Determination of ammonia in water samples using NDA fluorescence derivatization with different nucleophiles

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 This stud

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

Thai Nguyen, 9/2015

Thai Nguyen University of Agriculture and Forestry

Fluorescence Derivatization with Different Nucleophiles

- 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,

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

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

Ammonia, Cyanide, Sulfite

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 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

TABLE OF CONTENT

LIST OF FIGURES 1

LIST OF TABLE 3

PART I INTRODUCTION 5

1.1.Research rationale 5

1.2.Objectives of the research 6

1.3.Research questions and hypothesis 6

1.4.Limitations of research 6

PART II LITERATURE REVIEW 7

2.1 Ammonia 7

2.1.1 The properties of ammonia 7

2.1.2 Origins of ammonia pollution in water 8

2.2.Methods for determinate ammonia 9

2.2.1.Ion Chromatography 9

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

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 NDA- -sulfite product 28

4.1.3 Effect of reaction time on the fluorescence of NDA- -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 product 36 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

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

LIST OF FIGURES

Page

Figure 4 2 Effect Of Reaction Ph On The Fluorescence Of NDANH

Figure 4 8 Determine Ammonia By Fluorescence Using NDA-Sulfite 33

Figure 4.11 Effect Of Reaction Time On The Fluorescence Of NDANH

Figure 4.12 Effect Of Reaction Temperature On The Fluorescence Of

Figure 4.13 Effect Of Ratio Between NDA And Cyanide On The

Figure 4.16 Determine Ammonia By HPLC-Fluorescence Using

Figure 4.17 Determine Ammonia By HPLC-Fluorescence Using

Figure 4.18 Determine Ammonia By HPLC-Fluorescence Using

LIST OF TABLE

Table 1.1 Physical and chemical properties of ammonia

8

20

PART I INTRODUCTION

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 O-Phthaladehyde (OPA) has been the most widely used in this method because of its low cost, rapid reaction and highly fluorescent adduct (absorption maxima at 230 and 340 nm

and emission maximum at 455 nm) 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

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:

Because the thesis training time was too short, this research project is incomplete

PART II LITERATURE REVIEW.2.1 Ammonia

2.1.1 The properties of ammonia

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 0.2 mg / liter (from 21,5 µg/l to

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

The toxicity of ammonia is critically dependent on pH and temperature The un-ionized

Physical and chemical properties of ammonia:

Table 1.1 Physical and chemical properties of ammonia ( Chisholm, 1911)

2.1.2 Origins of ammonia pollution in water

The ammonium ion is the dominant form of ammonium, while ammonia is a minor

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)

2.2 Methods for determinate ammonia

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

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)

Moreover, ion chromatography is considered the method has many positive points such as:

reduced to about 10 minutes

the medium and lower ppb concentration range without pre-concentration

may be obtained within a short time

- Specific compound classes such as heavy metals, alkaline-earth metals, polyvalent anion, silicate, etc can be detected with high selectivity, this high degree of selectivity facilitates the identification of unknown sample components

Although, ion chromatography has some advantage on average analysis time, high sensitivities, simultaneous detection, but it also has some limited such:

reparation requires only three minutes Thus, quantitative results are obtained in a fraction

of the time previously necessary using traditional wet chemical methods, so increase sample throughput

some compound

toward organic solvent, so cannot be used for removal of organic contaminants.(Weiss, 2004)

Base on the advantage and disadvantage we recommend that ion chromatography method

Voltammetry is electro analytical methods used for analytical chemistry and various industrial processes In voltammetry, information about an analytic is obtained by measuring the current as the potential is varied (Thomas, 2001) Voltammetry techniques are based on the recording of the current, which flows between the working electrode and

an auxiliary electrode, due to the reduction or oxidation of the test element An important

factor which influences the efficiency of a particular voltammetry analysis (limit of detection, sensitivity and resolution of adjacent current signals) is the degree of reversibility of the electrode process

for determine chemical compound However, in determine ammonia the detection limit of electrode is quite ppb might need long time to balance, and we need large amount of

