3.1 Background: Chapter 3: Spectrophotometric Determination of Nitrate and Nitrite

Chapter 3: Spectrophotometric Determination of Nitrate and Nitrite
3.1 Background
Diazotization colorimetric methods [1] have been widely used for nitrite and
nitrate determination. These methods involve reduction of nitrate to nitrite first which
forms a colored dye with reagent which is measured spectrophotometrically. USEPA
methods 352.1, 353.1 and 353.2 utilize chemicals that are highly toxic and carcinogenic.
While performing these methods hazardous waste is generated and disposal is also costly
[2-4]. Many of these methods have difficulties and cannot be used for routine analysis.
Horita et al. (1997) were developed three spectrophotometric methods for the
determination of nitrate and nitrite in water and soil samples with composite
diazotization-coupling reagents containing sulfanilamide (SAM), sulfapyridine (SP) or
sulfathiazole (ST) as the diazotizable aromatic amine and sodium-1-naphthol-4-sulfonate
(NS) as the coupling agent using column preconcentration on naphthalene-
tetradecyldimethylbenzylammonium (TDBA)-iodide adsorbent. Nitrite ion reacts with
SAM in the pH range 2.0-5.0, SP in the pH range 2.0-2.5 and ST in the pH range 2.0-3.3
in HCl medium to form water-soluble colourless diazonium cations. These cations were
coupled with NS in the pH range 9.0-12.0 for the SAM system, 9.6-12.0 for the SP
system and 8.5-12.0 for the ST system to be retained on naphthalene-TDBA-I material
packed in a column. Absorbance was measured spectrometerically at 543 nm for SAM-
NS, 533 nm for SP-NS and 535 nm for ST-NS [5]. Torro et al. (1998) reported flow-
injection biamperometric determination of nitrate (by photoreduction) and nitrite. The
method was based on the NO2- and I- reaction, the tri-iodide thus formed was detected
biamperometrically in excess iodide by using a flow-cell furnished with two platinum
electrodes polarized at 100 mV. The limits of detection, throughput and reproducibility
thus achieved for nitrite and nitrate were 25 and 50 µg/L, 27 and 25 h-1 and 1.2 and 1.6
%, respectively [6]. Zater et al. (1999) reported a method for spectrophotometric
determination of nitrite and nitrate based on the reduction of phosphomolybdic acid to
phosphomolybdenum blue complex by sodium sulfide. The absorbance of the
phosphomolybdenum blue complex is monitored spectrophotometrically at 814 nm and
related to the concentration of nitrite present. The effect of different factors such as
acidity, stability of the complex, time, temperature, phosphate concentration,
molybdenum concentration, sodium sulfide concentration and the tolerance amount of
Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 45
other ions have been reported. The range of linearity using the conventional method is
0.5-2.0 ppm with molar absorptivity of 1.1×104 L mol-1 cm-1 and a relative standard
deviation of 2.6 % for five measurements. The range of linearity using the reaction rate
method is 0.2-3.6 ppm with a relative standard deviation of 2.4% for five measurements
[7]. Miranda et al. (2001) developed a method for simultaneous evaluation of nitrate and
nitrite concentrations in a microtiter plate format. The principle of this assay is reduction
of nitrate by vanadium (III) combined with detection by the acidic Griess reaction. This
assay is sensitive to 0.5 µM NO3− and is useful in a variety of fluids including cell culture
media, serum, and plasma. S-Nitrosothiols and l-arginine derivatives were found to be
potential interfering agents [8]. Geetha and Balasubramaniam (2001) reported a simple
and sensitive spectrophotometric method for the determination of nitrite using
phenosafranin. 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) [9]. Bai (2001) reported a kinetic spectrophotometric method for the
determination of nitrite, based on the catalytic oxidation of bromocresol purple with
KBrO3 in 0.45 mol mL-1 H3PO4 medium [10]. A method proposed by Padmarajah et
al. (2002) involved diazotization-coupling reaction between dapsone and N-(1-naphthyl)
ethylene diamine dihydrochloride in a hydrochloric acid medium. The molar absorptivity
and Sandell's sensitivity were found to be 7.20×104 L mol-1 cm-1 and 0.0006 µg cm-2,
respectively. The calibration graph was linear for 0.002-0.6 µg mL-1 of nitrite [11].
Sasry and et al. (2002) developed a spectrophotometric method for determination of
serum nitrite and nitrate by copper cadmium electrode using sulfanilamide and N-
naphthylethylenediamine in acidic medium which form a purple azo dye having an
absorption maximum at 545 nm. The concentration of nitrite should be subtracted
from the nitrite plus nitrate concentration to obtain the concentration of nitrate. The
detection limit of nitrate by this method was 1 mol/liter [12]. A method proposed by
Sreekumar et al. (2003) involved diazotization reaction between p-nitroaniline and
sulfanilamide with ethylacetoacetate as a coupling agent for the determination of nitrite.

