Chapter 3 __________________________________________________________________________

51

Aminophenyl Benzimidazole as a new reagent for the estimation

of nitrogen dioxide/nitrite/nitrate at trace level: Application to

environmental sample

3.1 Introduction

Nitrogen dioxide is one of the most hazardous pollutants among the oxides of nitrogen and

plays an important role in the formation of acid rain, photochemical smog and in the

generation of many secondary pollutants [1 - 3]. Nitrogen forms two environmental

important oxyacids, nitrous acid and nitric acid and its corresponding nitrite and nitrate

anions are commonly present in the environment [4]. The determination of nitrate ion is an

important factor in the analysis of food and natural water samples [5]. Nitrite and nitrate are

intimately involved in the fixation of overall nitrogen cycle in soil and plants [6]. Both nitrite

and nitrate represent important wide spread contaminants of aqueous environment and serve

as significant indicators of natural water quality. The increasing levels of nitrate in water

results mainly from agricultural application of fertilizers as well as from many industrial

processes [7]. Nitrate as one of the principal nutrients stimulates the growth of macrophytes

and phytoplankton causes eutrophication of water. Nitrite formed during the biodegradation

of nitrate and ammonical nitrogen or nitrogenous organic matter is important indicator of

faecal pollution of natural water [8]. The speciation of nitrite and nitrate in water and

foodstuffs has been emphasized in recent years because of their potential harmful impact on

human health. Nitrate in water is primarily low toxic but the converted nitrite due to

microbial or in vivo reduction when combines with hemoglobin to form methemoglobinemia.

This is quite significant in infants (blue baby syndrome). In addition the reaction between

nitrite and secondary or tertiary amines can result in the formation of nitroso compounds,

some of them are known to be carcinogenic, teratogenic and mutagenic [9]. The occurrence

of nitrate and nitrite in milk is generally at trace levels, with secretory and post secretory

contamination of bovine milk usually minimal. Therefore, unless post - secretory

contamination has occurred, dietary intake of these contaminants via diary foods is usually of

minor significance for adult humans. However newborn infants are especially susceptible to

the detrimental effects of these contaminants [10]. Leafy vegetables are an excellent source

Chapter 3 __________________________________________________________________________

52

of vitamins, minerals and biologically active compounds [11-12]. The incidence of coronary

heart disease, atherosclerosis and stroke can be reduced by increasing vegetable consumption

as that of the major cancers such as cancer of the stomach, lung, mouth, esophagus, colon

and rectum. Generally nitrates are abundant in foods because plants take up nitrogen from the

soil in this ionic form. The nitrates in foods then can be reduced to nitrite because of some

bacteria’s action [13]. Nitrite salts are used as preservatives in meat products to prevent the

growth of bacteria in meat curing process [14]. The reduction of nitrate to nitrite is possible

in the body of infants, where low acidity prevailing conditions causes the growth of nitrite

reducing microorganisms [15 - 17]. Due to the significant influence of nitrite and nitrate on

human environment and health, it is important to monitor their concentration levels and

examine the mechanisms involved in their production, transport and decomposition in

atmospheric condensed phase and surface waters [18]. Several techniques have been

developed for the determination of nitrite/nitrate/ammonia nitrogen as well as nitrogen

dioxide after fixing it as nitrite ion using a suitable trapping medium at trace level [19].

Among all these techniques spectophotometric methods are widely used due to their

simplicity, reproducibility and easy adoptability [20 - 22]. The method based on the diazo-

coupling reaction which is popularly known as Griess-Ilsovey reaction has been exploited for

the development of analytical procedures for the determination of nitrogen

dioxide/nitrite/nitrate from a variety of sample matrices due to its ease & high reproducibility

[23]. Among these diazocoupling methods only few methods find wide spread significance in

terms of detection limit and molar absorptivity. In this method the nitrite is diazotized with a

primary aromatic amine and coupled with a reagent to form an azo dye under suitable

reaction conditions [24 - 25]. Many of the the diazocoupling methods lack sensitivity,

selectivity and require a prolonged sampling periods in case of atmosphere sampling of

nitrogen dioxide. Methods based on the extraction of the formed azo dye into a suitable

organic solvent leads to lower detection limits but color stability and solvent properties

restrict its wide spread use. The proposed work describes a new reagent for the determination

of nitrogen dioxide in ambient air after fixing it as nitrite ion in sodium arsenite and

nitrite/nitrate in a variety of sample matrices. The fixed nitrite is diazotized with 2-(4-

aminophenyl)benzimidazole (APB) and coupled with N-(1-naphthyl)ethylenediamine

Chapter 3 __________________________________________________________________________

53

dihydrochloride (NEDA) in aqueous medium to form an azo dye with an absorption

maximum at 555 nm and the extraction procedure gives a very low detection limit.

3.2 Experimental Section

3.2.1 Apparatus and Reagents

Absorbance measurements were made using Shimadzu UV-VIS-NIR Scanning

Spectrophotometer (model UV-3101PC) with 1 cm quartz cuvettes, Miclins (Chennai)

peristaltic pump (model- PP 30) with suitable suction devices were used for sampling of

nitrogen dioxide from ambient air. Control Dynamics (Mumbai) digital pH meter (model

APX 175 E/C) was used for all pH measurements. All reagents used were analar grade

without further purification. Distilled water was used throughout the experiments.