Fluorescence derivatization-HPLC is method have widely application in chemical analysis It allows the separation of chemical constituents through the use of a mobile phase and a stationary phase

Base on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material Each component in the sample interacts slightly differently with the absorbent material, causing different flow rates for the different components and separating out as they move at different speeds The process can be completed in roughly 10 to 30 minutes, and it delivers high resolution Therefore, it versatile and extremely precise when apply to identifying and quantifying chemical components (Clare, 2015) Beside that advantage HPLC also have disadvantage is can be costly, requiring large quantities of expensive organics and it does have low sensitivity for certain compounds, some cannot be detected as they are irreversibly adsorbed In HPLC have three major variables affecting fluorescence are solvents, temperature, pH (Kuo, 2005), so we need optimize that factor of sample before inject to HPLC system

Nowadays, the application of HPLC to determine ammonia and organic matter

drawbacks make it less than an ideal method For example, the isoindole produced in the derivatization are relatively unstable and decompose to non-fluorescent products Furthermore, it has been reported that OPA fails to give fluorescent products with small

that affect, today in some research, scientist was change OPA by NDA one compound with structure 2-substituted Benz and 2 group CHO similar with OPA but it was anticipated that it would have greater aqueous solubility and more stably

Nowadays, to develop quality of derivatization reaction, many reagents have been used for derivatization of amines, amino acids and related compounds, for example, fluorescamine, dansyl Chloride, 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F), o-

reagent has some limitation such as need long reaction time, unstably, or high cost… Thus,

in this part we present about properties of two reagents have widely using in fluorescent to compare advantage and disadvantage of that reagent

The molecule is a dialdehyde, consisting of two formyl (CHO) groups attached to adjacent carbon centres on a benzene ring This pale yellow solid is a building block in the synthesis of heterocyclic compounds and a reagent in the analysis of amino acids OPA

dissolves in water solution at pH < 11.5 Its solutions degrade upon UV illumination and exposure to air The physical and chemical properties of OPA show in table 2.3.1

In reaction with amines OPA have outstanding advantages than other derivatization reagents is quickly and exhibits greater fluorescence quantum yields, the derivatization

have successful in determine ammonia using ion chromatography employing post-column

Although, OPA have some positive point in fluorescence and has become a standard method for the very low-level analysis of ammonia, amines and amino acids However, it also has some drawbacks negative For example, the isoindoles 2 produced in the derivatization are relatively unstable and decompose to non-fluorescent products Furthermore, it has been reported that OP fails to give fluorescent products with small peptides, thus limiting the applications to simple amino acids (Carlson, 1986) Moreover,

it is easy to determinate ammonia by ion chromatography(IC) But when we use IC to

method to determinate ammonia since Na+ cannot derivated with OPA

Table 2.1 Physical and chemical properties of o-phthaldialdehyde (Bill, 1954)

Naphthalene-2,3-dicarboxaldehyde(NDA) is the chemical compound with the formula C12H8O2, is shown to be a very useful reagent for the derivatization of primary amines, amino acids, and small peptides The reaction of these amino compounds with NDA and cyanide ion produces highly fluorescent, and relatively stable Moreover, based on some research about determine amines and acid amine by fluorescence using reagent is NDA with Nucleophile is CN recommend that NDA have fluorogenic properties as OPA and adding certain new features such as greater solution stability and higher fluorescence efficiency of the product isoindole, derivatization of small peptides, and greater efficiency

in the isoindole formation reactions

However, NDA is no longer commercially available and is not readily available, it was synthesized from 2,3-dimethylnaphthalene by the method of Ried.(the method to synthesis NDA show in figure2.3.2), Therefore, costs of experiment could be affected

Figure 2.1 synthesis NDA: (i) NaI, DMF; (ii) NaOH, HzO, H30+; (iii) LiAlH,, EtzO;

(iv) oxalyl chloride, Me,SO, CH2C12.(Carlson, 1986)