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 nitrite with molar
absorptivity of 1.59x104 Lmol-1cm-1; while that for sulfanilamide-ethyl acetoacetate
couple was found to be 0.2 to 3.0 µg mL-1 and 1.22x104 L mol-1cm-1 respectively [13].
Very simple, sensitive and selective spectrophotometric method for the determination of
nitrite in micellar media was described by Afkhami et al. (2004). The method is based on
the color reaction of nitrite with p-nitroaniline in the presence of diphenylamine in acid
media. In order to remove the extraction step, Triton X-100, a non-ionic surfactant was
used as micellar media. Regression equation for calibration graph was found to be
A=0.3098×C+0.008 with molar absorptivity of 1.425 × 10+4 L/ mol/ cm within a linear
range of (0.05-0.8 µg mL−1). A limit of detection of 0.01 µg mL−1 was obtained [14].
Suchandra Biswas et al. (2004) reported a novel spectrofluorimetric method for the ultra
trace analysis of nitrite and nitrate in aqueous medium and its application to air, water,
soil and forensic samples. The method is based on the quenching action of nitrite on the
native fluorescence of murexide (ammonium purpurate) in the acid range of 0.045-0.315
(M) H2SO4. The method is very precise and accurate S.D.= 0.4877 and R.S.D.=0.4878
for the determination of 0.1 µgml−1 of nitrite in 11 replicates. The fluorescence intensity
was measured at 444.5 nm against a corresponding reagent blank, keeping the excitation
wavelength at 349.0 nm under the same instrumental settings [15]. Adnan et al. (2005)
reported a rapid, simple, selective and sensitive method for the spectrophotometric
determination of nitrite in water. The method was based on reaction of nitrite with
barbituric acid in acidic solution to give the nitroso derivative, violuric acid and
absorbance was measured at the 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%. As well as the
method is sensitive (2.99×10−3 µg NO2 cm−2) and selective, it tolerates most of the
potential interferents [16]. Spectrophotometric determination of nitrite in water samples
with 4-(1-methyl-1-mesitylcyclobutane-3-yl)-2-aminothiazole was reported by Ozmen et
al. (2006). The developed azo dye, 4-(1-methyl-1-mesitylcylobutane-3-yl)-2-(p-N,N-
dimethylazobenzene)-1,3- thiazole was synthesized with the reaction of 4-(1- methyl-1-
mesitylcylobutane-3-yl)-2-aminothiazole and N,N-dimethyl aniline in acidic medium.
Obtained azo dye had been characterized by infrared (IR), H1 nuclear magnetic resonance
(NMR), and microanalysis methods. The dye showed an absorption maximum at 482 nm
[17]. Pasquali et al. (2007) developed a spectrophotometric simultaneous determination
of nitrite, nitrate and ammonium in soil by flow injection analysis. Nitrite, nitrate and
ammonium were determined within the range of 0.02-1.60, 0.02-1.60 and 0.05-1.40
µgmL−1, respectively by using the Griess-llosvay reaction. The R.S.D. values were 2.66,
1.41 and 3.58 for nitrate, nitrite and ammonium, respectively [18]. Chinchilla (2008)
developed an automated colorimetric method for nitrate analysis. Nitrate is reduced to
nitrite by a proprietary reagent “R1” at 50 °C. The reduced nitrate is then treated under
acidic conditions to form a highly colored soluble dye, which is measured
colorimetrically between 520 and 550 nm. Detection limits obtained were consistent and
ranged between 0.010-0.015 mg/L with a median detection limit (MDL) of
0.01119/10×100=0.11 percent and 0.00416/10×100= 0.04 percent, respectively [19]. Mir
(2008) reported a rapid technique for determination of nitrate and nitric acid by acid
reduction and diazotization at elevated temperature. The technique is based on
quantitative diazotization of sulfanilamide by nitrate on incubation in boiling water bath
for 3, 5 or 10 min in presence of high concentration of HCl, calculated 64.5 %. The
diazotized sulfanilamide is coupled at room temperature to N-1-(naphthyl)-
ethylenediamine dihydrochloride, and the chromophore evaluated spectrophotometrically
at 540 nm. The technique provides linear estimate of nitrate over detection range of 0.5-
10 µg N mL-1 and nitric acid as low as 35 µM even in presence of other acids. Recoveries
of nitrate have been, respectively, 1.99±0.03, 2.95±0.06 and 3.95±0.05 as µg N, with
overall percent recovery 99±1, compared to standard nitrate samples subjected to
identical treatment in absence of nitrite [20]. Nagaraja et al. (2010) reported a rapid
spectrophotometric method for the determination of trace amounts of nitrite. This method
was based on the diazotized intramolecular coupling of electrophilic diazonium cation
with the phenolic group of 4-amino-5-hydroxynaphthalene-2, 7-disulphonic acid
monosodium salt (AHNDMS) in a phosphate buffer solution of pH 7.5. The cyclic
product has a purple color with maximum absorbance at 560 nm and is stable for 6 h.
Optimum reaction conditions and other important analytical parameters for the maximum
color development were established. Beer’s law was found to obey for nitrite in the
concentration range of 0.1-1.6 µg ml-1 with molar absorptivity of 2.6×104 L mol-1 cm-1
and Sandell’s sensitivity of 0.0075 µg cm-2 [21]. Badiadka and Kenchaiah (2009) were
developed a spectrophotometric method for the determination of nitrite and nitrate. The
range of linearity for sulfanilic acid-methyl anthranilate couple was found to be 0.2-8.0
µg/mL of nitrite with molar absorptivity is 1.03x104 L mol-1 cm-1 and Sandell’s
sensitivity 4.5x10-3 µg cm-2 and azo dye was measured at 493 nm. The detection limit and
quantitation limit of the nitrite determination was found to be 0.93 and 2.82 µg ml-1
respectively [22]. Two spectrophotometric ﬂow injection methods were developed for the
individual determination of nitrite or nitrate and for the simultaneous determination of
nitrite and nitrate, in soil samples by Pasquali (2010). Nitrite was determined directly
using a modiﬁed version of the Griess–Ilosvay diazo-coupling reaction, measuring at 543
nm the absorbance of the azo–dye complex formed. Simultaneous nitrite and nitrate
determinations were based on on-line nitrate reduction in a micro column containing
copperised cadmium. The optimized conditions gave a linear calibration range between
0.05 and 1.6 µg ml-1 for N-NO2- and between 0.05 and 7.0 µg ml-1 for N-NO3- and the
detection limits for nitrite and nitrate were 0.022 µg ml-1 and 0.044 µg ml-1 respectively
[23]. Pasha (2010) reported a spectrophotometric method for the determination of
mosapride. This method was based on the reaction of mosapride with nitrite in acid
medium to form diazonium ion, which is coupled with acetylacetone or ethyl acetoacetate
in basic medium to form azo dyes, which shows absorption maxima at 402 or 422 nm.
The method obeys Beer’s law in the concentration range of 0.4-13.5 µg ml-1 of mosapride
with acetylacetone and 0.5-17.0 µg ml-1 of mosapride with ethyl acetoacetate. The molar
absorptivity and Sandell’s sensitivity of mosapride-acetylacetone and mosapride-ethyl
acetoacetate azo dyes are 1.026x104 l mol-1cm-1, 4.484 x 10-3 µg cm-2 and 1.097x104 l
mol-1cm-1, 4.193x10-3 µg cm-2 respectively [24]. Moldovan (2012) developed kinetic-
spectrophotometric method for the determination of trace amounts of nitrite. The method
is based on its catalytic effect on the oxidation of congo red (CR) by potassium bromate
in acidic solution. The oxidation reaction is monitored spectrophotometrically by
measuring the decrease in the absorbance of CR at a suitable λmax which was 570 nm for
the first 10-40 s from the start of the reaction. Under the optimum experimental
conditions (sulfuric acid, 0.3 M; CR, 0.75×10-4 M; potassium bromate, 5×10-4 M and 25
o
C), nitrite can be determined in the range of 0.015-0.75 µg mL-1 with the detection limit
of 0.006 µg mL−1. The relative standard deviation of five replicate determination of 0.25
µg mL-1 nitrite was 2.5 % [25]. Shah et al. (2005) developed a simple and rapid
spectrophotometric method for the determination of furosemide. The method was based
upon simple diazotization reaction. The absorbance was measured at 480 nm. The
method is optimized for acidity, amount of reagents required and heating time. The range
of linearity was 0.4-10 µg mL-1 of furosemide with molar absorptivity of 1.0×104 L mol-1
cm-1. Standard deviation, coefficient of variance, limit of detection and limit of
quantification were calculated using authentic standard of furosemide and were found to
0.0013, 12.7%, 0.16 ppm and 0.52 ppm, respectively [26].
3.2 Materials and Methods
3.2.1 Equipment
Spectral measurements were made with Shimadzu-1800 double beam
spectrophotometer with 1.00 cm quartz cuvette. A digital pH meter (Electronic
Corporation of India Ltd., model pH 5651) was used for the measurements of pH.
Electronic balance (Shimadzu, type AUY 120) was used for weighing the chemicals.
3.2.2 Reagents and Materials

