chapter 2 - Shodhganga

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In an acid medium, nitrite reacts with primarily aromatic amines to form diazonium salts, the salt is then coupled with suitable aromatic compounds to yield azo ...
CHAPTER 2 SPECTROPHOTOMETRIC DETERMINATION OF NITRITE BY DIAZOTIZATION METHOD

2.1

INTRODUCTION

2.2

ANALYTICAL CHEMISTRY

2.3

APPARATUS

2.4

REAGENTS AND SOLUTIONS

2.5

PROCEDURES

2.6

RESULTS AND DISCUSSION

2.7

APPLICATIONS

2.8

CONCLUSIONS

2.9

REFERENCES

20

2.1 INTRODUCTION Nitrogen compounds are of interest to scientists because they are essential nutrients ie, beneficial to living organisms, but at the same time they can be pollutants in the environment, with potentially harmful effects [1-5]. In the environment, nitrogen exists in different oxidation states including ammonium (NH4+), nitrates (NO3-) and nitrites (NO2-). Thus nitrate and nitrite are nitrogen species, which occur naturally in water and soil and are essential nutrients for plants [6-8]. Nitrate and nitrite contain a nitrogen atom joined to three and two oxygen atoms respectively. Both are anions and carry one unit of negative charge. Hence they very easily associated with cations or ions with a positive charge to achieve neutral state. Sodium nitrite is used industrially [9] for the synthesis of hydroxylamine and its derivatives, organic nitriles and nitro-compounds and particularly in diazotization reaction leading to azo dyes and pharmaceuticals. Nitrate and nitrite are found commonly in ground water than in surface water. They are more commonly detected as contaminants in well water. Principal sources of nitrate or nitrite contamination are fertilizers, septic tank waste, livestock manure and erosion of natural deposits. Surface water run-offs greatly increase nitrate/nitrite concentrations above the admissible levels for drinking water quality [10]. The nitrite is of major concern because it is significantly more toxic than ammonia or nitrate. [11-15]. It is an environmental concern due to its harmful effects to aquatic plants, animals and to human health [13]. The main source of nitrite in the environment is the microbial oxidation of ammonium ions, particularly of the genus nitrosomonas, equation (1), or the reduction of nitrates in water bodies by the denitrification bacteria (Pseudomonas and Achromobactor), equation (2) [16]. +

2NH 4 + 3O 2

C6H12+ 12NO3

-

+

2NO 2 + 4H + 2H 2O

(1)

-

(2)

-

12NO2 + 6CO 2 + 6H 2

Nitrites are mainly used as a food preservative. Both, nitrates and nitrites are used to enhance the color and extend the shelf life of processed meat. It is also a component of salt mixtures, which is used for curing bacon and ham in the food

21

industry [17]. Nitrite combines with myoglobin to form nitrosohaemoglobin, which is responsible for the characteristic red color in cured meat and hence it is some time used as meat preservative. These compounds are also used in various chemical production and separation. Industrial effluents and the nitrogen cycle add to contamination of the environment with nitrite [13]. Nitrite is undesirable in water owing to its toxicity. The maximum permissible concentration of nitrite in potable water prescribed by the US Public Health Service is 0.06 mg L-1 (21, 22). Under ordinary conditions nitrite levels in water are low, generally < 0.1 µg mL-1. However, the increasing use of nitrite as a preservative in the food industry and as a corrosion inhibitor in industrial processes has resulted in the increased levels of nitrite to water bodies (18-21). Concern about higher nitrate (nitrite) intake is based on a concern for infant health; as the infant feed made with water containing more than 50 mg nitrate (nitrite) per liter is believed to have been the cause of methamoglobinemia in infants. Methaemoglobinemia is a disease characterized by cyanosis, a bluish coloration of the skin, hence is also known as “blue baby” syndrome [11]. This is due to the fact that nitrite at high levels can cause mathamoglobinaemia which causes reduction in the ability of hemoglobin in red blood cells to carry oxygen [11]. Also, the cattle have digestive systems capable of supporting nitrate-reducing bacteria and they too can suffer from methaemoglobinaemia, if nitrate (nitrite) levels are high in their drinking. Thus nitrite is considered potentially hazardous to health due to its interaction with hemoglobin in blood preventing oxygen carrying ability of the hemoglobin [22]. Nitrite is not a direct toxicant, but may be dangerous in the form of N-nitroso compounds produced upon its interaction with proteins. In the recent years there has been an increasing about the role of nitrite, as an important precursor in the formation of N-nitrosamines (3) that possesses tetratogenic and mutagenic activities and hence are potential carcinogens (23-24). Owing to its toxicity, nitrite is a characteristic pollutant and toxicant [25]. R2NH + HCl + NaNO 2

( Dialkylamine; R=alkyl group)

R2N-N=O + NaCl + H 2O

(N-Nitrosodialkylamine) 22

(3)

The formation of N-nitrosamines, generally takes place at physiological pH in a variety of animals and is suspected of causing cancer in humans as well. Thus the second concern is that nitrate (nitrite) may react with the natural amines in food components in the stomach to form carcinogenic compounds affecting, especially stomach, liver and esophagus. Generally, humans are exposed to nitrite through the ingestion of vegetables, water and cured meats (6,14,26). The occcurance of nitrite in the drinking water and vegetables has been a matter of growing concern that has attracted the attention of analytical chemists. It is therefore important that sensitive methods are devised for the determination of trace concentrations of nitrite in environmental water and vegetable samples. Nitrite originates in the sub surface through the microbiological degradation of organic nitrogenous material including human and animal wastes, as well as agricultural, fertilizers and plant refuses. The ammonium ion, NH4+, generated from microbiological decomposition, is oxidizes to yield NO2- and NO3-. The three main sources for nitrite and nitrate in most soil are: 1.

The microbiological breakdown of soil organic matter, organic manures and plant residues.

2.

Nitrogenous fertilizers, which add nitrate and that formed by the microbiological oxidation of NH4+ from ammonium fertilizers or urea.

3.

