THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY PHẠM THỊ HẢI VÂN TOPIC TITLE: DETERMINATION OF AMMONIA IN WATER SAMPLES USING NDA FLUORESCENCE DERIVATIZATION WITH DIFFERENT NUCLEOPHILES BACHELOR THESIS Study Mode : Full-time Major : Environmental Science and Management Faculty : International Training and Development Center Batch : 2011-2015 Thai Nguyen, 9/2015 i 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): - Assoc.Prof Dr Do Thi Lan Abstract: Fluorescence has been in use by physical chemists for many years Based on emission capacity of substance that has absorbed light or other electromagnetic radiation, fluorescence seems as one method with highest applicability in chemical analysis An attraction of fluorescence is its sensitivity, which helps scientists save time in research Based on that advantage, fluorescence has widely application for determining ammonia and other possible nitrogen-containing photoproduct compounds (amine, amino acid ) with derivatizing reagent, OPA (o-phthaldialdehyde) On the other hand, naphthalene-2,3-Dicarboxaldehyde (NDA) has been estimated as another popular derivatizing reagent, that can form much more stable derivatives with amines than OPA derivatives(Carlson, 1986) This study was to optimize the derivatization conditions including concentration of NDA, maximum excitation and emission wavelengths, reaction pH, reaction temperature and time, and choice of nucleophiles for analysis of ammonia in aqueous solution Keywords: Fluorescence, Naphthalene-2,3-dicarboxaldehyde (NDA), Ammonia, Cyanide, Sulfite Number of Pages: Date of Submission: 53 September, 30, 2015 ii 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, National Tsing Hua University for accepting me to working in this wonderful place Furthermore, express my sincere deepest gratitude to Assoc Prof Wu Chien Hou, from Biomedical Engineering & Environmental Science Department, National Tsing Hua University and Assoc.Prof Dr Do Thi Lan from Thai Nguyen University of Agriculture and Forestry, who directly guided and created favorable conditions for me during the implementation of this thesis 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 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 beside me all the time, giving spiritual help for me complete the tasks assigned during learning and research doing this thesis experiment In the process of implementing the project, my thesis might have inevitable shortcomings Therefore, would like to receive the attention and feedback from teachers and friends to this thesis is more complete Sincerely, Pham Thi Hai Van iii 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 1.4.Limitations of research PART II LITERATURE REVIEW 2.1 Ammonia 2.1.1 The properties of ammonia 2.1.2 Origins of ammonia pollution in water 2.2.Methods for determinate ammonia 2.2.1.Ion Chromatography 2.2.2.Voltammetry 10 2.2.3Fluorescence 11 2.3.Overview of research and application of NDA in fluorescence 12 2.3.1.o-phthaldialdehyde (OPA) 12 2.3.2.Naphthalene-2,3-dicarboxaldehyde (NDA) 14 PART III METHODS 16 3.1 Material 16 3.1.1 Chemicals 16 iv 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 4.1.1 Optimization Excitation and Emission wavelength 27 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 28 -sulfite product 29 4.1.4 Effect of reaction temperature on the fluorescence of NDA- -sulfite product 30 4.1.5 Effect of ratio between NDA and sulfite on the fluorescence of NDA- -sulfite product 31 4.1.6 Apply Pertinent Parameters on determine ammonia 33 4.2 NDA-ammonia-cyanide reaction 34 4.2.1 Optimization Excitation and Emission wavelength 34 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- -Cyanide product36 4.2.4 Effect of ratio between NDA and cyanide on the fluorescence of NDA- -sulfite product 37 4.2.5 Apply Pertinent Parameters on determine ammonia 38 4.3 Determine ammonia using HPLC-fluorescence system 40 v 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 vi LIST OF FIGURES Figures Page Figure 2.1 Synthesis NDA 15 Figure3.1 Fluorescence Spectrophotometer System 17 Figure 3.2 HPLC Fluorescence System Diagram 18 Figure 3.3 Warm Up Sample Solution 20 Figure4.1 Excitation And Emission Of NDA-Ammonia-Sulfite Reaction 27 Figure Effect Of Reaction Ph On The Fluorescence Of NDA-NH 28 Sulfite Product Figure Effect Of Reaction Time On The Fluorescence Of NDA-NH - 29 Sulfite Product Figure 4 Effect Of Reaction Temperature On The Fluorescence Of NDA- 30 NH -Sulfite Product Figure Effect Of Ratio Between NDA And Sulfite At NDA=0.5mm 31 Figure Effect Of Ratio Between NDA And Sulfite At NDA=1mm 32 Figure Effect Of