Base on the advantage and disadvantage of NDA and OPA in derivatization reaction

to determine amines and amino acids, NDA are suitable to apply in analysis ammonium using fluorescence But as mentioned in part 2.2,3 that HPLC system can be effect by temperature and reaction time and pH so optimization the concentration of method after inject to HPLC system to analysis are necessary

PART III METHODS

- A vortex machine

Figure3.1 Fluorescence Spectrophotometer system

High Performance Liquid Chromatography – HPLC system:

allows including a low-pressure gradient unit Flow rate range: 0.001 to 10.000 mL/min

collection interface card and related software (Model 0224-2, chromatography data station for HPLC, Scientific information service corporation)

Figure 3.2 HPLC fluorescence system diagram

3.2 Methods

3.2.1 Determine the optimum concentration

Preparation of stock solution

better when stored solution in refrigerate

solution better when stored in refrigerate and using around 5day to 1 week, after that should prepare new solution

0.01694g of NDA in 9.197 mL of MeOH stored this solution in glasses jar 10ml enclose outside by Foil paper, then put this solution in refrigerate

• Prepare Cyanide

Prepared Cyanide(10mM) by dissolving 0.0245g of sodium cyanide (NaCN) in 50 mL of DIW and adjusting the pH to the desired value with either perchloric acid (0.1 N) or sodium hydroxide (0.1 N), in this solution

This buffer prepare by dissolved 0.06183g H BO in about 80 mL DIW, then adjust pH to 9.00 with 7.0 N NaOH(aq), and graduate to 100mL (the reaction as shown in Eq 3-1 and the final concentration is 10mM) The solution is filtered with a 0.22 m PVDF filter to remove the impurities Stored this solution in refrigerate

solution is stored in refrigerate and filtered through a 0.45 m membrane to remove the impurities before using

Experimental procedures

solutions

HPLC system

- To warm up temperature of sample to be temperature we need setup diagram include Magnetic stirrer, Magnetic ball, Thermometer to boil water like figure 3.2.1 diagram

Figure 3.3 warm up sample solution

Stock

solution

Initial conc

Adding volume (µL)

Ratio:

add/total

Dilution solvent

Dilute fold (x)

Final conc

Total volume

distilled

Table 3 1 dilute working solution

Other compound can use stock solution to be working solution

3.2.2 Fluorescence Spectrophotometer

In this research to using fluorescence spectrophotometer for determine ammonia-NDA with different Nuclephiles, the optimized condition of Ex/Em wavelength of NDA derivative NDA reagent pH, reaction time and buffer concentration need to normalizing

Cary Eclipse and calibrate to zero

is 2000 &

fluorescence spectrophotometer Put glasses tubes into holder and start scan from 200 to 800nm to find out Ex/ Em wavelength with auto scan mode of the spectrophotometer

spectrophotometer system for experiment

+ Click set up → auto store → ASCII (CSV)

+ Scan set up → set up Ex/Em

+ Option → medium, over lay → OK

record the intensity of sample after 1 minute

1mM, 2mM and blank)

add different ratio of sodium sulfite with NDA (1:0.1, 1:0.2, 1:1, 1:5, 1:10) from sodium

solution is 2000 &

solution then warm up at 60 ° and start find time reaction with auto scan mode of the

spectrophotometer record the intensity of sample after 5 minute (5, 10 ,15, 20 ,25, 30, 35,

40 minute)

Optimization of Pertinent Parameters for NDA-(# Reaction

is 2000 &

fluorescence spectrophotometer system and find Ex/Em like NDA-NH3 Reaction

spectrophotometer system

add 2mM NDA and add 1mM of ammonia Mix solution then put to spectrophotometer machine and set up auto model record the intensity of sample after 5 minute

(10mM) stock solution and add different ratio of sodium cyanide with NDA (1:0.1, 1:2.5,1:5, 1:10, 1:20) from sodium sulfite (20mM) stock solution, then add 2mM=400