Additions from atmosphere, deposited as NH3, NH4+ and NO3-. According to this source, the fatal dose of potassium nitrate for adult

humans is in the range of 30 to 35 g consumed as a single dose; the fatal dose of sodium nitrite is in the range of 22 to 23 mg kg-1 of body weight. Lower doses of sodium

or

potassium

nitrate

or

sodium

nitrite

have

caused

acute

methemoglobinemia (when hemoglobin loses its ability to carry oxygen) particularly in infants, resulting from conversion of nitrate to nitrite after consumption. There is no confirmable evidence in the literature on the carcinogenicity (cancer-causing capacity) of nitrate as such. The determination of nitrite in water and food samples is fraught with a number of difficulties related primarily to the reactivity of nitrous acid. As nitrite readily undergoes diazotization, it forms the basis of the most common method for

23

nitrite determination involving UV-Vis spectrophotometric determination of the dye thus formed [27]. In an acid medium, nitrite reacts with primarily aromatic amines to form diazonium salts, the salt is then coupled with suitable aromatic compounds to yield azo dye, which is the basis of the UV-Vis spectrophotometric determination. [28, 29]. The classical Griess method 1879 is based on this principle. [29-31]. More than fifty different combinations based on formation of azo dye have been reported. Obviously, the more the reaction step involved greater is the chance of loss of nitrite (or reaction intermediates) due to possible side reactions, leading to lesser amount of nitrite determined. These methods are highly sensitive and specific to nitrite but are not applicable if too much nitrite is present due to occurance of side reactions. Modification of this method suffers with draw backs of the toxicity due to the reagents used (1- napthylamine as well as its other derivatives are carcinogens and their use is not recommended). Despite the simplicity and ease of application of the technique it is essentially time consuming, which makes it unsuitable for routine analysis of large numbers of samples. In addition

to

spectrophotometry,

chemiluminescence,

ion

chromatography,

fluorometry, capillary electrophoresis, potentiometry, amperometry, voltametry and flow injection analysis have also been used for determination of nitrite. However, some of these methods suffer from complicated and expensive instrumentation and costly chemicals, while others involve difficult and timeconsuming separation procedure, certain methods require high temperature. The properties of nitrite determine its usage and its chemical, analytical identification in the environment. Physically, nitrite is a colorless and odorless ion. Nitrite is a conjugate base of weak acid HNO2; pKa =3.4. It is highly soluble in water and mobile in the environment. Under normal conditions, nitrites are rapidly protonated and yield nitrous acid, HNO2. Nitrous acid further decomposes to nitric oxide and other nitrogen oxides as below: -

NO2 + H

+

Nitrite

2HNO2

Nitrous acid

HNO2

(pKa =3.4)

(4)

Nitrous acid

N 2O 3 + H 2O

(5)

Dinitrogen trioxide 24

N2O 3

Dinitrogen trioxide

NO 2

+

NO

Nitric oxide

(6)

Nitrogen dioxide

Generally, the presence of chlorides, some metals and organic material destabilize both nitrates and nitrites. Ammonium nitrite is unstable and decomposes to N2 and H2O. Nitrites oxidize slowly to nitrates when exposed to air. NH4NO2 -

2NO2 + O 2

N2

2H2O

+

2NO3

-

(7) (8)

Nitrates and nitrites have high potential for entering surface water when it rains as excess rain water flows in to streams or lakes. They also have high potential for entering ground water through leaching. Unlike ammonia, nitrites do not evaporate and remain in water until they are taken up by plants or consumed by microorganism [32]. Chemically, nitrite is moderately reactive. The nitrite ion contains nitrogen in a relatively unstable oxidation state. Chemical and biological processes can further reduce nitrite to various compounds or oxidize it to nitrate [33]. In other words it is unstable in the environment. It form compounds with organic or inorganic ionic species. In nature nitrites are readily converted to nitrates and vice versa. Nitrite oxidizes antioxidants, Fe2+ of hemoglobin to Fe3+. A review of the chemistry of nitrogen shows that nitrite reacts with primary and secondary amines to form different products such as alcohol and nitrogen gas, nitrosamines respectively. Similarly nitrites react with primary aromatic amine to form diazonium salt. The formation of diazonium salt with primary amine is the important feature of many types of analytical methods for nitrite and nitrate or aromatic amines. 2.2. ANALYTICAL CHEMISTRY Various methods have been reported for the quantitative determination of nitrite as well as nitrate. These include kinetic [34-37], chromatographic [38-40], potentiometric [41,42], amperometric [43], electrophoretic techniques [44], polarography [45], voltametry [46], fluorometry [47], biamperometry [48], flow injection spectrophotometry [49-51] and spectrophotometry [52-57]. AOAC

25

official method of analysis for nitrite and nitrate determination was based on the diazocoupling reaction between sulphanilamide and N-(1-napthyl) ethylene diamine dihydrochloride [58]. Nitrite was normally determined by the diazocoupling reaction using various

reagents

spectrophotometrically.

Fox

[59]

reviewed

many

spectrophotometric methods for the determination of nitrite. A glassy carbon electrode modified with alternated layers of iron(III) tetra(N-methyl-4-pyridyl)-porphyrin and copper tetrasulfonated phthalocyanine was employed for nitrite determination by differential pulse voltametry [60]. This modified electrode showed excellent catalytic activity for the nitrite oxidation. After optimizing the operational conditions, a linear response range from 0.5 to 7.5 -1

-1

was obtained.

Two spectrophotometric methods were developed for the determination of nitrite using dapsone

-naphthol and 4-amino-5-hydroxynapthalene-2,7-

disulphonic acid monosodium salt as chromogenic reagents with maximum absorbance wavelength at 540 and 520 nm respectively [61]. For the method that utilizes dapsone

-naphthol, the Beer's law range was obeyed between 0.05-

-1

0.8 µg mL with molar absorptivity of 5.74 ×104 L mol-1 cm-1. The second method that used dapsone with 4-amino-5-hydroxynapthalene-2,7-disulphonic acid monosodium salt, Beer's law was valid over the range 0.2-1.4 µg mL-1 with molar absorptivity of 2.44 × 104 L mol-1 cm-1. Rincol et al. reported the roles of pH extraction and colloidal protein solubility in the optimization of spectrophotometric nitrite determination in meat products via response surface methodology [62]. The influence of four critical factors such as sample weight/ borax reagent ratio (BR factor), ascorbic acid content (AR factor), neutralization with HCl 1 N (N R factor) and stirring extraction time (SET factor), was investigate in order to find the best conditions to develop the official ISO 2.918 spectrophotometric method to determine the residual nitrite content in meat products, using the response surface methodology as optimization tool.

26

Somnam et al. developed an automated hydrodynamic sequential injection system with spectrophotometric detection [63]. The determination of nitrite and nitrate in water samples by employing the Griess reaction was chosen as a model. Calibration graphs with linearity in the range of 0.7 - 4 2