Ratio Between NDA And Sulfite At NDA=2mm 32 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 Figure 4.12 Effect Of Reaction Temperature On The Fluorescence Of NDA- 37 NH -Cyanide Product Figure 4.13 Effect Of Ratio Between NDA And Cyanide On The Fluorescence Of NDA-NH -Cyanide Product 38 Figure 4.14 Determine Ammonia By Fluorescence Using NDA-Cyanide 39 Figure 4.15 Calibration Curve Of NH + From Standard Solution 40 Figure 4.16 Determine Ammonia By HPLC-Fluorescence Using NDA41 Sulfite With Solvent DIW And Methanol Figure 4.17 Determine Ammonia By HPLC-Fluorescence Using NDASulfite With Solvent Acetate Buffer And Methanol 42 Figure 4.18 Determine Ammonia By HPLC-Fluorescence Using NDACyanide With Solvent Acetate Buffer And Methanol Figure 4.19 Calibration Curve Of NH + From Standard Solution 43 44 LIST OF TABLE Table Page Table 1.1 Physical and chemical properties of ammonia Table 2.1 Physical and chemical properties of o-phthaldialdehyde Table 3.1 dilute NH working solution 14 20 Table 3.2 HPLC-Fluorescence instrument setting condition 25 LIST OF ABBREVIATIONS HPLC High Performance Liquid Chromatography IC Ion chromatography NDA Naphthalene-2,3-dicarboxaldehyde OPA o-phthaldialdehyde DIW Deionized water PPB Parts Per Billion 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 30 20 10 460 480 500 520 540 560 580 600 W a v e le n g th (n m ) 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 figure 4.15 was y = 8.5683x + 64.295 and a correlation coefficient = 0.9743 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 to determine ammonia by fluorescence Spectrophotometer with advantage is easy to prepare high intensity and stably 39 120 Intensity (a.u.) 100 y = 8.5683x + 64.295 R² = 0.9881 80 60 40 20 0 Concentration (mM) Figure 4.15 Calibration curve of + from standard solution 4.3 Determine ammonia using HPLC-fluorescence system Base on the result of experiment determine ammonia by fluorescence Spectrophotometer using ammonia with different nucleophiles we apply for HPLC fluorescence system, on method emerged as the most in chemical analysis with advantage over other techniques are their high selectivity, sensitivity, reliability, versatility and generally low cost of operation 4.3.1 NDA-ammonia-sulfite reaction 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 concentration for determine ammonia using NDA-sulfite by fluorescence Spectrophotometer include Buffer (pH9) 10mM, NDA 2mM, sulfite 2mM, ammonium M, M, m and blank at temperature = 60 ℃ and reaction time = 20 minute However for HPLC fluorescence system we use the total volume of sample = 1mM 40 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 Relative fluorescnce intensity(mv) 1000 800 600 400 200 0 10 15 20 T im e ( m in u te ) Figure 4.16 Determine ammonia by HPLC-fluorescence using NDA-sulfite with solvent DIW and methanol To make sure application capacities of sulfite in determine ammonia by HPLC system 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 experiment was present in figure 4.17 According the graph, we also get signals and at signal (10.43 minute and 13.15 minute) intensity of solution seem stable The intensity of blank sample is lower than other concentration However, the intensity of other concentration over scale, so it impossible to identify the signal of ammonia 41 1000 b la n k intensity (a.u) 800 600 400 200 0 10 15 20 tim e (m in ) Figure 4.17 Determine ammonia by HPLC-fluorescence using NDA-sulfite with solvent Acetate buffer and methanol Compare the result from graph 4.3.1a and 4.3.1b realize that NDA-ammonia-sulfite is not suitable for HPLC fluorescence system Its might cause by sulfite is weaker nucleophile than organic thiol so the compound easy to break bonds then separation when inject to HPLC system or by some other reason from system of method 4.3.2 NDA-ammonia-cyanide reaction The experiment determine ammonia by HPLC fluorescence system using NDA-cyanide follow experiment on section 3.2.3 is the collecting result of concentration for determine ammonia using NDA-sulfite by fluorescence Spectrophotometer include Buffer (pH9) 10mM, NDA 2mM, Cyanide 5mM, ammonium M, M, m and blank at temperature = 40℃ and reaction time = 20 minute However for HPLC fluorescence system we use the total volume of sample = 1mM 42 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 20 0 10 15 20 ammonia (mM) time Figure 4.18 Determine ammonia by HPLC-fluorescence using NDA-Cyanide with solvent Acetate buffer and methanol 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 increase According to vary of solution intensity we get a typical calibration curve of