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 n 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. WU ,Chien-Hou Supervisor (s): - Assoc. 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: 53 Date of Submission: September, 30, 2015 ii n 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 do 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.
Wu Chien Hou, from Biomedical Engineering & Environmental Science Department, National Tsing Hua University and Assoc. 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 n TABLE OF CONTENT LIST OF FIGURES. 1 LIST OF TABLE .Objectives of the research .Research questions and hypothesis .Limitations of research .1 The properties of ammonia.
Origins of ammonia pollution in water .Methods for determinate ammonia .Overview of research and application of NDA in fluorescence. Determine the optimum concentration. NDA-ammonia-sulfite reaction .1 Optimization Excitation and Emission wavelength. Effect of pH value on the fluorescence of NDA- -sulfite product.
Effect of reaction time on the fluorescence of NDA- -sulfite product. Effect of reaction temperature on the fluorescence of NDA- -sulfite product. Effect of ratio between NDA and sulfite on the fluorescence of NDA- -sulfite product .6 Apply Pertinent Parameters on determine ammonia. NDA-ammonia-cyanide reaction.1 Optimization Excitation and Emission wavelength .2 Effect of reaction time on the fluorescence of NDA- -Cyanide product .3 Effect of reaction temperature on the fluorescence of NDA- -Cyanide product36 4.
Effect of ratio between NDA and cyanide on the fluorescence of NDA- -sulfite product .5 Apply Pertinent Parameters on determine ammonia .3 Determine ammonia using HPLC-fluorescence system .1 NDA-ammonia-sulfite reaction .2 NDA-ammonia-cyanide reaction. DISCUSSION AND CONCLUSION. 47 vi n 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 4. 2 Effect Of Reaction Ph On The Fluorescence Of NDA-NH - 28 Sulfite Product Figure 4.
3 Effect Of Reaction Time On The Fluorescence Of NDA-NH - 29 Sulfite Product Figure 4. 4 Effect Of Reaction Temperature On The Fluorescence Of NDA- 30 NH -Sulfite Product Figure 4. 5 Effect Of Ratio Between NDA And Sulfite At NDA=0. 6 Effect Of Ratio Between NDA And Sulfite At NDA=1mm 32 Figure 4.
7 Effect Of Ratio Between NDA And Sulfite At NDA=2mm 32 1 n Figure 4. 8 Determine Ammonia By Fluorescence Using NDA-Sulfite 33 Figure 4. 9 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 38 Fluorescence Of NDA-NH -Cyanide Product.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 NDA- 41 Sulfite With Solvent DIW And Methanol Figure 4.17 Determine Ammonia By HPLC-Fluorescence Using NDA- 42 Sulfite With Solvent Acetate Buffer And Methanol Figure 4.18 Determine Ammonia By HPLC-Fluorescence Using NDA- 43 Cyanide With Solvent Acetate Buffer And Methanol Figure 4.19 Calibration Curve Of NH + From Standard Solution 44 2 n LIST OF TABLE Table Page Table 1.1 Physical and chemical properties of ammonia 8 Table 2.1 Physical and chemical properties of o-phthaldialdehyde 14 Table 3.1 dilute NH working solution 20 Table 3.2 HPLC-Fluorescence instrument setting condition 25 3 n 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 4 n PART I. Research rationale Ammonium ion is an important micronutrient significantly contributing to the nutritional needs for terrestrial organisms and intermediate of the nitrogen cycle in aquatic ecosystem.
Ammonia also plays an important role in fertilizer industry, providing fuel for production of nitrogenous fertilizers about 83% of total consumption of ammonia. However, Ammonium is a common alkaline pollutant in water; it directly impacts the habitat of aquatic organisms in water. Increasing amount of ammonia in water leads to ammonia excretion in organisms and level of ammonia in blood and tissue will increase. Therefore, increase blood pH, has negative impact to enzyme catalyzed reaction and fastness of cell membrane.