-

3

-

, respectively, with a sample throughput of 20 h-1 for consecutive determination of both the species. Pons et al. reported a multi-pumping flow system for the spectrophotometric determination of nitrite and nitrate [64]. The method was based on the Griess-Ilosvay reaction. Calibration was linear up to 3.0 mg NO2- L-1 with a limit of detection (3sb/S) of 0.013 mg NO2- L-1 an injection throughput of 55 injections h-1 and a repeatability of 0.5% for the direct determination of nitrite. Two calibration graphs within the ranges 0.039-7.0 mg NO3- L-1 and 0.026-5.0 mg NO2- L-1 were run for the determination of nitrate and nitrite under reducing conditions, respectively. A limit of detection of 0.039 mg NO3- L-1 was obtained. Dayanand

and

Revanasiddappa

reported

a

method

for

the

spectrophotometric determination of nitrite [65]. The method was based on the reaction of nitrite with 4-aminoazobenzene under acidic conditions in the presence of a bromide ion allowing to complete the diazotization reaction almost instantaneously. The formed diazonium ion was then coupled with acetyl acetone to give bisazo dye in an aqueous alkaline medium having maximum absorption at 500 nm. The system obeyed the Beer's law within the concentration range of 0.19.0 µg of nitrite in the final sample volume of 10 cm3. Nine spectrophotometric methods based on new reactions for the determination of tracer amounts of nitrite in environmental samples were developed [66]. The methods were based on the oxidation of sulfanilamide, sulfadoxine or sulfamethoxazole by nitrite in hydrochloric acid medium and coupling with phenoxazine, 2-chlorophenoxazine or 2-trifluoromethylphenoxazine which yielded red colored derivatives having an absorbance maximum in the range 530-540 nm and were stable for about 4 h. Beer's law was obeyed for nitrite in the concentration range 0.13-

-1

.

Abdul Galil et al. reported a method for the spectrophotometric determination of nitrite by its decolorizing effect on peroxovanadate complex [67]. The method was optimized for effect of concentrations of ammonium

27

metavanadate, hydrogen peroxide, various acids, concentrations of sulphuric acid, order of reagents addition and color stability. The color of the complex was found to be stable for about 2 days, and the stability constant of the complex was also calculated by modified Job's method. The linearity range of the calibration graph was over 6.67-66.7 µg mL-1 of nitrite with molar absorptivity, 0.28×103 mol L-1 cm-1 and Sandell's sensitivity, 0.167 µg cm-2. Yu et al. reported study and spectrophotometric determination of nitrite with sulfanilamide and N-phenyl J-acid system [68]. At room temperature, in the presence of potassium bromide, nitrous acid reacted with sulfanilamide in the medium of thin hydrochloric acid. Then diazonium salt reacted with N-phenyl J-acid in the aqueous solution of sodium carbonate, forming colored azo compounds. The apparent molar absorptivity was 4.63 × 104 L mol-1 cm-1. The Beer's law was obeyed in the range of 0.003-0.7 mg L-1 Novel reagents for facile spectrophotometric determination of trace amount of nitrite in water and soil samples were reported [69]. The methods were based on the diazotization of either dapsone or metoclopramide, followed by coupling with max

of 437 and 411

nm, respectively. The molar absorptivity and Sandell's sensitivity were found to be 4.31 × 104 L mol-1 cm-1 and 1.07 × 10-3 µg cm

-2

for dapsone-benzoylacetone,

while that for metoclopramide- benzoylacetone was found to be 3.18 × 104 L mol-1 cm-1 and 1.45 × 10-3 µg cm-2. Mubarak et al. reported a method for the determination of trace levels of nitrite based on its catalytic effect on the oxidation of perphenazine with bromate in a phosphoric acid medium [70]. The reaction rate was monitored spectrophotometrically by tracing the formation of the red-colored oxidized product of perphenazine at 525 nm within 30 sec of mixing. The optimum reaction

-1

perphenazine, 0.4 mol L

-1

H3PO4 and 30

-1

mmol L bromate at 25°. A flow injection analysis system was built with a liquid core waveguide detector using an 80 cm Teflon AF-1600 capillary tube (2,2-bistrifluoromethyl-4,5difluoro-1,3-dioxole/tetrafluoroethylene) [71]. The system was applied to determine nitrite ion in river water samples. The lower limit of detection for nitrite was 2.1 nmol dm-3 (0.1 ng dm-3 as NO2-) and the relative standard deviation of

28

measurements was typically 0.56% (n = 5) at 0.21

-3

C. Cherian and

Narayana reported a spectrophotometric method for the determination of nitrite [72]. It was based on the reaction of nitrite with p-nitroaniline in acid medium to form diazonium ion, which was coupled with ethoxyethylenemaleic ester or ethylcyanoacetate in basic medium to form azo dyes, showing absorption maxima at 439 and 465 nm respectively. Spectrophotometric determination of nitrite in water samples with 4-(1-methyl-1-mesitylcyclobutane-3-yl)-2-aminothiazole was reported [73]. The azo dye, 4-(1-methyl-1-mesitylcylobutane-3-yl)-2-(p-N,Ndimethylazobenzene)-1,3- thiazole was synthesized with the reaction of 4-(1methyl-1-mesitylcylobutane-3-yl)-2-aminothiazole and N,N-dimethyl aniline in acidic medium. Obtained azo dye had been characterized by infrared (IR), 1H nuclear magnetic resonance (NMR), and microanalysis methods. The dye showed an absorption maximum at 482 nm. An overview was given of an experiment involving the reaction of the azo dye 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride with nitrite, which decreased the color of the dye [74]. The experiment was a powerful tool to teach many aspects involving optimization, spectrophotometry and kinetic analysis of real samples. Aydin et al. reported a method for the spectrophotometric determination of nitrite in water [75]. Nitrite reacts with barbituric acid in acidic solution to give the nitroso derivative, violuric acid. At analytical wavelength of 310 nm, Beer's law was obeyed over the concentration range 0.00-3.22 ppm of nitrite. The molar absorptivity was 15330 ± 259.7 (95%) with pooled standard deviation of 355.57 and R.S.D. of 2.32%. Spectrophotometric determination of trace amounts of nitrite in water and soil samples was reported using p-nitroaniline and citrazinic acid [76]. The Beer's law was obeyed over the concentration range of nitrites of 0.5-14.0 µg in the final volume of 10 mL. Centelles et al. reported spectrophotometric determination of nitrite in biological samples using 1,2-diaminoanthraquinone, potential application to the determination of nitric oxide synthase activity [77]. Absorbance was seen to change linearly with increasing nitrite concentrations in the (0.05-50 µM) range. Sreekumar et al. reported a simple diazotization reaction involving pnitroaniline and sulfanilamide with ethylacetoacetate as a coupling agent for the 29