All reagents were of analytical reagent grade and water used was milli-Q obtained
from Millipore water purification system throughout the study. A stock solution of nitrate
1000 µg/ml and nitrite 1000 µg/ml were prepared from potassium nitrate and sodium
nitrite respectively. These stock solutions were diluted appropriately to get the working
concentrations. Other reagents used were as follows:
Mosapride (4-amino-5-chloro-2 ethoxy-{N-{4-[4-fluorobenzyl)morpholin-2-yl] methyl}-
benzamide citrate dihydrate) (0.025 % in ethanol) (MSP)
Metoclopramide [(4-amino-5-chloro-N-(2-(diethylamino)ethyl)-2-methoxybenzamide]
(0.1 % in water) (MTCP)
Sulphanilamide (SAM) and N-(1-napthyl)-ethyenediamine dihydrochloride (NEDA)
solution: Added 105 mL concentrated HCl, 5.0 gm sulphanilamide and 0.5 gm NEDA to
250 mL distilled water and stirred until dissolved. Added 136 gm sodium acetate to it and

stirred again until dissolved. The solution was diluted to 500 mL with distilled water and
stored in dark. This solution was used as a buffered color reagent for the reference
method [27].
Ethyl acetoacetate (EAA) (2 & 0.5 % in water)
Sodium hydroxide (2 M aqueous)
Hydrochloric acid (0.5 & 2 M aqueous).
Inorganic ions aqueous solutions of Cu2+, K+, Na+, Ca2+, Fe3+, Cl-, CO32-, HPO42- and
SO42- prepared from their A.R. grade water soluble salts.
3.3 Results and Discussion

3.3.1 Selection of Common Solvent (Diluent)
Distilled water was selected as common solvent for prepration of stock solutions
and developing spectral characteristics of nitrite and nitrate, further dilutions from stock
solutions were made in distilled water. The selection was made after assessing the
solubility of both nitrite and nitrate in different solvents.
3.3.2 Wavelength of Maximum Absoption for Spectrophotometric Detection

Preparation of Nitrite Stock Solution (1000 µg/ml): An accurately weighed quantity of
sodium nitrite 0.1500 g was dissolved in Milli-Q water and transferred to the 100 ml
volumetric flask and diluted with Milli-Q water and final volume was made up to the
mark with distilled water. The aliquot portions of stock solution of nitrite were diluted
appropriately with Milli-Q water to obtain 0.2 µg/ml of nitrite.
3.3.2.1 Determination of λmax of Azo Dye using MSP-EAA as Reagents

1 mL of 0.2 µg/ml nitrite solution was transferred to 10 ml volumetric flask, 0.5
ml of 2 M HCl was added to make it acidic and then diazonium ion was formed by
adding 2 ml of 0.025 % mosapride reagent and was shaken for 5 min. After that 1ml of 2
% ethyl acetoacetate was added as a coupling agent and 2 ml of 2 M NaOH solution was
added to get yellow coloured azo dye which is diluted to 10 ml using distilled water. The
azo dye solution was then scanned in the range of 200-800 nm. From the spectrum the
wavelength of maximum absorption selected for determination of nitrite was 429 nm.

The absorbance spectrum of azo dye and diazonium ion using MSP
MSP-EAA
EAA reagents is
shown in Figure 3.1
Figure 3.1: Absorbance S

Spectrum of Azo Dye and Diazonium Ion
on using MSP-EAA
MSP
3.3.2.2 Determination of λmax of Azo Dye using MTCP-EAA as Reagents

eagents
1 mL of 0.2 µg/ml nitrite solution was transferred to 10 ml volumetric flask, it
was made acidic on addition of 1 ml of 0.5 M HCl to form diazonium ion by adding 0.5
ml of 0.1 % metoclopramide reagent and was shaken for 5 min. After that 1ml of 2 %
ethyl acetoacetate was added as a coupling agent and 2 ml of 2 M NaOH solution was
added to get yellow coloured azo dye which is diluted to 10 ml using distilled
dis water. The
azo dye solution was then scanned in the range of 200
200-800
800 nm. From the spectrum the
wavelength of maximum absorption selected for determination of nitrite was 437.5 nm.
The absorbance spectrum of azo dye and diazonium ion using MTCP
MTCP--EAA reagents is
shown in Figure 3.2.
Figure 3.2: Absorbance S

Spectrum of Azo Dye and Diazonium Ion
on using MTCP-EAA
MTCP

3.4 Optimization of UV-Visible Spectrophotometric Parameters

In order to fix optimum conditions for spectrophotometric determination of
nitrite, some experiments were performed as follows.
3.4.1 Effect of Acidity: In the present method hydrochloric acid provides a wide range of
acidity for the maintenance of optimum pH. The effect was studied for solutions
containing fixed concentration of nitrite and by varying the concentration and volume of
HCl. The results obtained showed that 0.5 ml of 2 M HCl gave maximum absorbance for
MSP-EAA reagents and 0.5 ml of 1 M HCl gave maximum absorbance using MTCP-
EAA reagents. The absorbance was observed in the presence of 0.5-2.5 M hydrochloric
acid in a total 10.0 ml of solution as shown in Figure 3.3 (for MSP-EAA) and Figure 3.4
(for MTCP-EAA). Above this range a decrease in absorbance was observed.
0.8
0.7
0.6 0.5 M
Absorbance
0.5 1.0 M
0.4
0.3 1.5 M
0.2 2M
0.1
0
0 0.5 1 1.5 2 2.5
volume of HCl in ml
Figure 3.3: Absorbance Variation with Varying Concentration and Volume of HCl for
MSP-EAA
1.2
1 0.5 M
0.8 1.0 M
Absorbance
0.6 1.5 M
0.4 2.0 M
0.2
0
0 0.5 1 1.5 2 2.5
Volume of HCl (ml)
Figure 3.4: Absorbance Variation with Varying Concentration and Volume of HCl for
MTCP-EAA
The optimum concentration of HCl was fixed to be 2 M and volume of HCl

required was 0.5 ml for MSP-EAA reagents. The optimum concentration of HCl was
fixed to be 0.5 M and volume of HCl required was 1 ml for MTCP-EAA reagents. This
acidity was sufficient for protonation in diazotization reaction of nitrite using MSP-EAA
and MTCP-EAA reagents [28-30].
3.4.2 Effect of Substrate Concentration

