower than other om l.c concentration However, the intensity of other concentration over scale, so it impossible to identify the signal of ammonia an Lu n va a th 41 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 1000 b la n k intensity (a.u) 800 600 400 200 an lu 0 10 15 20 va tim e (m in ) n gh tn to Figure 4.17 Determine ammonia by HPLC-fluorescence using NDA-sulfite with solvent p ie Acetate buffer and methanol d oa nl w Compare the result from graph 4.3.1a and 4.3.1b realize that NDA-ammonia-sulfite is not v an lu suitable for HPLC fluorescence system Its might cause by sulfite is weaker nucleophile fu an than organic thiol so the compound easy to break bonds then separation when inject to m ll HPLC system or by some other reason from system of method oi 4.3.2 NDA-ammonia-cyanide reaction nh at The experiment determine ammonia by HPLC fluorescence system using NDA-cyanide z z @ follow experiment on section 3.2.3 is the collecting result of concentration for determine gm l.c ammonia using NDA-sulfite by fluorescence Spectrophotometer include Buffer (pH9) om 10mM, NDA 2mM, Cyanide 5mM, ammonium M, M, m and blank at temperature Lu = 40℃ and reaction time = 20 minute However for HPLC fluorescence system we use the an a th 42 n va total volume of sample = 1mM c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 3500000 260 t7.7 t11.3 240 blank 1uM 3uM 6uM 220 200 Intensity (a.u.) Intensity (a.u.) 180 3000000 160 140 120 100 80 2500000 2000000 1500000 60 40 1000000 lu 20 an 0 10 15 20 ammonia (mM) n va time gh tn to Figure 4.18 Determine ammonia by HPLC-fluorescence using NDA-Cyanide with solvent Acetate buffer and methanol p ie d oa nl w Base on the result presented in the graph 4.18 we get signals and from that signal at 7.7 minute and 11.3 minute the intensity of solution increase when concentration of ammonia v an lu increase According to vary of solution intensity we get a typical calibration curve of fu an signal at 7.7 minute figure 4.3.2 b was = 109631x + 848744 and a correlation coefficient oi m ll = 0.9785 The typical calibration curve of signal at 11.3 minute was Y = 297203x + 1E+ 06 and a correlation coefficient = 0.9992 Compare the slope of the curve and nh at correlation coefficient of this method, the result of relative error at signal 7.7 and 11.3 z z @ respectively 2.15% and 0.08% gm l.c Both of that are suitable to be a graph of ammonia However to support the real graph of om ammonia of that we need prepare more experiment with other concentration of ammonia an Lu and developed that with some technique to analysis the result n va a th 43 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 3500000 Intensity (a.u.) 3000000 y = 297203x + 1E+06 R² = 0.9992 2500000 2000000 T 7.7 Series1 1500000 1000000 y = 109631x + 848744 R² = 0.9785 500000 T11.3 Series2 lu an n va Concentration (mM) gh tn to Figure 4.19 Calibration curve of + from standard solution p ie d oa nl w oi m ll fu an v an lu nh at z z @ om l.c gm an Lu n va a th 44 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 PART V DISCUSSION AND CONCLUSION 5.1 Discussion In this study, the analytical factors to determine ammonia by fluorescence using NDA was optimize with concentration: Buffer (pH9) 10mM, NDA 2mM, sulfite 2mM, at temperature = 60℃, reaction time = 20 minute for nucleophile is sulfite and NDA= 2mM, borate buffer (pH 9) = 10mM, cyanide = 5mM at reaction temperature = 40 ℃, reaction time = 25 minute for nucleophile is cyanide lu an The application method for determine ammonia in water sample by fluorescence n va spectrophotometer using NDA was successfully with the result of relative error = 2.57% to gh tn for nucleophile is sulfite and 1.19% for cyanide Base on the result of comparison p ie between the slope of the curve and correlation coefficient in figure 4.1.6 and 4.2.5 this d oa nl w research was demonstrable capacity of NDA in determine ammonia by fluorescence with advantage are highly sensitive save time, reproducible, and selective v an lu By the result of relative error in determine ammonia using fluorescence fu an spectrophotometer system higher than sulfite of cyanide and the capacity apply in HPLC method oi m ll system Cyanide seem more suitable to react with NDA than sulfite in determine ammonia nh at Although this method might have some negative point made the result of solution not z z @ clear enough but it provide information to other method intended for develop using NDA gm to determine ammonia in other method or with other purpose om l.c an Lu n va a th 45 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 5.2 Conclusion This method might have good result when apply to fluorescence spectrophotometer system, but when using HPLC system to determine that solution the result seem unstable and difficult to identify the real signal of ammonia So, it offers doubt about analysis capacity of that method in determine ammonia Therefore, to evaluate specific and accurate