determination of nitrite [78]. The absorbance was measured at 507 and 356 nm, respectively. The range of linearity for p-nitroaniline-ethylaceto acetate couple was 0.05-6.0 µg mL-1 of nitrite with molar absorptivity of 1.59x104 L mol-1cm-1; while that for sulfanilamide-ethyl acetoacetate couple was found to be 0.2-3.0 µg mL-1 and 1.22 x 104 L mol-1cm-1 respectively. A method proposed by Nagaraj et al. involved diazotization-coupling reaction between dapsone and N-(1-naphthyl) ethylene diamine dihydrochloride in a hydrochloric acid medium [79]. The molar absorptivity and Sandell's sensitivity were found to be 7.20×104 L mol-1 cm-1 and 0.0006 µg mL-1, respectively. The calibration graph was linear for 0.002-0.6 µg mL-1 of nitrite. Geetha and Balasubramaniam reported a simple and sensitive spectrophotometric method for the determination of nitrite using phenosafranin [80]. Diazotization of the primary amino group in the dye phenosafranine resulted in a decrease in absorbance at 520 nm. This decrease in absorbance was directly proportional to nitrite concentration, obeyed Beer's law in the range 0-12.0 µg of nitrite in a final aqueous volume of 25 mL with a relative standard deviation of 2.9% at 8 µg of nitrite (n = 10). A kinetic spectrophotometric method for the determination of nitrite was developed, based on the catalytic oxidation of bromocresol purple with KBrO 3 in 0.45 mol mL-1 H3PO4 medium [81]. Dong and Lu reported spectrophotometric determination of nitrite ion with acridine red based on the nitrosation reaction [82]. The method was based on the nitrosation reaction of acridine red with nitrite ion in hydrochloric acid medium. The decolorization of acridine red by the reaction with nitrite was used for the spectrophotometric determination at 525 nm. Dong et al. reported a method for the spectrophotometric determination of nitrite [83]. The method was based on the diazo-reaction of methylene violet with nitrite in hydrochloric acid medium. The L-1 NO2-. Gonçalves et al. reported a spectrophotometric method for the determination of nitrite using safranin as color reagent [84]. The reaction between nitrite and safranin produces a safranin-HNO2 species, which exhibits absorption peaks at 280, 349, 420 (shoulder) and 610 nm. The Lambert-Beer law was obeyed in the concentration range 7.0 x 10 -6 – 5.0 x10-5 M.

30

The present work describes the spectrophotometric determination of nitrite using nevirapine, frusemide and 5-methyl-4-{[(1E)-phenylmethylene]amino}-2,4 dihydro-3H-1,2,4-triazole-3-thione as coupling agents. The method is based on the reaction of nitrite with p-nitroaniline or p-phenylenediamine in acid medium to form diazonium ion, which is coupled with nevirapine or frusemide or 5-methyl-4{[(1E)-phenylmethylene]amino}-2,4 dihydro-3H-1,2,4-triazole-3-thione in basic medium to form azo dyes. The method has been successfully applied to the determination nitrite in water samples, soil samples and dietary supplements. Comparison of the spectrophotometric method with the earlier methods are shown in table 2. 2.3 APPARATUS 2.3.1 Spectrophotometer A SHIMADZU (Model No: UV-2550) UV-Visible spectrophotometer with 1 cm matching quartz cells were used for the absorbance measurements. 2.4 REAGENTS AND SOLUTIONS All reagents used were of analytical reagent grade and distilled water was -1

was prepared

the solution was diluted appropriately to get the working concentration. To this 2 mL sodium hydroxide solution was added to prevent nitrite decomposition and 1 mL of chloroform to prevent bacterial growth. Other reagents used were pnitroaniline (PNA) (0.05% in 2.5 M HCl), p-phenylenediamine (PPD) (0.05% in 2.5 M HCl), nevirapine (NEV) (0.2 and 0.3%) in 50 % ethanol, frusemide (FRU) (0.2% and 0.25%) in 50% ethanol, 5-methyl-4-{[(1E)-phenylmethylene]-amino-2,4 dihydro-3H-1,2,4 triazole-3-thione (MPAT) (0.2% and 0.3%) in 50% ethanol, sodium hydroxide (2M), EDTA (0.02 M) and sodium carbonate (1%). 2.5 PROCEDURE 2.5.1 Using p-Nitroaniline-Nevirapine as Reagents To an aliquot of sample containing 0.40-1.40 µg mL-1 of nitrite was transferred in to series of 10 mL calibrated flasks. To this solution 1 mL of pnitroaniline was added and the solution was shaken thoroughly for 2 minutes to allow the diazotization reaction for completion. Then 1 mL of 0.2% nevirapine and 1mL of ~2M NaOH solution were added and the contents were diluted to 10 mL 31

with distilled water in a standard flask. The absorbance of the colored dye was measured at 492 nm against the reagent blank. 2.5.2. Using p-Phenylenediamine-Nevirapine as Reagents To an aliquot of sample containing 1.00-9.00 µg mL-1 of nitrite was transferred in to series of 10 mL calibrated flasks. To this solution 1 mL of pphenylene diamine was added and the solution was shaken thoroughly for 2 minutes to allow the diazotization reaction for completion. Then 1 mL of 0.3% nevirapine and 1mL of ~2M NaOH solution were added and the contents were diluted to 10 mL with distilled water in a standard flask. The absorbance of the colored dye was measured at 678 nm against the reagent blank. 2.5.3. Using p-Nitroaniline-Frusemide as Reagents To an aliquot of sample containing 0.02-0.60 µg mL-1 of nitrite was transferred in to series of 10 mL calibrated flasks. To this solution 1 mL of pnitroaniline was added and the solution was shaken thoroughly for 2 minutes to allow the diazotization reaction for completion. Then 1 mL of 0.2% frusemide and 1mL of ~2M NaOH solution were added and the contents were diluted to 10 mL with distilled water in a standard flask. The absorbance of the colored dye was measured at 680 nm against the reagent blank. 2.5.4. Using p-Phenylenediamine-Frusemide as Reagents To an aliquot of sample containing 0.10-0.60 µg mL-1 of nitrite was transferred in to series of 10 mL calibrated flasks. To this solution 1 mL of pphenylene diamine was added and the solution was shaken thoroughly for 2 minutes to allow the diazotization reaction for completion. Then 1 mL of 0.25% frusemide and 1mL of ~2M NaOH solution were added and the contents were diluted to 10 mL with distilled water in a standard flask. The absorbance of the colored dye was measured at 692 nm against the reagent blank. 2.5.5. Using p-Nitroaniline- 5-Methyl-4-{[(1E)-phenylmethylene]-amino2,4 dihydro-3H-1,2,4 triazole-3-thione as Reagents To an aliquot of sample containing 0.40-2.00 µg mL-1 of nitrite was transferred in to series of 10 mL calibrated flasks. To this solution 1 mL of pnitroaniline was added and the solution was shaken thoroughly for 2 minutes to allow the diazotization reaction for completion. Then 1 mL of 0.2% 5-methyl-432