3.4.2.1 Mosapride Reagent Concentration: In order to fix the amount of mosapride
concentration required for diazotizitaion of nitrite and to achieve maximum absorbance,
experiments were done by using different volumes (0.5 to 2.5 ml) of 0.025 % of
mosapride using nitrite solutions in the range of 50-500 mg/L to form diazonium salt.
The results are presented in Figure 3.5.
3.4.2.2 Metoclopramide Reagent Concentration: In order to fix the amount of

metoclopramide concentration required for diazotization of nitrite and to achieve
maximum absorbance, experiments were done by using different volumes (0.5 to 1 ml) of
0.1 % of metoclopramide in the range of 0.05 to 0.1 % to form diazonium salt. The
results are presented in Figure 3.6.
0.8 50 mg/L
Absorbance
0.6 100
0.4 250
0.2 500
0
0 1 2 3
Volume of MSP (ml)
Figure 3.5: Absorbance Variation with Varying Concentration of Nitrite and Volume of
MSP Solutions

1
0.8
0.05%
Absorbance
0.6 0.10%
0.4
0.2
0
0 0.5 1 1.5
Volume of MTCP (ml)
Figure 3.6: Absorbance Variation with Varying Concentration of Nitrite and Volume of
MTCP Solutions
The maximum absorbance was observed for mosapride at 2 ml of 250 mg/L of
mosapride reagent solution. The maximum absorbance was observed for 0.5 ml of 0.1 %
metoclopramide solution. The yellow colored diazonium salt is very reactive and acts as
an electrophile. Aryl diazonium cation undergoes electrophilic substitution reaction with
coupling agent to produce an azo derivative [28].
3.4.3 Effect of Coupling Agent (EAA) Concentration

3.4.3.1 Effect of EAA Concentration on Mosapride Diazonium Cation:
The mosapride diazonium cation couples with different concentrations and
different volumes of EAA solution to get yellow coloured azo dye in basic medium.
These experiments were done using 0.5-2.5 % concentrations and 0.5-2.5 ml volumes of
EAA solutions and the amount of EAA solution required for maximum absorbance was
examined. The results are represented in Figure 3.7.
3.4.3.2 Effect of EAA Concentration on Metoclopramide Diazonium Cation:

The amount of EAA solution required for maximum absorbance was examined.
Experiments were done using 0.5-2.0 % concentrations and 0.5-2.0 ml volumes of EAA
solutions. The results are represented in Figure 3.8.

1
0.8 0.50%
Absorbance
0.6 1%
0.4 1.50%
0.2 2%
0
0 0.5 1 1.5 2 2.5
Volume of EAA (ml)
Figure 3.7: Absorbance Variation with Varying Concentration and Volume of EAA
Solutions for MSP
0.8
Absorbance
0.6 0.50%
1%
0.4
1.50%
0.2 2%
0
0 0.5 1 1.5 2 2.5
Volume of EAA (ml)
Figure 3.8: Absorbance Variation with Varying Concentration and Volume of EAA
solutions for MTCP
1 ml of 0.5 % EAA was found enough to get maximum absorbance with MSP and was
selected in all subsequent experiments. The diazonium cation of mosapride couples with
hydroxyl group of EAA in alkaline medium. The protons in EAA, alpha to carbonyl
carbon are acidic and resulting carbanion undergo nucleophilic substitution. Enolate of
EAA serves as a bidentate ligand, thus it forms yellow colour azo dye. Enolate anion of
EAA is a strong nucleophile. 1 ml of 2 % EAA was found enough to get maximum
absorbance with MTCP and was selected in all subsequent experiments. The diazonium
cation of metoclopramide couples with hydroxyl group of EAA in alkaline medium
[28-30].

3.4.4 Effect of NaOH

3.4.4.1 MSP-EAA Dye
Nitrite reacts with mosapride in HCl medium to form diazonium cation, which
gets coupled with ethyl acetoacetate to form yellow color in basic medium. Sodium
hydroxide was used to form this color. Effect of sodium hydroxide solutions at varying
concentrations and varying volume was studied. The results showed that, the use of 2 ml
of 2 M sodium hydroxide was found to give maximum absorbance with good linearity
and was used for the determination of nitrite in the recommended procedure. The effect
of NaOH on the color development is shown in Figure 3.9.
3.4.4.2 MTCP-EAA Dye
Nitrite reacts with metoclopramide in HCl medium to form diazonium cation,
which gets coupled with ethyl acetoacetate to form yellow color in basic medium.
Sodium hydroxide was used to form this color. Effect of sodium hydroxide solutions at
varying concentrations and varying volume was studied. The results showed that, the use
of 2 ml of 2 M sodium hydroxide was found to give maximum absorbance with good
linearity and was used for the determination of nitrite in the recommended procedure.
The effect of NaOH on the color development is shown in Figure 3.10.
0.9
0.8
0.7
0.6 0.5 M
Absorbance
0.5 1.0 M
0.4
1.5 M
0.3
0.2 2.0 M
0.1 2.5 M
0
0 1 2 3
Volume of NaOH (ml)
Figure 3.9: Absorbance Variation with Varying concentration and volume of NaOH for
MSP Reagent

1.2
1
0.5M
Absorbance
0.8
1M
0.6 1.5M
0.4 2M
0.2
0
0 1 2 3
Volume of NaOH (ml)
Figure 3.10: Absorbance Variation with Varying concentration and volume of NaOH for
MTCP Reagent
It was observed from absorbance values that the color intensity and the stability
maximum with 2 M NaOH. The diazonium cation couples with hydroxyl group in EAA.
Isomeric structure of compound changes with change in properties of solvent. Formed
azo dye shows increased absorption in NaOH because percentage of enolic form
increases [28].
3.4.5 Effect of Reaction Time

The effect of reaction time on diazotization was studied. Variation in time was
experimented from 2-10 minutes. The results obtained are presented in Figure 3.11 and
Figure 3.12.
1.2
1
Absorbance
0.8
0.6
0.4
0.2
0
0 2 4 6 8 10 12
Time (min)
Figure 3.11: Absorbance Variation with Varying Time of Diazotization for MSP

1.2
1
0.8
Absorbance
0.6
0.4
0.2
0
0 2 4 6 8 10 12
Time (min)
Figure 3.12: Absorbance Variation with Varying Time of Diazotization for MTCP
It was observed that the time required for diazotization with shaking was 5
minutes at room temperature and yellow colored azo dye stability was up to 45 minutes.
Increase or decrease of the time for diazotization reaction affects the change in color
intensity and absorbance value.
3.5 The Diazotization Reactions

3.5.1 MSP-EAA Reaction Scheme
Mosapride reacts with nitrite in acidic medium to form diazonium cation which is
coupled with EAA in basic media to form yellow coloued azo dye [24, 29].
Nitrite ion reacts with diazotizable aromatic amines such as metoclopramide in
HCl to form diazonium ions. These ions produced strongly colored azo dyes with coupling
reagent like EAA to form yellow colored azo dye [30].

