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

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY lu an n va to p ie gh tn PHẠM THỊ HẢI VÂN TOPIC TITLE: DETERMINATION OF AMMONIA IN WATER SAMPLES nl w USING NDA FLUORESCENCE DERIVATIZATION WITH DIFFERENT d oa NUCLEOPHILES an lu oi lm ul nf va BACHELOR THESIS : Full-time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2011-2015 z at nh Study Mode z m co l gm @ an Lu Thai Nguyen, 9/2015 n va ac th i 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): lu - Assoc.Prof Dr Do Thi Lan an n va Abstract: Fluorescence has been in use by physical chemists for many years tn to Based on emission capacity of substance that has absorbed light or other electromagnetic radiation, fluorescence seems as one method with highest gh p ie applicability in chemical analysis An attraction of fluorescence is its sensitivity, helps scientists save time in research Based on that w which oa nl advantage, fluorescence has widely application for determining ammonia and other d possible nitrogen-containing photoproduct compounds (amine, amino acid ) lu OPA (o-phthaldialdehyde) On the other hand, va an with derivatizing reagent, naphthalene-2,3-Dicarboxaldehyde (NDA) has been estimated as another popular nf OPA derivatives(Carlson, 1986) oi lm ul derivatizing reagent, that can form much more stable derivatives with amines than z at nh This study was to optimize the derivatization conditions including concentration of NDA, maximum excitation and emission wavelengths, reaction pH, reaction z l solution Keywords: gm @ temperature and time, and choice of nucleophiles for analysis of ammonia in aqueous Fluorescence, Naphthalene-2,3-dicarboxaldehyde (NDA), September, 30, 2015 n ac th ii va Date of Submission: 53 an Lu Number of Pages: m co Ammonia, Cyanide, Sulfite 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, lu an 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 nl w and Forestry, who directly guided and created favorable conditions for me during the d oa implementation of this thesis an lu Next, i would like spend special thanks to Mr Bill (Wang Chin Yi) PhD student nf va and my friends in the laboratory facilitated and provided the information and data oi lm ul necessary for my implementation process and helped me finish this thesis Finally, I would like to sincerely thank my family, all of my friends who always z at nh beside me all the time, giving spiritual help for me complete the tasks assigned during learning and research doing this thesis experiment z @ In the process of implementing the project, my thesis might have inevitable gm m co l shortcomings Therefore, would like to receive the attention and feedback from teachers and friends to this thesis is more complete an Lu Sincerely, ac th iii n va Pham Thi Hai Van 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 tn to 2.1 Ammonia gh p ie 2.1.1 The properties of ammonia nl w 2.1.2 Origins of ammonia pollution in water d oa 2.2.Methods for determinate ammonia lu va an 2.2.1.Ion Chromatography ul nf 2.2.2.Voltammetry 10 oi lm 2.2.3Fluorescence 11 z at nh 2.3.Overview of research and application of NDA in fluorescence 12 2.3.1.o-phthaldialdehyde (OPA) 12 z gm @ 2.3.2.Naphthalene-2,3-dicarboxaldehyde (NDA) 14 m co l PART III METHODS 16 3.1 Material 16 an Lu 3.1.1 Chemicals 16 n va ac th iv si 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 lu an 4.1.1 Optimization Excitation and Emission wavelength 27 n va -sulfite product 28 tn to 4.1.2 Effect of pH value on the fluorescence of NDA- -sulfite product 29 ie gh 4.1.3 Effect of reaction time on the fluorescence of NDA- -sulfite product 30 p 4.1.4 Effect of reaction temperature on the fluorescence of NDA- w -sulfite oa nl 4.1.5 Effect of ratio between NDA and sulfite on the fluorescence of NDA- d product 31 an lu 4.1.6 Apply Pertinent Parameters on determine ammonia 33 nf va oi lm ul 4.2 NDA-ammonia-cyanide reaction 34 4.2.1 Optimization Excitation and Emission wavelength 34 z at nh 4.2.2 Effect of reaction time on the fluorescence of NDA- -Cyanide product 35 4.2.3 Effect of reaction temperature on the fluorescence of NDA- z -Cyanide product36 @ -sulfite l gm 4.2.4 Effect of ratio between NDA and cyanide on the fluorescence of NDA- m co 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 ac th v si 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 lu an n va p ie gh tn to d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th vi si LIST OF FIGURES Figures Page lu an 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 lu an Figure Effect Of Reaction Time On The Fluorescence Of NDA-NH 29 ul nf va Sulfite Product 30 z at nh NH -Sulfite Product oi lm Figure 4 Effect Of Reaction Temperature On The Fluorescence Of NDA- 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 