{[(1E)-phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3-thione and 1mL of ~2M NaOH solution were added and the contents were diluted to 10 mL with distilled water in a standard flask. The absorbance of the colored dye was measured at 395 nm against the reagent blank. 2.5.6. Using p-Phenylenediamine- 5-Methyl-4-{[(1E)-phenylmethylene]-amino2,4 dihydro-3H-1,2,4 triazole-3-thione as Reagents To an aliquot of sample containing 1.00-3.00 µg mL-1 of nitrite was transferred in to series of 10 mL calibrated flasks. To this solution 1 mL of pphenylene diamine was added and the solution was shaken thoroughly for 2 minutes to allow the diazotization reaction for completion. Then 1 mL of 0.3% 5methyl-4-{[(1E)-phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3-thione and 1mL of ~2M NaOH solution were added and the contents were diluted to 10 mL with distilled water in a standard flask. The absorbance of the colored dye was measured at 668 nm against the reagent blank. 2.5.7. Nitrite in Water Samples Aliquots of water samples were treated with 0.5 mL 1M NaOH and 0.5 mL 0.2M EDTA and shaken well. The precipitate formed was removed by after centrifugation. The centrifugate was transferred to a 10 mL calibrated flasks. They all tested negative. To these samples a known amount of nitrite (containing not more than 1.40 µg mL-1 of nitrite for PNA-

-1

of nitrite for

-1

PPD-NEV system, 0.60 µg mL of nitrite for PNA-FRU system, 0.60 µg mL-1 of nitrite for PPD-FRU system, 2.00 µg mL-1 of nitrite for PNA-MPAT system, 3.00 -1

of nitrite for PPD-MPAT system) was added. Aliquots of the made up

solution containing nitrite was determined directly according to the proposed methods and and also by the reference method [78]. The results are summarized in the table 2.1A, 2.1B, 2.1C, 2.1D, 2.1E and 2.1F. 2.5.8. Nitrite in Soil Samples About 1.0 g of soil sample was weighed and placed in a 50 mL beaker and extracted three times with 5 mL portions of 1% sodium carbonate solution. The extract was filtered through Whatman 41 filter paper and suitable aliquots of the sample solution were analyzed. All the tested samples gave negative results. To these samples a known amount of the nitrite was added and analyzed for nitrite by 33

the proposed method and also by the reference method [78]. The results are summarized in the table 2.1A, 2.1B, 2.1C, 2.1D, 2.1E and 2.1F. 2.5.9. Nitrite Determination in Pharmaceutical Samples Isosorbide dinitrate (0.05 g) sample was taken, and was reduced to nitrite using Zn/NaCl and the clear solution was made up to 100 mL using distilled water. Known amount of this solution was taken and analyzed for nitrite content following the procedure described for the analysis of water sample. The results are summarized in the table 2.1A, 2.1B, 2.1C, 2.1D, 2.1E and 2.1F. 2.6. RESULTS AND DISCUSSION 2.6.1. Absorption Spectra 2.6.1.1 Using p-Nitroaniline- Nevirapine or Frusemide or 5-Methyl-4-{[(1E)phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3-thione as Reagents This method involves the diazotization of p-nitroaniline in acid medium followed by coupling with nevirapine or frusemide or 5-methyl-4-{[(1E)phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3-thione in alkaline medium to give a colored dyes (scheme 2.3A, 2.3B and 2.3C). The azo dye formed by p-nitroaniline and

nevirapine or frusemide or 5-methyl-4-{[(1E)-

phenylmethylene]-amino-

2,4 dihydro-3H-1,2,4 triazole-3-thione has an

absorption maximum at 492 or 680 or 395 nm respectively against the reagent blank.

2.6.1.2 Using p-phenylenediamine- Nevirapine or Frusemide or 5-Methyl-4{[(1E)- phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3thione as Reagents This method involves the diazotization of p-phenylenediamine in acid medium

followed by the coupling with nevirapine or frusemide or 5-methyl-4-

{[(1E)- phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3-thione in alkaline medium to give a coloured dye. The azo dye formed by pphenylenediamine

and

nevirapine

or

frusemide

or

5-methyl-4-{[(1E)-

phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3-thione has an absorption maximum at 678 or 692 or 669 nm respectively against the reagent blank and the reaction stoichiometry was found to be 1:2 [85].

34

2.6.2 Effect of Acidity and Reagent Concentration Preliminary investigations show that hydrochloric acid is better than sulphuric, phosphoric or acetic acid. The effect of acidity on the diazotization is studied in the range of 0.1-0.5 M of HCl, and constant absorbance is observed in this range. It is found that maximum color developed within 2 minutes and remained almost stable for about 1 h. Diazotization is carried out at room temperature and optimum acidity for the formation of diazonium chloride is fixed to be 0.2 M. It is found that 1 mL of 0.05% solutions of p-nitroaniline or pphenylenediamine is sufficient for complete diazotization. Nevirapine or frusemide or 5-methyl-4-{[(1E)- phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3thione is used as coupling reagents and 1 mL of nevirapine or frusemide or 5methyl-4-{[(1E)-

phenylmethylene]-amino-

2,4 dihydro-3H-1,2,4 triazole-3-

thione is sufficient for maximum color development. There is a decrease in absorbance at lower concentrations of nevirapine or frusemide or 5-methyl-4{[(1E)- phenylmethylene]-amino-2,4 dihydro-3H-1,2,4 triazole-3-thione where as higher concentration does not give good results. The effect of sodium hydroxide concentration on the absorbance is also studied, effects of addition of 0.5-2.0 mL of 2M sodium hydroxide are examined. The investigation shows that 1-2 mL of sodium hydroxide give maximum absorbance and 1 mL is chooses for the procedure. 2.7 Analytical Data 2.7.1. Using p-Nitroaniline- Nevirapine or Frusemide or 5-Methyl-4-{[(1E)phenylmethylene]-amino- 2,4 dihydro-3H-1,2,4 triazole-3-thione as Reagents Key parameters that influence the performance of the method are studied to arrive at the optimum working configurations. All the optimization steps are carried out with a chosen nitrite configuration. The range of linearity for PNANEV system, PNA-FRU system and PNA-MPAT system are found to be 0.40– 1.40 µg mL 1, 0.02–0.60 µg mL

1

and 0.40–2.00 µg mL

1

of nitrite respectively.

The molar absorptivity, Sandell’s sensitivity, detection limit and quantitation limit are found to be 6.60×103 L mol-1cm-1

-2

35

, 0.025 µg mL-1, 0.078 µg mL-

1

for PNA-NEV system, 1.46×103 L mol-1cm-1

-2

, 0.485 µg mL-1,

1.470 µg mL-1 for PNA-FRU system and 3.31×103 L mol-1cm-1 -1

-2

,

-1

0.559 µg mL , 1.695 µg mL for PNA-MPAT system respectively. Adherences to Beer’s law for the determination of above methods are shown in figure 2A, 2B and 2C. The absorption spectrums of azo dyes formed are presented in fig. II.A, II.B and II.C. 2.7.2 Using p-phenylenediamine- Nevirapine or Frusemide or 5-Methyl-4{[(1E)-phenylmethylene]-amino-2,4 dihydro-3H-1,2,4 triazole-3-thione as Reagents Key parameters that influence the performance of the method are studied to arrive at the optimum working configurations. All the optimization steps are carried out with a chosen nitrite configuration. The range of linearity for PPD-NEV system, PPD-FRU system and PPD-MPAT system are found to be 1.00-9.00 µg mL 1, 0.10–0.60 µg mL 1 and 1.00–3.00 µg mL 1 of nitrite respectively. The molar absorptivity, Sandell’s sensitivity, detection limit, quantitation limit are found to be 1.49×104 L mol-1cm-1