The reaction system is presented in the following scheme in Figure 3.12.
Cl
Cl
NH 2 + -
O N N CI
O
N NH + NO 2
-
H+
N NH
O CH 3
O O CH 3
O
+
O
F Mosapride C 2H 5
F
(Ethyl acetoacetate)
OCH 3
O
-
OH
C 2 H 5
O
Cl OCH 3
N
O N
NH O
N
O CH 3
O
F Yellow coloured azo dye (429 nm)
Figure 3.13: Diazotization between Nitrite and MSP and its coupling with EAA
3.5.2 MTCP-EAA Reaction Scheme

The diazotization coupling reactions scheme is given in Figure 3.13.
O N H C H 3
O N H C H 3
N
N
C H 3
OCH 3
C H 3
OCH 3
-
+ NO 2 HCI
C l
H N C l
2 + -
Metoclopramide N 2 CI
O
C 2 H 5
(Ethyl acetoacetate)
OCH 3
O -
OH
O N H C H 3
N
C H 3
OCH 3
C l O
N C H
2 5
N
OCH 3
Yellow coloured azo dye (437.5 nm)
Figure 3.14: Diazotization between Nitrite and MTCP and its coupling with EAA

3.6 Preparation of Calibration Curves

3.6.1 Calibration Curves for MSP and MTCP Reagents
The calibration graph was prepared using eight different levels of calibration
standards from 0.01 to 0.4 mg/l NO2-N using 1000 mg/l of nitrite solution. The
absorbance was measured at 429 nm for MSP-EAA reagents and 437.5 nm for MTCP-
EAA reagents against a blank and plotted against the concentration of nitrite solution.
The results are represented in Figure 3.15 and Figure 3.16 below.
1.200
1.000 y = 2.433x + 0.0018
R² = 0.999
Absorbance
0.800
0.600
0.400
0.200
0.000
0 0.1 0.2 0.3 0.4 0.5
Nitrite Concentration (µg/ml)
Figure 3.15: Calibration Curve of Nitrite using Diazotization with MSP-EAA Reagents
0.3
0.25 y = 0.6218x + 0.0016
Absorbance
0.2 R² = 0.997
0.15
0.1
0.05
0
0 0.1 0.2 0.3 0.4 0.5
Nitrite Concentrations (mg/l)
Figure 3.16: Calibration Curve of Nitrite using Diazotization with MTCP-EAA Reagents
The series of concentrations from 0.01-0.4 µg/ml of nitrite solution forming azo
dye using MSP-EAA reagents are shown in Figure 3.17 and that of MTCP-EAA reagents
are shown in Figure 3.18. The solutions were scanned at 429 nm for MSP-EAA and
437.5 nm for MTCP-EAA and absorbance was recorded.

Figure 3.17: Calibration Standards of Nitrite for MSP-EAA Reagents
Figure 3.18: Calibration Standards of Nitrite for MTCP-EAA Reagents
3.6.2 Calibration Curve for Reference Method using Sulphanilamide-n-naphthyl

ethylene diamine (SAM-NEDA) Reagents
To a series of 10 ml volumetric flasks containing 0.01-0.4 mg/L of standard nitrite
solutions, added 2.0 mL sulphanilamide-NEDA reagent solution and was shaken for 5
min and kept aside for 10 minutes. The absorbance was measured on spectrophotometer
at wavelength 540 nm. The concentration of nitrite was determined with the help of a
standard calibration graph [27]. The calibration curve of nitrite using diazotization with
SAM-NEDA reagent represented in Figure 3.19.

0.070
0.060 y = 0.1582x + 0.003
R² = 0.998
0.050
Absorbance
0.040
0.030
0.020
0.010
0.000
0 0.1 0.2 0.3 0.4 0.5
Nitrite Concentration (µg/ml)
Figure 3.19: Calibration Curve of Nitrite using Diazotization with SAM-NEDA

The series of concentrations from 0.01-0.4 µg/ml of nitrite solution forming azo
dye using SAM-NEDA reagents are shown in Figure 3.20. The solutions were scanned at
540 nm for SAM-NEDA reagent and absorbance was recorded.
Figure 3.20: Calibration Standards of Nitrite for SAM-NEDA Reagents
3.7 Application of Proposed Spectrophotometric methods

Proposed Methods:
MSP-EAA Method
MTCP-EAA Method
Reference Method:
SAM-NEDA Method- Sulphanilamide (SAM) and N-(1-naphthyl) ethylenediamine
dihydrochloride (NEDA) method [27]. Filtered clear real samples were treated for nitrite

determination by MSP-EAA and MTCP-EAA proposed methods and SAM-NEDA

reference method.
3.7.1 Determination of Nitrate and Nitrite in Laboratory Mixture

Suitable aliquots of nitrite and nitrate mixture at different mass ratios were taken
separately in 10 ml beakers and the nitrate was reduced to nitrite with a pinch of granular
mixture of zinc dust/NaCl and conc. HCl. Excess zinc dust was filtered and filtrate was
used to determine the concentrations of total nitrate plus nitrite by following the proposed
and reference procedures. The absorbance of equal volume of nitrite and nitrate mixture
was measured without reduction of nitrate. This gives the conc. of nitrite originally
present in the mixture. Subtracting this from the total gives the concentration of nitrate
present in the mixture [31]. The results are presented in Table 3.1 & 3.2.
3.7.2 Determination of Nitrate and Nitrite in Water and Soil Samples