m co l 32 Figure Effect Of Ratio Between NDA And Sulfite At NDA=2mm an Lu 32 n va ac th si 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 lu Figure 4.12 Effect Of Reaction Temperature On The Fluorescence Of NDA- an 37 n va NH -Cyanide Product tn to Figure 4.13 Effect Of Ratio Between NDA And Cyanide On The 38 p ie gh Fluorescence Of NDA-NH -Cyanide Product Figure 4.14 Determine Ammonia By Fluorescence Using NDA-Cyanide 39 oa nl w Figure 4.15 Calibration Curve Of NH + From Standard Solution d 40 an lu va Figure 4.16 Determine Ammonia By HPLC-Fluorescence Using NDA- nf 41 oi lm ul Sulfite With Solvent DIW And Methanol Figure 4.17 Determine Ammonia By HPLC-Fluorescence Using NDA- z at nh 42 Sulfite With Solvent Acetate Buffer And Methanol z Figure 4.18 Determine Ammonia By HPLC-Fluorescence Using NDA- @ 43 m co l gm Cyanide With Solvent Acetate Buffer And Methanol Figure 4.19 Calibration Curve Of NH + From Standard Solution 44 an Lu n va ac th si 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 lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th si 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 lu HPLC an n va p ie gh tn to d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th si 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 lu 30 an 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 According to standard of method we get result of a typical calibration curve of + at nl w d oa figure 4.15 was y = 8.5683x + 64.295 and a correlation coefficient = 0.9743 va an lu Base on the slope of the curve and correlation coefficient of this method, the result of relative error = 1.19% Compare with intensity change at figure 4.14, cyanide are suitable ul nf z at nh prepare high intensity and stably oi lm to determine ammonia by fluorescence Spectrophotometer with advantage is easy to z m co l gm @ an Lu n va ac th 39 si 120 Intensity (a.u.) 100 y = 8.5683x + 64.295 R² = 0.9881 80 60 40 20 0 lu Concentration (mM) an 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 nl w using ammonia with different nucleophiles we apply for HPLC fluorescence system, on d oa method emerged as the most in chemical analysis with advantage over other techniques lu low cost of va an are their high selectivity, sensitivity, reliability, versatility and generally ul nf operation oi lm 4.3.1 NDA-ammonia-sulfite reaction z at nh The result of experiment determine ammonia by HPLC fluorescence system using NDA 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 by fluorescence gm @ concentration m co l Spectrophotometer include Buffer (pH9) 10mM, NDA 2mM, sulfite 2mM, ammonium M, M, m and blank at temperature = 60 ℃ and reaction time = 20 minute an Lu However for HPLC fluorescence system we use the total volume of sample = 1mM n va ac th 40 si 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 lu Relative fluorescnce intensity(mv) 1000 800 600 an n va 400 gh tn to 200 0 10 15 20 ie T im e ( m in u te ) p Figure 4.16 Determine ammonia by HPLC-fluorescence using NDA-sulfite with solvent w d oa nl DIW and methanol lu va an To make sure application capacities of sulfite in determine ammonia by HPLC system oi lm ul nf using NDA We use the condition similar as experiment at 4.16 However, at that experiment we change solvent of HPLC system to acetate and methanol The result of that z at nh experiment was present in figure 4.17 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 m co l concentration However, the intensity of other concentration over scale, so it impossible to identify the signal of ammonia an Lu n va ac th 41 si 1000 b la n k intensity (a.u) 800 600 400 200 lu an 10 15 20 va tim e (m in ) n tn to ie gh Figure 4.17 Determine ammonia by HPLC-fluorescence using NDA-sulfite with solvent p Acetate buffer and methanol nl w d oa Compare the result from graph 4.3.1a and 4.3.1b realize that NDA-ammonia-sulfite is not an lu suitable for HPLC fluorescence system Its might cause by sulfite is weaker nucleophile ul nf va than organic thiol so the compound easy to break bonds then separation when inject to oi lm HPLC system or by some other reason from system of method 4.3.2 NDA-ammonia-cyanide reaction z at nh The experiment determine ammonia by HPLC fluorescence system using NDA-cyanide z gm @ follow experiment on section 3.2.3 is the collecting result of concentration for determine m co l ammonia using NDA-sulfite by fluorescence Spectrophotometer include Buffer (pH9) 10mM, NDA 2mM, Cyanide 5mM, ammonium M, M, m and blank at temperature an Lu = 40℃ and reaction time = 20 minute However for HPLC fluorescence system we use the ac th 42 n va total volume of sample = 1mM si 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 tn to Figure 4.18 Determine ammonia by HPLC-fluorescence using NDA-Cyanide with solvent