-2

, 0.015 µg mL-1, 0.045 µg mL-1 for PPD-NEV

system,. 7.670×103 L mol-1cm-1

-2

, 0.240 µg mL-1, 0.728 µg mL-1 for

p-phenylenediamine-frusemide system, 1.08×104 L mol-1cm-1 µg

mL-1,

1.396

µg

mL-1

phenylmethylene]-amino-2,4

for

p-phenylenediamine-

dihydro-3H-1,2,4

-2

, 0.460

5-methyl-4-{[(1E)-

triazole-3-thione

system

respectively Adherences to Beer’s law for the determination of above methods are shown in figure 2D, 2E and 2F. The absorption spectrums of azo dyes formed are presented in fig. II.D, II.E and II.F. 2.7.3 Effect of Interfering Species The selectivity of the proposed method is studied by determining the effect of various chemical species on the estimation of nitrite. The tolerance limit is defined as the concentration of added ion causing less than ±2% relative error for the nitrite determination. The present method is based on the oxidation pnitroaniline or p-phenylenediamine with nitrite then coupled with coupling agents. Therefore strong oxidizing or reducing species are expected to interfere. The results indicates that Hg2+, Ce4+, Cu2+ and Co2+ shows severe interference. However the tolerance level of these ions may be increased by the addition of 2mL

36

of 2% EDTA. Molybdate, thiocyanate and hexacyanoferrate are found to interfere and are masked by masking agents. The effects of diverse ions on the determination of nitrite are shown in table 2.2A and 2.2B. 2.8 APPLICATIONS The proposed method is applied to the determination of nitrite in water samples, soil samples and dietary supplements. The water samples are collected from different sources and are filtered before analysis. As the samples that are available are found to devoid of nitrite, synthetic samples are prepared by the addition of nitrite and then analyzed according to the proposed procedure. The performance of the proposed methods are compared statistically in terms of student’s t-values and the variance ratio F-test. At 95% confidence level, the calculated t value and F-value do not exceed the theoretical values for the two methods. The theoretical t- value is 2.31 and F-value is 6.39. The detection limit and quantitation limit are determined with good results. 2.9 CONCLUSIONS The interaction of nitrite with amino group of p-nitroaniline or pphenylenediamine to form diazonium salt is almost specific, which provides the determination of nitrite with good sensitivity. The introduction of nevirapine, frusemide and 5-methyl-4-{[(1E)-phenylmethylene] amino}-2,4-dihydro-3H-1,2,4triazole-3-thione, as new coupling agents provide a simple rapid and selective method for the determination of nitrite. The developed method does not involve any stringent reaction conditions and offers the advantages of color stability about more than 2 hours. The statistical analysis of the results by t- and F- tests show that, there is no significant difference in accuracy and precision between the proposed method and reference method. The proposed method has been successfully applied to the determination of trace amount of nitrite in water samples, soil samples and dietary supplements.

37

TABLE 2: COMPARISON OF THE SPECTROPHOTOMETRIC METHOD WITH THE EARLIER METHODS Reagent

Range

-1

)

max (nm)

PVC

6.67-66.7

470

PNA-DPA

2.00-40.00

FAI AMS

Remarks

References

Low sensitivity

[67]

-

Extraction required

[53]

0-0.40

495

Extraction required

[54]

0.01-0.08

530

Extraction required

[55]

0-0.75

382

Extraction required

[56]

0.04-0.36

485

Extraction required

[57]

PNA-NEV

0.40-1.40

492

PPD-NEV

1.00-9.00

678

PNA-FRU

0.02-0.60

680

Simple, rapid, moderately

PPD-FRU

0.10-0.60

692

sensitive and non-extractive

PNA-MPAT

0.40-2.00

395

PPD-MPAT

1.00-3.00

668

DC Ferron Proposed method

PVC-Peroxovanadate complex, DPA-Diphenylamine, FAI- Fluorescein amine isomer I, AMS-1-Aminonaphthalene-2-sulphonic acid, DC- 4,5- Dihydroxy coumarin.

38

TABLE 2.1A: DETERMINATION OF NITRITE IN WATER SAMPLES, SOIL SAMPLES AND DIETARY SULLPEMENTS (PNA-NEV SYSTEM)

Sample

Water sample 1 Water sample 2 Soil sample ID

Nitrite added mL-1 0.40 0.80 0.40 0.80 0.40 0.80 0.40 0.80

Reference method Nitrite Relative error found in mL-1 % 0.402 0.50 0.804 0.50 0.418 4.50 0.810 1.25 0.402 0.50 0.826 3.25 0.402 0.50 0.801 0.12

Proposed method Nitrite Relative error found in % mL-1± SDa 0.401 0.25 0.798 0.25 0.404 1.00 0.812 1.50 0.406 1.50 0.810 1.25 0.404 1.00 0.801 0.12

t-testb

F-testc

2.236 0.559 0.894 2.683 1.341 2.236 0.894 2.236

2.25 6.25 1.00 1.00 1.56 1.00 1.23 2.25

a

Mean±Standard deviation (n = 5)

b

Tabulated t-value for 5 degrees of freedom at 95% probability level is 2.31

c

Tabulated F-value for (4, 4) degrees of freedom at 95% probability level is 6.39. ID- Isosorbidinitrate tablets, Nichoals Piramal Intra Limited, Himachal Pradesh

TABLE 2.1B: DETERMINATION OF NITRITE IN WATER SAMPLES, SOIL SAMPLES AND DIETARY SUPPLEMENTS (PPD-NEV SYSTEM)

Sample

Water sample 1 Water sample 2 Soil sample ID

Nitrite added mL-1 1.00 1.40 1.00 1.40 1.00 1.40 1.00 1.40

Reference method Nitrite Relative error found in mL-1 % 1.034 3.40 1.416 1.10 1.026 2.60 1.398 0.14 1.010 1.00 1.406 0.43 0.99 -1.00 1.398 0.14

Proposed method Nitrite Relative error found in -1 a % mL ± SD 1.039 3.90 1.398 0.14 1.004 0.40 1.405 0.35 1.039 3.90 1.406 0.43 0.98 -2.00 1.405 0.35

t-testb

F-testc

2.250 0.447 0.894 1.118 2.900 1.340 0.370 1.118

2.25 1.00 4.00 1.00 2.25 4.00 1.08 1.00

a

Mean±Standard deviation (n = 5)

b

Tabulated t-value for 5 degrees of freedom at 95% probability level is 2.31

c

Tabulated F-value for (4, 4) degrees of freedom at 95% probability level is 6.39. ID- Isosorbidinitrate tablets, Nichoals Piramal Intra Limited, Himachal Pradesh