The proposed method has been applied for the determination of nitrite and nitrate
in tap water and surface water samples. The interference of chloride was removed by
precipitating the chloride with silver sulphate [32, 33]. Suitable aliquots were taken and
analyzed for nitrite and nitrate following the proposed and reference methods.
About 1.0 g of soil sample was taken in a 25 ml beaker and extracted with 3 ml
portions of 0.5 % sodium carbonate solution. The extract was filtered through Whatman
No. 41 filter paper [32]. The filtrate was collected and diluted to 25 ml. Appropriate
aliquots of 1-2 ml of the solution were transferred in to a 10 ml calibrated flasks and
analyzed according to the proposed procedure. They were tested negative for nitrite
concentration. To these samples known amounts of nitrite and nitrate solutions were
added and analyzed for nitrate and nitrite following the proposed and reference
procedures. The results are presented in Table 3.3 & 3.4.
3.7.3 Determination of Nitrate and Nitrite in Vegetable Samples

Vegetable samples were removed from their package and the outside leaves,
ends of leaves and stems/stalks removed. Samples were rinsed in distilled, deionizer
water, allowed to drain and the excess water spun off using a salad spinner (10 pumps of
pressure, spun for 30 s). Rinsed samples were cut into smaller pieces and weighed to a

mass 200 g sample. Sample pieces were placed in a food processor and homogenized for
15 s. The sides of the food processor were scraped and the sample homogenized for an
additional 15 s. This process was repeated until the sample was homogenized for a total
of 60 s (4 iterations). The results are presented in Table 3.5 & 3.6.
3.7.4 Determination of Nitrate and Nitrite in Pharmaceutical Samples

Isosorbide dinitrate (0.05 g) sample was taken, and nitrate was reduced to nitrite
using zinc dust/NaCl with HCl 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 proposed and reference procedures described for the analysis of water and
soil samples. The results are presented in Table 3.7 & 3.8.
Table 3.1: Determination of Nitrate and Nitrite in Laboratory Mixtures using MSP-EAA
Sr. No. Nitrite Nitrate Nitrite Nitrate % Recovery

taken taken found found Nitrite Nitrate
µg/ml µg/ml µg/ml µg/ml
1 0.4 -- 0.399 -- 99.75 --
2 0.3 0.1 0.299 0.098 99.67 98
3 0.2 0.2 0.201 0.198 100.5 99
4 -- 0.4 -- 0.397 -- 99.25
Table 3.2: Determination of Nitrate and Nitrite in Laboratory Mixtures using MTCP-
EAA
Sr. No. Nitrite Nitrate Nitrite Nitrate % Recovery

taken taken found found Nitrate Nitrate
µg/ml µg/ml µg/ml µg/ml
1 0.35 -- 0.344 -- 98.29 --
2 0.3 0.05 0.296 0.051 98.67 102
3 0.25 0.1 0.247 0.099 98.80 99

4 -- 0.35 -- 0.347 -- 99.14

Table 3.3: Determination of Nitrite in Water and Soil Samples using Proposed MSP-EAA
Method and Reference Method (n=6)
Sample Nitrite MSP-EAA method Reference Method [27] t-test

added Nitrite found % Nitrite found %
µg/ml µg/ml±SD Recovery µg/ml±SD Recovery
Tap 0.04 0.0397±0.0015 99.04 0.0396±0.001 98.96 0.4696
water
0.06 0.059±0.002 98.19 0.058±0.002 97.25 0.3315
0.08 0.079±0.0016 98.35 0.077±0.0028 95.71 0.1152
Surface 0.04 0.039±0.0016 97.04 0.0393±.0019 98.42 0.3280
water
0.06 0.058±0.002 96.69 0.059±0.002 98.22 0.3126
0.08 0.077±0.0029 96.52 0.079±0.002 98.19 0.0399
Soil 0.04 0.039±0.002 97.92 0.0388±0.001 97.13 0.3742
sample
0.06 0.057±0.0015 95.78 0.0599±.0028 99.75 0.0658
0.08 0.758±0.0017 94.71 0.0768±0018 96 0.1978
Table 3.4: Determination of Nitrite in Water and Soil Samples using Proposed MTCP-
EAA Method and Reference Method (n=5)
Sample Nitrite MTCP-EAA method Reference Method [27] t-test

(mg/l) (mg/l±SD) Recovery (mg/l±SD) Recovery
Tap 0.1 0.093±0.003 93 0.095±0.004 95.8 0.0223
Water 0.2 0.189±0.005 94.3 0.190±0.012 95.2 0.395
0.3 0.288±0.01 96 0.301±0.011 100.3 0.0689
Surface 0.1 0.101±0.011 101.4 0.098±0.001 98.2 0.2636
Water 0.2 0.197±0.026 98.6 0.204±0.021 102.1 0.3593
0.3 0.291±0.01 97.07 0.297±0.008 99.1 0.1331
Soil 0.1 0.092±0.003 92 0.092±0.004 91.6 0.466
sample 0.2 0.188±0.006 94.2 0.193±0.008 96.3 0.2684
0.3 0.294±0.01 97.93 0.293±0.007 97.7 0.4695

Table 3.5: Determination of Nitrite in Vegetable Samples using Proposed MSP-EAA

Sample Nitrite MSP-EAA method Reference method [27] t-test

µg/ml µg/ml ± SD Recovery µg/ml ± SD Recovery
Spinach 0.04 0.041± 0.005 103 0.042 ± 0.005 106 0.3056

leaves
0.06 0.061 ± 0.005 101 0.062 ± 0.005 103.3 0.2677
0.08 0.084 ± 0.01 104 0.084 ± 0.009 104.5 0.4714
Coriander 0.04 0.042 ± 0.005 104.5 0.04 ± 0.004 101 0.2684

leaves
0.06 0.061 ± 0.003 102 0.06 ± 0.004 99.67 0.1955
0.08 0.079 ± 0.01 99 0.083 ± 0.007 103.3 0.4822
Tabulated t-value for 5 degrees of freedom at P (0.05) is 2.015
Table 3.6: Determination of Nitrite in Vegetable Samples using Proposed MTCP-EAA

Sample Nitrite MTCP-EAA method Reference method [27] t-test

Spinach 0.1 0.103±0.007 103.4 0.104±0.005 104.2 0.4231

leaves
0.2 0.189±0.005 99.4 0.198±0.005 99 0.4405
0.3 0.297±0.01 98.87 0.298±0.004 99.2 0.3574
Coriander 0.1 0.096±0.005 95.8 0.104±0.005 101.4 0.1245
leaves
0.2 0.189±0.005 94.3 0.190±0.012 95.2 0.395
0.3 0.293±0.007 97.6 0.296±0.003 98.5 0.2091

Table 3.7: Determination of Nitrite in Pharmaceutical Preparation using Proposed MSP-

EAA Method and Reference Method (n=6)
Sample Nitrite MSP-EAA method Reference method [27] t-test