capability of NDA application, the experiment need more time for research the factor effect to value of ammonia in that method an lu n va p ie gh tn to d oa nl w oi m ll fu an v an lu nh at z z @ om l.c gm an Lu n va a th 46 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 REFERENCES Anne, M H & Constant, M G B (1993) Determination of ammonia in seawater using catalytic cathodic stripping voltammetry American Chemical Society, pp 3411–3416 Bill J C & Tarbell, D S (1954) o-Phthalaldehyde Organic Syntheses, Vol 4, pp 87 California Environmental Protection Agency (2011) Ammonia Retrieved from: http://www.waterboards.ca.gov/water_issues/programs/swamp/docs/cwt/guidance/3310en pdf (accessed on 17/06/2015) lu an Carlson, R G., Srinivasachar, K., Givens, R S & Matuszewski, B K (1986) New Derivatizing va Agents for Amino Acids and Peptides.1.Facile Synthesis of N-Substituted n gh tn to 1Cyanobenz[f]isoindoles and Their Spectroscopic Properties American Chemical Society, pp 3978-3983 p ie Press Clare, S d oa nl w Chisholm, H (1911) Ammonia Encyclopædia Britannica (11th ed.) Cambridge University (2015) Disadvantages Advantages of an HPLC Retrieved v an lu http://www.ehow.com/list_5911530_disadvantages-advantages-hplc.html (accessed on m ll fu an 13/06/2015) from: Erika, P F., & Arnaldo, A (2012) Cardoso A Method for Determination of Ammonia in Air oi nh using Oxalic Acid-Impregnated Cellulose Filters and Fluorimetric Detection, Journal of at z the Brazilian Chemical Society 23(1), pp 142- 147 z @ Irina, T , Ilnur, Kh , Mihail, K , Aleksey, M , & Andrey, B (2015) Automated procedure for gm l.c determination of ammonia in concrete with headspace single-drop micro-extraction by om stepwise injection spectrophotometric analysis Talanta , pp 34-37 an Lu n va a th 47 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 Kuo, C T., Wang, P Y., & Wu, C H (2005) Fluorometric determination of ammonium ion by ion chromatography using postcolumn derivatization with o-phthaldialdehyde Journal of Chromatography A, pp 91-97 Laura, A.A., Ginny, S.M, & Ronald, E.S (1984) The o-phthalaldehyde derivatives of amines for high-speed liquid chromatography.electrochemistry American Chemical Society, 56 (7), pp 1089–1096 Mark, C R & Marlin, D H (1987) Determination of Amino Acids at Subfemtomole Levels by High-Performance Liquid Chromatography with Laser-Induced Fluorescence Detection an lu American Chemical Society, pp 411-415 n va Monica, Z B (2006) Ion Chromatography Retrieved from: gh tn to http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html (accessed p ie on 7/08/2015) w Petr, Z (2004) Reactions of Orthophthalaldehyde with Nucleophiles American Chemical Society, d oa nl pp 3217–3238 v an lu Pierre de, M , John F S , Richard, S G , Robert, G C , & Takeru, H (1987) Naphthalene-2,3 dicarboxaldehyde/Cyanide Ion: A Rationally Designed Fluorogenic Reagent for Primary fu an Amines American Chemical Society, pp 1096 - 1101 m ll oi Thomas, F.G & Henze, G (2001) Introduction Voltammetry analysis theory and practice nh at Retrieved z z from:https://books.google.com.vn/books?id=yoFKXm0ssIC&pg=PA60&dq=voltammetry @ gm +%C3%A2nlysis&hl=en&sa=X&ved=0CCoQh3QFg2M#v=onepage&q&f=false om l.c (accessed on 19/07/2015) an Lu n va a th 48 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 Weiss, J (2004) Ion chromatography Retrieved from: https://books.google.com.vn/books?hl=en&lr=&id=PP2&dq=ion+chromatography+book &ots=6N6rHEh4Mi&sig &redir_esc=y#v=onepage&q&f=false (accessed on 7/08/2015) Zaifang, Z., Joann, J L., M Inês G S A , Qiaosheng P., Spas D K & Shaorong L A (2015) microfabricated electroosmotic pump coupled to a gas-diffusion microchip for flow injection analysis of ammonia Microchimica Acta, pp 1063-1070 Zhaolai, D , Zhenlong, W , Sichao, J & Guoyao, W (2014) Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde an lu derivatives by HPLC with fluorescence detection Journal of Chromatography B, pp 116 - va n 127 gh tn to Zotou, A & Notou, M (2012) Study of the naphthalene-2,3-dicarboxaldehyde pre-column p ie derivatization of biogenic mono- and diamines in mixture and fluorescence-HPLC w determination Euroanalysis XVI, pp 1039–1048 d oa nl oi m ll fu an v an lu nh at z z @ om l.c gm an Lu n va a th 49 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 APPENDICES an lu n va p ie gh tn to Fluorescence spectrophotometer machine d oa nl w oi m ll fu an v an lu nh at z z @ om l.c gm HPLC fluorescence machine an Lu n va a th 50 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.2237.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.66 an lu n va p ie gh tn to Solvent of HPLC system d oa nl w oi m ll fu an v an lu nh at z z Warm up system @ om l.c gm an Lu n va a th 51 c si 37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 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37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.99 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