39

TABLE 2.C: DETERMINATION OF NITRITE IN WATER SAMPLES, SOIL SAMPLES AND DIETARY SULLPEMENTS (PNA-FRU SYSTEM)

Sample

Nitrite added mL-1

Water sample 1

Reference method

Proposed method Nitrite Relative error found in -1 a % mL ± SD

Nitrite found in mL-1

Relative error %

0.20 0.40

0.198 0.418

-0.90 4.50

0.221 0.407

Water sample 2

0.20 0.40

0.208 0.403

4.00 0.75

Soil sample

0.40 0.60

0.402 0.606

ID

0.40 0.60

0.402 0.602

t-testb

F-testc

0.25 1.80

2.236 2.000

1.00 1.20

0.200 0.402

0.20 0.50

0.089 2.940

1.00 1.10

0.50 1.00

0.403 0.596

1.50 0.66

1.630 1.400

4.00 2.70

0.50 0.66

0.401 0.602

0.25 0.50

2.236 2.790

1.00 1.00

a

Mean±Standard deviation (n = 5)

b

Tabulated t-value for 5 degrees of freedom at 95% probability level is 2.31

c

Tabulated F-value for (4, 4) degrees of freedom at 95% probability level is 6.39. ID- Isosorbidinitrate tablets, Nichoals Piramal Intra Limited, Himachal Pradesh

TABLE 2.1D: DETERMINATION OF NITRITE IN WATER SAMPLES, SOIL SAMPLES AND DIETARY SUPPLEMENTS (PPD-FRU SYSTEM)

Sample

Water sample 1 Water sample 2 Soil sample ID

Nitrite added mL-1 0.40 0.60 0.40 0.60 0.40 0.60 0.40 0.60

Reference Method Nitrite Relative error found in -1 mL % 0.402 0.50 0.604 0.66 0.418 4.50 0.606 1.00 0.402 0.50 0.606 1.00 0.402 0.50 0.602 0.66

Proposed method Nitrite Relative error found in -1 a % mL ± SD 0.402 0.25 0.598 0.33 0.404 1.00 0.596 0.66 0.403 0.85 0.606 1.00 0.401 0.25 0.604 0.73

t-testb

F-testc

2.236 0.550 2.600 1.267 3.800 1.340 2.230 3.000

2.25 5.22 5.00 2.70 4.00 1.00 1.00 6.00

a

Mean±Standard deviation (n = 5)

b

Tabulated t-value for 5 degrees of freedom at 95% probability level is 2.31

c

Tabulated F-value for (4, 4) degrees of freedom at 95% probability level is 6.39. ID- Isosorbidinitrate tablets, Nichoals Piramal Intra Limited, Himachal Pradesh

40

TABLE 2.1E: DETERMINATION OF NITRITE IN WATER SAMPLES, SOIL SAMPLES AND DIETARY SULLPEMENTS (PNA-MPAT SYSTEM)

Sample

Water sample 1 Water sample 2 Soil sample ID

Nitrite added mL-1 0.40 0.60 0.40 0.60 1.00 2.00 1.00 2.00

Reference method Nitrite Relative error found in -1 mL % 0.402 0.50 0.604 0.66 0.418 4.50 0.606 1.00 1.010 1.00 1.983 -0.85 0.990 -1.00 2.060 3.00

Proposed method Nitrite Relative error found in -1 mL % 0.401 0.25 0.596 0.66 0.404 1.00 0.598 -0.90 1.002 0.20 2.406 0.42 1.004 0.40 2.039 1.95

t-testb

F-testc

2.230 1.400 0.894 0.479 0.447 1.340 0.894 2.900

1.00 4.00 1.23 1.56 4.00 1.56 6.25 2.25

a

Mean±Standard deviation (n = 5)

b

Tabulated t-value for 5 degrees of freedom at 95% probability level is 2.31

c

Tabulated F-value for (4, 4) degrees of freedom at 95% probability level is 6.39. ID- Isosorbidinitrate tablets, Nichoals Piramal Intra Limited, Himachal Pradesh

TABLE 2.1.F: DETERMINATION OF NITRITE IN WATER SAMPLES, SOIL SAMPLES AND DIETARY SUPPLEMENTS (PPD-MPAT SYSTEM)

Sample

Water sample 1 Water sample 2 Soil sample ID

Nitrite added mL-1 1.00 2.00 1.00 2.00 1.00 2.00 1.00 2.00

Reference method Nitrite Relative error found in mL-1 % 1.034 3.40 2.060 3.00 1.026 2.60 1.983 -0.85 1.010 1.00 1.983 -0.85 0.990 -1.00 2.060 3.00

Proposed method Nitrite Relative error found in mL-1 % 1.003 0.30 2.034 1.70 1.024 2.40 2.002 0.10 1.020 2.00 2.002 0.10 1.024 2.40 2.053 2.65

t-testb

F-testc

0.670 1.520 2.680 0.447 2.236 0.447 2.680 2.320

2.25 4.00 6.25 1.56 6.25 1.60 4.00 1.00

a

Mean±Standard deviation (n = 5)

b

Tabulated t-value for 5 degrees of freedom at 95% probability level is 2.31

c

Tabulated F-value for (4, 4) degrees of freedom at 95% probability level is 6.39. ID-Isosorbidinitrate tablets, Nichoals Piramal Intra Limited, Himachal Peadesh

41

TABLE 2.2A: THE EFFECT OF DIVERSE IONS ON THE DETERMINATION OF NITRITE (PNA-NEV OR FRU OR MPAT SYSTEM)

Foreign Ion

Tolerence Limit (

Using PNA-NEV as Reagents Oxalate, tartarate, citrate

>800

Fluoride, iodide, carbonate

1500

Cd2+, Mn2+, Ba2+

>500

Na+, K+

>1200

Hg2+*, Mo6+*, Co2+*

25

Cu2+*, Ce4+*

30

Using PNA-FRU as Reagents Oxalate, tartarate, citrate

>1300

Fluoride, iodide, carbonate

1500

Cd2+, Mn2+, Ba2+

>800

Na+, K+ , SCN-*

>200

[Fe(CN)6]4- *

50

Hg2+*, Mo6+*, Co2+*

25

Cu2+*, Ce4+*

30

Using PNA-MPAT as Reagents Oxalate, tartarate, citrate

>1000

Fluoride, iodide, carbonate

1000

Cd2+, Mn2+, Ba2+

>800

Na+, K+ , SCN- *

100

[Fe(CN)6]4- *

50

Hg2+*, Mo6+*, Co2+*

25

Cu2+*, Ce4+*

100

* Masked by masking agents

42

-1

)

TABLE 2.2B: THE EFFECT OF DIVERSE IONS ON THE DETERMINATION OF NITRITE (PPD-NEV OR FRU OR MPAT SYSTEM)