µg/ml
µg/ml±SD Recovery µg/ml±SD Recovery
Isosorbidinitrate 0.04 0.039±0.003 99.75 0.041±0.001 102.5 0.0328
0.06 0.061±0.002 101.66 0.059±0.001 99.83 0.3315
0.08 0.079±0.03 99.5 0.079±0.001 99.75 0.2137
Table 3.8: Determination of Nitrite in Pharmaceutical preparation using Proposed

MTCP-EAA Method and Reference Method (n=6)
Sample Nitrite MTCP-EAA method Reference method [27] t-test
Isosorbidinitrate 0.1 0.098±0.001 97.8 0.095±0.004 95.8 0.1373
0.2 0.194±0.001 96.9 0.196±0.004 97.9 0.1597
0.3 0.293±0.006 97.53 0.299±0.006 99.6 0.0954
3.8 Statistical Analysis

Statistical analysis of result by Student’s t-test reveals that there is close
agreement between the results obtained by the proposed method and reference method for
the results of water and soil samples. The results of pharmaceutical samples show that the
recovery values obtained are close to 100 %. The recovery experiments are performed on
water and soil samples in presence of Cu2+, K+, Na+, Ca2+, Fe2+, Cl-, CO32-, HPO42- and
SO42- etc. naturally present in the samples.
3.9 Validation of Developed UV- Visible Spectrophotometric Methods

3.9.1 Accuracy
To test the accuracy of the method, recovery experiments were performed on
synthetic mixtures of nitrite in presence of ions such as Cu2+, K+, Na+, Ca2+, Fe3+, Cl-,

CO32-, HPO42- and SO42- etc. by using proposed methods and the recoveries obtained are
in the range of 99.75 to 100.5 % for MSP-EAA reagents and 98.34 to 99.53 % for
MTCP-EAA reagents. Recovery study was also performed by spiking 0.1, 0.2 and 0.3
mg/l nitrite in real samples like water, soil, vegetable and pharmaceutical samples in
triplicate. The recovery result for nitrite were given in Table 3.1-3.8.
3.9.2 Precision
The precision of the proposed method was evaluated by replicate analysis of
samples containing nitrate and nitrite at five different concentrations. Precision study was
carried out using parameter like method repeatability, intra-day and inter-day precision
showed that the results were within acceptable limit i.e. % R.S.D. below 2.0 indicating
that the method is reproducible. Intra-day and Inter-day Precision are represented in
Table 3.9 for MSP-EAA and in Table 3.10 for MTCP-EAA.
Table 3.9: Intra-day and Inter-day Precision Data using MSP-EAA Reagents
Nitrite Intra-day Inter-day
Spiked % Average SD % RSD % Average SD % RSD
level Recovery Recovery
µg/ml
0.04 97.48 0.002 3.918 98.85 0.001 2.529
0.06 96.95 0.002 3.578 100.15 0.003 4.403
0.08 95.49 0.002 2.000 93.78 0.002 2.036
Average 96.64 3.165 97.59 2.989
Table 3.10: Intra-day and Inter-day Precision Data using MTCP-EAA Reagents
Nitrite Intra-day Inter-day
Spiked % Average SD % RSD % Average SD % RSD
level Recovery Recovery
µg/ml
0.1 99.82 0.002 1.530 99.82 0.002 2.09
0.2 99.62 0.002 1.020 99.76 0.004 2.09
0.3 99.57 0.004 1.350 99.39 0.006 2.04
Average 99.67 1.300 99.66 2.07

3.9.3 Specificity
In order to assess the possible applications of the proposed method, the effect of
interfering substances that often present with nitrite in complex sample matrices were
studied by adding various amounts of cations and anions to 0.2 µg/ml of nitrite and 0.5
µg/ml of nitrate for MSP-EAA and 0.2 µg/ml of nitrite and 0.3 µg/ml of nitrate for
MTCP-EAA. During the interference studies, any insoluble substances that formed were
simply removed by centrifugation and the absorbance was measured with the
centrifugates. The results are given in Table 3.11 (for MSP-EAA) and Table 3.12 (for
MTCP-EAA).
Table 3.11: Effect of Foreign Ions on the Determination of Nitrite and Nitrate using
MSP-EAA Reagents
Foreign ion Added as Tolerance limit

( µg/ml)
For Nitrite For Nitrate
(0.2 µg/ml) (0.5µg/ml)
Cu2+ CuSO4.5H2O 130 130
K+ KCl 2000 2000
Na+ NaCl 300 300
Ca2+ CaCl2.2H2O 2000 50
Fe3+ FeSO4.7H2O 500 500
Cl- NaCl 3000 3000
CO32- Na2CO3 1000 1000
HPO42- Na2HPO4 200 200
SO42- Na2SO4 1000 1000

Table 3.12: Effect of Foreign Ions on the Determination of Nitrite and Nitrate using
MTCP-EAA Reagents
Foreign ion Added as Tolerance limit

(µg/ml)
For Nitrite For Nitrate
(0.2 µg/ml) (0.3 µg/ml)
Cu2+ CuSO4.5H2O 100 100
K+ KCl 1500 1500
Na+ NaCl 200 200
Ca2+ CaCl2.2H2O 1000 50
Fe2+ FeSO4.7H2O 500 500
Cl - NaCl 3000 3000
2-
CO3 Na2CO3 1000 1000
HPO42- Na2HPO4 300 300
SO42- Na2SO4 1000 1000
The tolerance level of interfering ions may be increased by the addition of 2 ml

of 2 % EDTA solution. Selectivity of the proposed method was improved by converting
nitrite into its azo derivative which absorbs at longer wavelength in the presence of
interfering cations and anions and there tolerance limits were measured by adding ions of
known concentration in present procedure which causes ±2 % relative error [27, 34].
3.9.4 Limit of Detection (LOD) and Limit of Quantitation (LOQ)
Nitrite sample solution was subjected to LOD and LOQ studies using MSP-
EAA and MTCP-EAA reagents. The results are summerised in Table 3.13.
Table 3.13: LOD and LOQ of Nitrate and Nitrite
Component Proposed method LOD (µg/ml) LOQ (µg/ml)
Nitrite MSP-EAA 0.1 0.35
Nitrite MTCP-EAA 0.02 0.07
3.9.5 Linearity and Range

For nitrite determination the concentration of the nitrite is 0.1600 µg/ml. So the
working range of analyte was set between 0.01 to 0.2 µg/ml. To show the linearity and

range of sample solutions approximately 60 % to 140 % of the test concentrations were

prepared as per described method. The results are tabulted in Table 3.14.
Table 3.14: Linearity and Range Study Data of Nitrite Estimation by Spectrophotometric
Method for MSP-EAA and MTCP-EAA reagents (n=5)
Sr. Wt. of working Concentration Mean Mean