Acetate buffer and methanol p ie gh nl w Base on the result presented in the graph 4.18 we get signals and from that signal at 7.7 d oa minute and 11.3 minute the intensity of solution increase when concentration of ammonia an lu increase According to vary of solution intensity we get a typical calibration curve of ul nf va signal at 7.7 minute figure 4.3.2 b was = 109631x + 848744 and a correlation coefficient oi lm = 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 z at nh correlation coefficient of this method, the result of relative error at signal 7.7 and 11.3 z gm @ respectively 2.15% and 0.08% m co l Both of that are suitable to be a graph of ammonia However to support the real graph of 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 ac th 43 si 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) tn to Figure 4.19 Calibration curve of + from standard solution p ie gh d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th 44 si 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 nl w research was demonstrable capacity of NDA in determine ammonia by fluorescence with d oa advantage are highly sensitive save time, reproducible, and selective an lu By the result of relative error in determine ammonia using fluorescence ul nf va spectrophotometer system higher than sulfite of cyanide and the capacity apply in HPLC method oi lm system Cyanide seem more suitable to react with NDA than sulfite in determine ammonia z at nh Although this method might have some negative point made the result of solution not z gm @ clear enough but it provide information to other method intended for develop using NDA to determine ammonia in other method or with other purpose m co l an Lu n va ac th 45 si 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 lu an n va p ie gh tn to d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th 46 si 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 S (2015) oa nl Disadvantages Advantages d Clare, Press w Chisholm, H (1911) Ammonia Encyclopædia Britannica (11th ed.) Cambridge University of an HPLC Retrieved lu on oi lm ul nf 13/06/2015) (accessed va an http://www.ehow.com/list_5911530_disadvantages-advantages-hplc.html from: Erika, P F., & Arnaldo, A (2012) Cardoso A Method for Determination of Ammonia in Air z at nh using Oxalic Acid-Impregnated Cellulose Filters and Fluorimetric Detection, Journal of the Brazilian Chemical Society 23(1), pp 142- 147 z gm @ Irina, T , Ilnur, Kh , Mihail, K , Aleksey, M , & Andrey, B (2015) Automated procedure for l determination of ammonia in concrete with headspace single-drop micro-extraction by m co stepwise injection spectrophotometric analysis Talanta , pp 34-37 an Lu n va ac th 47 si 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 lu an American Chemical Society, pp 411-415 Z n va Monica, B (2006) Ion Chromatography Retrieved from: (accessed gh tn to http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html p ie on 7/08/2015) Petr, Z (2004) Reactions of Orthophthalaldehyde with Nucleophiles American Chemical Society, nl w d oa pp 3217–3238 an lu Pierre de, M , John F S , Richard, S G , Robert, G C , & Takeru, H (1987) Naphthalene-2,3 nf va dicarboxaldehyde/Cyanide Ion: A Rationally Designed Fluorogenic Reagent for Primary oi lm ul Amines American Chemical Society, pp 1096 - 1101 Thomas, F.G & Henze, G (2001) Introduction Voltammetry analysis theory and practice z at nh Retrieved z from:https://books.google.com.vn/books?id=yoFKXm0ssIC&pg=PA60&dq=voltammetry @ l gm +%C3%A2nlysis&hl=en&sa=X&ved=0CCoQh3QFg2M#v=onepage&q&f=false (accessed on 19/07/2015) m co an Lu n va ac th 48 si 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 lu an derivatives by HPLC with fluorescence detection Journal of Chromatography B, pp 116 - va n 127 tn to Zotou, A & Notou, M (2012) Study of the naphthalene-2,3-dicarboxaldehyde pre-column gh p ie derivatization of biogenic mono- and diamines in mixture and fluorescence-HPLC determination Euroanalysis XVI, pp 1039–1048 d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ an Lu n va ac th 49 si APPENDICES lu an n va p ie gh tn to Fluorescence spectrophotometer machine d oa nl w oi lm ul nf va an lu z at nh z m co l gm @ HPLC fluorescence machine an Lu n va ac th 50 si lu an n va p ie gh tn to Solvent of HPLC system d oa nl w oi lm ul nf va an lu z at nh z Warm up system m co l gm @ an Lu n va ac th 51 si lu an n va Stored Working Solution with Ice p ie gh tn to d oa nl w oi lm ul nf va an lu z at nh z gm @ Borate buffer working solution m co l NDA working solution an Lu n va ac th 52 si lu an va Deionized water n Sodium sulfite working solution p ie gh tn to d oa nl w oi lm ul nf va an lu z at nh z gm @ Sodium cyanide working solution m co l Ammonium chloride working solution an Lu n va ac th 53 si