Foreign Ion

Tolerence Limit (

Using PPD-NEV as Reagents Oxalate, tartarate, citrate

>2000

Fluoride, iodide, carbonate

1500

Cd2+, Mn2+, Co2+*

>500

Na+, K+

500

Hg2+*, Mo6+*

25

Using PPD-FRU as Reagents Oxalate, tartarate, citrate

>1300

Fluoride, iodide, carbonate

1000

Cd2+, Mn2+, Co2+*

>500

Na+, K+, SCN- *

100

Hg2+*, Mo6+*

25

Using PPD-MPAT as Reagents Oxalate, tartarate, citrate

>1500

Fluoride, iodide, carbonate

1000

Cd2+, Mn2+, Co2+*

>300

Na+, K+, SCN- *

100

Hg2+*, Mo6+*

25

* Masked by masking agents

43

mL-1)

FIGURE 2A: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF NITRITE (PNA-NEV SYSTEM)

0.20

Absorbance

0.15

0.10

0.05

0.00 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Concentration (µg/ml)

FIGURE 2B: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF NITRITE (PPD-NEV SYSTEM)

2.5

Absorbance

2.0

1.5

1.0

0.5

0.0 0

2

4

6

8

Concentration (µg/ml)

44

10

12

FIGURE 2C: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF NITRITE (PNA-FRU SYSTEM).

0.018

Absorbance

0.016

0.014

0.012

0.010 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Concentration (µg/ml)

FIGURE. 2D: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF NITRITE (PPD-FRU SYSTEM).

0.10

Absorbance

0.08

0.06

0.04

0.02

0.00 0.0

0.1

0.2

0.3

0.4

0.5

Concentration (µg/ml)

45

0.6

0.7

0.8

FIGURE 2E: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF NITRITE (PNA-MPAT SYSTEM) 0.16 0.14

Absorbance

0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.0

0.5

1.0

1.5

2.0

2.5

Concentration (µg/ml)

FIGURE. 2F: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF NITRITE (PPD-MPAT SYSTEM)

0.35

0.30

Absorbance

0.25

0.20

0.15

0.10

0.05

0.00 0

1

2

Conce ntra tion (µg/ml)

46

3

4

FIGURE II.A: ABSORPTION SPECTRA OF AZO DYE (PNA-NEV SYSTEM)

3.0

2.5

Absorbance

2.0

1.5

1.0

0.5

0.0

-0.5 350

400

450

500

550

600

Wavelength (nm)

FIGURE II.B: ABSORPTION SPECTRA OF AZO DYE (PPD-NEV SYSTEM)

1.8

1.6

Absorbance

1.4

1.2

1.0

0.8

0.6

0.4 350

400

450

500

550

600

Wavelength (nm)

47

650

700

750

FIGURE II.C: ABSORPTION SPECTRA OF AZO DYE (PNA-FRU SYSTEM)

0.9 0.8

Absorbance

0.7 0.6 0.5 0.4 0.3 0.2 0.1 600

620

640

660

680

700

720

Wavelength (nm)

FIGURE II.C: ABSORPTION SPECTRA OF AZO DYE (PPD-FRU SYSTEM)

2.5

Absorbance

2.0

1.5

1.0

0.5

0.0 600

620

640

660

680

Wavelength (nm)

48

700

720

FIGURE II.E: ABSORPTION SPECTRA OF AZO DYE (PNA-MPAT SYSTEM)

3.0

2.5

Absorbance

2.0

1.5

1.0

0.5 a 0.0 300

400

500

600

700

800

W avelength (nm)

FIGURE II.F: ABSORPTION SPECTRA OF AZO DYE (PPD-MPAT SYSTEM)

1.6 1.4

Absorbance

1.2 1.0 0.8 0.6 0.4 0.2 0.0

350

400

450

500

550

600

Wavelength (nm)

49

650

700

750

REACTION SCHEMES 2.3A:

H2N

NH2

+

NO2

-

HCl

-

+

+

N2 Cl

Cl N2

-

N

NH -

Cl N2

+

+

-

N2 Cl

+

2

N

O

O

N

CH3

N N H3C

N N

CH3 N

N

N

O

N

N

O2N

NH2

+

HCl

-

NO2

+

O2N

O

O2N

+ N2 Cl

-

N2 Cl

NO2

O NH

CH3

N

+ N N

50

N

N N

N CH3

REACTION SCHEMES 2.3B:

H2 N

NH2

+

+

-

2 NO2

-

-

HCl

Cl N2

Cl N2

+

N2 Cl

+

N2 Cl

-

Cl

O -

+

+

Cl

O O

2 NH

O

S

O

N N

NH2

S

N

O

O

NH2 O

OH

OH HO O NH2

O

N N

S

N

O O

Cl

O 2N

NH 2

O 2N

N 2 Cl

+

NO 2

HCl

-

Cl

O +

-

+

N 2 Cl

H 2N

-

Cl

O O

+ NH

S NH 2

O O

N N

O

S

N

NH 2 O

OH

OH

NO 2

51

O

REACTION SCHEMES 2.3C:

HCl

NH2 + NO 2 -

H2 N

-

Cl N2

-

+

+

N2 Cl

-

+ 2 H3 C

+

N2 Cl

-

N

H N

N Cl N2

+

CH3 N N

S

S

N N

N N

N

H3 C N N

N N N

N

S

NH2

O2N

+ NO2

HCl

-

+

N2 Cl

O2N

-

NO2 H N

N O2N

+

N2 Cl

-

+

H3C

N

H3C S

N N

N N

N

N S

52

N

2.9 REFERENCES 1.

M. Radojecvic & V. N. Bashkin, Practical Environmental Analysis, Royal Society of Chemistry, UK (1999).

2.

A. A. Ensafi & M. S. Kolgar, Anal. Lett., 27 (1994) 169.

3.

D. Pobel, E. Riboli, J. Cornee, B. Hemon & M. Guyader, Eur. J. Epidemiol, 11 (1995) 67.

4.

B. C. Andrea, C. O. Viana, S. G. Guadagnin, F. G. R. Reyes & S. Rata, Food Chem., 50 (2003) 597.

5.

B. M. Thomsom, C. J. Nokes & P. Cressy, Food Addit. Contam., 24 (2007) 113.

6.

M. J. Hill, Nitrates and Nitrites in Food and Water, Ist Edn., Woodhead Publishing Limited, England (1996).

7.

A. Afkhami, S. Masahi & M. Baharam, Bull. Kor. Chem. Soc., 25 (2004) 1009.

8.

A. A. Ensafi, B. Rezaei & S. Nouroozi, Anal. Sci., 20 (2004) 1749.

9.

C. Matasa & E. Matasa, L’ Industries Modern des Produits Azoles, Dunod, Paris (1968) 567

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