No. standard (mg) added of Nitrite absorbance absorbance
and its dilution (ml) (µg/ml ) at 429 nm for at 437.5 nm for
MSP-EAA MTCP-EAA
1 0.16/100/0.1/10/0.01/10 0.01 0.015 0.01
2 0.16/100/0.1/10/0.05/10 0.05 0.119 0.028
3 0.16/100/0.1/10/0.1/10 0.1 0.252 0.063
4 0.16/100/0.1/10/0.15/10 0.15 0.364 0.091
5 0.16/100/0.1/10/0.2/10 0.2 0.496 0.121
Y ----- ----- Y = 2.513x - Y = 0.594x +
0.007 0.001
(r2) ----- ----- 0.999 0.997
3.9.6 Robustness
The real sample of nitrite was analyzed using proposed method after a deliberate
change in detection wavelength for estimation by ±2 nm.
Table 3.15: Robustness Study Data of Nitrite Estmation by Spectrophotometric Method
for MSP-EAA and MTCP-EAA reagents (n=3)
Sr. Change Nitrite Sr. Change Nitrite

No. in λ estimation No. in λ estimation
for % for %
MSP- MTCP-
EAA EAA
(±2 nm) (±2 nm)
1 427 97.49 1 435.5 97.14
2 429 101.19 2 437.5 101.96
3 431 98.31 3 439.5 98.75
Mean 99 Mean 99.28
±SD 1.94 ±SD 2.46

3.9.7 Ruggedness
Six standard solutions of nitrite (0.2 µg/ml) were prepared and treated with MSP-
EAA and MTCP-EAA reagents seperately and analysed by two different analysts using
same make of different Spectrophotometers. The concentration of nitrite was calculated
and reported along with the standard deviation(sample) and % RSD of the six samples.
Table 3.16: Ruggedness Study Data for Working Reference Standard using MSP-EAA
and MTCP-EAA reagents (n=6)
Analyst Concentration Amount of Nitrite Found (%)

of Nitrite
(µg/ml) Using MSP-EAA Using MTCP-EAA
Analyst I 0.2 99.27 95.88
Analyst II 0.2 98.24 96.55
3.10 Conclusion
1. The reagents provide a simple and sensitive method for the spectrophotometric
determination of nitrite and nitrate from environmental samples.
2. The developed method does not involve any stringent reaction conditions and offers
the advantages of color stability about more than 4 hours.
3. The results obtained for MSP-EAA and MTCP-EAA methods compare favorably
with the reference method [27].
4. A statistical analysis of the results by Student’s t-test at 95 % confidence level
showed no significant difference in accuracy and precision between proposed and
reference methods.
5. The proposed method has been successfully applied to the determination of trace
amounts of nitrite and nitrate in water, soil, vegetables and pharmaceutical
preparations.
6. The developed method was found to be sensitive, accurate, simple, precise and
economical and can be used as an alternative to the existing methods for routine
quality control analysis of nitrite in pure form, pharmaceutical preparations and
environmental water, soil and food samples.

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3.1 Background: Chapter 3: Spectrophotometric Determination of Nitrate and Nitrite

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3.1 Background: Chapter 3: Spectrophotometric Determination of Nitrate and Nitrite

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Chapter 3: Spectrophotometric Determination of Nitrate and Nitrite

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 46

3.2 Materials and Methods

3.2.2 Reagents and Materials

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 50

3.3 Results and Discussion

3.3.2 Wavelength of Maximum Absoption for Spectrophotometric Detection

3.3.2.1 Determination of λmax of Azo Dye using MSP-EAA as Reagents

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 51

Figure 3.1: Absorbance S

3.3.2.2 Determination of λmax of Azo Dye using MTCP-EAA as Reagents

Figure 3.2: Absorbance S

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 52

3.4 Optimization of UV-Visible Spectrophotometric Parameters

The optimum concentration of HCl was fixed to be 2 M and volume of HCl

3.4.2 Effect of Substrate Concentration

3.4.2.2 Metoclopramide Reagent Concentration: In order to fix the amount of

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3.4.3 Effect of Coupling Agent (EAA) Concentration

3.4.3.2 Effect of EAA Concentration on Metoclopramide Diazonium Cation:

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 55

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 56

3.4.4 Effect of NaOH

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3.4.5 Effect of Reaction Time

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3.5 The Diazotization Reactions

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 59

The reaction system is presented in the following scheme in Figure 3.12.

F Yellow coloured azo dye (429 nm)

3.5.2 MTCP-EAA Reaction Scheme

Yellow coloured azo dye (437.5 nm)

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 60

3.6 Preparation of Calibration Curves

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Figure 3.17: Calibration Standards of Nitrite for MSP-EAA Reagents

Figure 3.18: Calibration Standards of Nitrite for MTCP-EAA Reagents

3.6.2 Calibration Curve for Reference Method using Sulphanilamide-n-naphthyl

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 62

Figure 3.19: Calibration Curve of Nitrite using Diazotization with SAM-NEDA

Figure 3.20: Calibration Standards of Nitrite for SAM-NEDA Reagents

3.7 Application of Proposed Spectrophotometric methods

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determination by MSP-EAA and MTCP-EAA proposed methods and SAM-NEDA

3.7.1 Determination of Nitrate and Nitrite in Laboratory Mixture

3.7.2 Determination of Nitrate and Nitrite in Water and Soil Samples

3.7.3 Determination of Nitrate and Nitrite in Vegetable Samples

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 64

3.7.4 Determination of Nitrate and Nitrite in Pharmaceutical Samples

Sr. No. Nitrite Nitrate Nitrite Nitrate % Recovery

Sr. No. Nitrite Nitrate Nitrite Nitrate % Recovery

3 0.25 0.1 0.247 0.099 98.80 99

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 65

Sample Nitrite MSP-EAA method Reference Method [27] t-test

0.08 0.758±0.0017 94.71 0.0768±0018 96 0.1978

Sample Nitrite MTCP-EAA method Reference Method [27] t-test

Ph. D Thesis: North Maharashtra University, Jalgaon, 2015 Page 66

Table 3.5: Determination of Nitrite in Vegetable Samples using Proposed MSP-EAA

Sample Nitrite MSP-EAA method Reference method [27] t-test

Spinach 0.04 0.041± 0.005 103 0.042 ± 0.005 106 0.3056

0.08 0.084 ± 0.01 104 0.084 ± 0.009 104.5 0.4714

Coriander 0.04 0.042 ± 0.005 104.5 0.04 ± 0.004 101 0.2684

0.08 0.079 ± 0.01 99 0.083 ± 0.007 103.3 0.4822