signal at 7.7 minute figure 4.3.2 b was = 109631x + 848744 and a correlation coefficient = 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 correlation coefficient of this method, the result of relative error at signal 7.7 and 11.3 respectively 2.15% and 0.08% 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 and developed that with some technique to analysis the result 43 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 0 Concentration (mM) Figure 4.19 Calibration curve of 44 + from standard solution 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 The application method for determine ammonia in water sample by fluorescence spectrophotometer using NDA was successfully with the result of relative error = 2.57% for nucleophile is sulfite and 1.19% for cyanide Base on the result of comparison between the slope of the curve and correlation coefficient in figure 4.1.6 and 4.2.5 this research was demonstrable capacity of NDA in determine ammonia by fluorescence with advantage are highly sensitive save time, reproducible, and selective By the result of relative error in determine ammonia using fluorescence spectrophotometer system higher than sulfite of cyanide and the capacity apply in HPLC system Cyanide seem more suitable to react with NDA than sulfite in determine ammonia method Although this method might have some negative point made the result of solution not clear enough but it provide information to other method intended for develop using NDA to determine ammonia in other method or with other purpose 45 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 46 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) Carlson, R G., Srinivasachar, K., Givens, R S & Matuszewski, B K (1986) New Derivatizing Agents for Amino Acids and Peptides.1.Facile Synthesis of N-Substituted 1Cyanobenz[f]isoindoles and Their Spectroscopic Properties American Chemical Society, pp 3978-3983 Chisholm, H (1911) Ammonia Encyclopædia Britannica (11th ed.) Cambridge University Press Clare, S (2015) Disadvantages Advantages of an HPLC Retrieved http://www.ehow.com/list_5911530_disadvantages-advantages-hplc.html from: (accessed on 13/06/2015) Erika, P F., & Arnaldo, A (2012) Cardoso A Method for Determination of Ammonia in Air using Oxalic Acid-Impregnated Cellulose Filters and Fluorimetric Detection, Journal of the Brazilian Chemical Society 23(1), pp 142- 147 Irina, T , Ilnur, Kh , Mihail, K , Aleksey, M , & Andrey, B (2015) Automated procedure for determination of ammonia in concrete with headspace single-drop micro-extraction by stepwise injection spectrophotometric analysis Talanta , pp 34-37 47 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 American Chemical Society, pp 411-415 Monica, Z B (2006) Ion Chromatography Retrieved http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html from: (accessed on 7/08/2015) Petr, Z (2004) Reactions of Orthophthalaldehyde with Nucleophiles American Chemical Society, pp 3217–3238 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 Amines American Chemical Society, pp 1096 - 1101 Thomas, F.G & Henze, G (2001) Introduction Voltammetry analysis theory and practice Retrieved from:https://books.google.com.vn/books?id=yoFKXm0ssIC&pg=PA60&dq=voltammetry +%C3%A2nlysis&hl=en&sa=X&ved=0CCoQh3QFg2M#v=onepage&q&f=false (accessed on 19/07/2015) 48 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 derivatives by HPLC with fluorescence detection Journal of Chromatography B, pp 116 127 Zotou, A & Notou, M (2012) Study of the naphthalene-2,3-dicarboxaldehyde pre-column derivatization of biogenic mono- and diamines in mixture and fluorescence-HPLC determination Euroanalysis XVI, pp 1039–1048 49 APPENDICES Fluorescence spectrophotometer machine HPLC fluorescence machine 50 Solvent of HPLC system Warm up system 51 Stored Working Solution with Ice Borate buffer working solution NDA working solution 52 Deionized water Sodium sulfite working solution Sodium cyanide working solution Ammonium chloride working solution 53 ... quantum yields Ammonia have similar configuration with amino acid Thus, this project conducted Determination of Ammonia in Water Samples Using NDA Fluorescence Derivatization with Different Nuclephiles”... Standard Solution 40 Figure 4.16 Determine Ammonia By HPLC -Fluorescence Using NDA4 1 Sulfite With Solvent DIW And Methanol Figure 4.17 Determine Ammonia By HPLC -Fluorescence Using NDASulfite With. .. absorption maxima of solution, analyzing the determination capacity of solution by using Excel 3.3 HPLC system In this part to using HPLC to determine ammonia we also use pertinent parameters of concentration