Ammonia increases the oxygen demand in the tissue, injured membrane and reduced oxygen transport capacity of blood. So control status of ammonia discharged into the environment and the development of a sensitive and selective analytic method for ammonium is one matter interested in protect aquatic ecosystem. In recent years, different methods and techniques have been developed for the analysis of ammonium ion, such as fluorescence, voltammetry, and ion chromatography. Especially, the fluorescence method has been in use by physical chemists in general and analytical chemistry in particular for many years by advantage of the intrinsically high sensitivity and help scientist save time in study.
Numerous useful reagents have also been developed to take advantage of the intrinsically high sensitivity of fluorescence detection such as dansyl chloride, fluorescamine, nitrobenzoxadiazoles , and o-phthaladehyde. Nevertheless, precolumn derivatization methods are used the reported detection limits with OPA and conventional (nonlaser) fluorescence detection are in the vicinity of 100 nmol. One such reagent which appears to have excellent potential for primary amino acids and peptides is 2,3-naphthalenedialdehyde (NDA), which when used in the presence of cyanide as the nucleophile, yields adducts of excellent thermal stability and high 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” to find which nucleophile have high performance in NDA- Reaction. Objectives of the research The aim of this work was to evaluate the analytical performance of NDA in determine ammonia using fluorescence system and to find out which nucleophile are suitable with that method. The specific objectives of this study are: − To optimize of pertinent parameters of The NDA- Reaction − To evaluate applicability of NDA- Reaction in HPLC system 1. Research questions and hypothesis 1.
Does fluorescence can determine ammonia by using NDA reagent? 2. How to optimize of pertinent parameters of the NDA- reaction? 1. Limitations of research Because the thesis training time was too short, this research project is incomplete.1 The properties of ammonia Ammonia is a compound of nitrogen and hydrogen with the formula. Ammonium is present at the environment have origin from conversion process, agriculture, industry and from sterilization of water by chloramine.
The extent of its toxicity to aquatic life depend upon the degree of ionization, which in turn depends upon the temperature, the pH, and the concentration of dissolved salts in the water Natural amount of ammonium in surface water and ground water are usually lower than 0. Ammonia can enter environmental water as a product of anaerobic decomposition of nitrogen-containing compounds or from waste streams containing ammonia. The livestock large scale can increase ammonia amount in surface water. Ammonium contamination may increase due to pipe connection by cement mortar.
The amount of ammonia in the environment compared with the synthesis inside body is negligible. It’s just affect to body when dosage over 200 mg / kg body weight. The toxicity of ammonia is critically dependent on pH and temperature. The un-ionized form ( ) is more toxic than the ionized form ( ).
As pH increases, is converted to ( ) and the toxicity increases. Higher temperatures also favor the more toxic form. (California Environmental Protection Agency, 2011) Physical and chemical properties of ammonia: 7 n Table 1.1 Physical and chemical properties of ammonia (Chisholm, 1911) Property Value Melting point −77.81 K) Density of vapor 0.6 g/L at 20 °C Water solubility 421 g/L at 20 °C Vapor pressure 882 kPa at 20 °C Equilibrium constant 1. Origins of ammonia pollution in water The ammonium ion is the dominant form of ammonium, while ammonia is a minor component.
Ammonia dissolve in water become ammonium hydroxide ( OH) and then separate to ammonium ion ( ) and hydroxide ions (OH-). There are many causes of the contamination status of ammonium and organic substances in water but one of the mainsprings is because using large amount organic fertilizer, pesticide, chemicals, vegetable in agricultural activities, livestock. It has impact seriously to water resources. The decomposition of organic compounds and that Substance can promote ammonia contamination process in ground water.
Moreover, sewage from daily life and food industry also is Emission sources ammonia to environment, concentration of ammonia in that water really high (approximate 100 mg/L). Methods for determinate ammonia Nowadays several methods have been developed to determine ammonia. In this part, first I will review some methods widely used in determine analytical chemistry in general and determine ammonia in particular. Next, the advantages and disadvantages of these methods will be compared with method we use in this study to see which methods are more suitable 2.
Ion Chromatography Ion Chromatography is method use for water chemistry analysis. It’s used to separate the individual cations and anions in solution and quantitatively determine their concentrations. Ion chromatographs are able to measure concentrations of major anions, such as fluoride, chloride, nitrate, nitrite, and sulfate, also major cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium in the parts-per-billion (ppb) range.