Standard sodium nitrite stock solution (1000 µgmL-1): It has been prepared by dissolving

0.15 g of pre-dried sodium nitrite (at 105 ± 5˚C for an hour) in distilled water and diluted to

100 mL. Working standards were prepared from stock solution on the day of use.

Sodium arsenate absorber solution: Prepared by dissolving 4 g of NaOH and 1g of sodium

arsenite in 1litre of water.

2-(4-aminophenyl) benzimidazole (APB) (0.05 %): Prepared by dissolving 0.05g of APB

in 5 mL of acetonitrile and diluted to 100 mL with distilled water.

Diaminobenzene (DAB) (0.05 %): Prepared by dissolving 0.05g of DAB in 5 mL of 2N

HCl and diluted to 100 mL with distilled water.

2-Aminobenzoic acid (ABA) (0.05 %): Prepared by dissolving 0.05 g of ABA in 3 mL of 2

N HCl and diluting to 100 mL with distilled water.

N-(1-naphthyl) ethylenediamine dihydrochloride (NEDA) (0.05 %): Prepared by

dissolving 0.05 g of NEDA in distilled water and diluted to 100 mL.

Methanolic Hydrochloric acid (3.7 N): Prepared by mixing 50 mL of methanol with 25 mL

of hydrochloric acid (Sp.gr.1.18).

NH3-NH4Cl buffer solution (pH = 10): It has been prepared by dissolving 0.531 g of NH4Cl

in 80 mL of water, adjusting the pH to 10 with 1:1 ammonia (V/V) and diluted to 100 mL

with distilled water.

Chapter 3 __________________________________________________________________________

54

NH3-NH4Cl buffer solution (pH = 8.5): Prepared by dissolving 0.531g of NH4Cl in 80 mL

of water, adjusting pH to 8.5 with 1:1 ammonia (vol/vol) and diluted to 100 mL with distilled

water.

Acetate buffer (pH: 3.5): Dissolve 6.8 g of sodium acetate in 3 mL of acetic acid and adjust

the pH to 3.5 with acetic acid and diluting to 100 mL with distilled water.

Sodium Carbonate (0.5 %): It has been prepared by dissolving 0.5 g of sodium carbonate

in 100 mL using distilled water.

Formaldehyde (0.5 %): Prepared by diluting 1.3 mL of formaldehyde (38 %) to 100 mL

with water.

Trichloroacetic acid (TCA 10 %): It has been prepared by diluting 10 mL of TCA to 100

mL using distilled water.

Zinc sulphate (30 %): It has been prepared by dissolving 30 g of zinc sulphate in 100 mL

using distilled water.

Lead acetate (0.01 %): It has been prepared by dissolving 0.01 g of lead acetate in 100 mL

using distilled water.

Ascorbic acid (0.01 molL-1): It has been prepared by dissolving exactly 0.176 g of ascorbic

acid in 100 mL using distilled water.

Solvents for extraction: Isoamylalcohol, isoamyl acetate, MIBK, MBK and 1-butanol.

3.2.2 Copperized cadmium reductor column

Wash 25 g of 20 - 100 mesh Cd granules with 6N HCl and rinse with water. Swirl Cd

granules with 100 mL of 2 % CuSO4 solution for 5 minutes or until blue color partially fades.

Decant and repeat with fresh CuSO4 until a brown colloidal precipitate begins to develop.

Gently flush with water to remove all precipitated Cu. Insert a glass wool plug into the

bottom of a glass column (30 cm long × 5 mm id) and fill with water. Add sufficient Cu-Cd

granules to produce a column of 18.5 cm long. Maintain water level above Cu - Cd granules

to prevent entrapment of air. Wash column with 200 mL of dilute NH4Cl - EDTA buffer

solution. The column was activated by passing NH4Cl - EDTA buffer solution at a flow rate

7-10 mLmin-1. The flow rate was adjusted in such a way that the nitrate solution

quantitatively reduces to nitrite after passing through the reductor column. The column was

Chapter 3 __________________________________________________________________________

55

stored using NH4Cl - EDTA solution. The column should not be allowed to dry. Under these

conditions the column can be used for several months. All the column conditions were

optimized according to the standard method [23].

3.2.3 Recommended procedure

Aqueous: 10 mL aliquots of sodium arsenite solution containing 0 -10 µg nitrite were added

to series of 25 mL standard flasks containing 1 mL of 0.05 % 2-(4-aminophenyl)

benzimidazole and 1 mL of 2N HCl. The contents were mixed well and allowed to stand for

2 min. Then 1 mL of 0.05 % N-(1-naphthyl) ethylenediamine dihydrochloride was added and

diluted to 25 mL with distilled water and the absorbance values were measured at 555 nm

using 1cm quartz cuvette.

Extraction: 10 mL aliquots of sodium arsenite solution containing 0 - 2 µg nitrite were

added into a series of 25 mL standard flasks containing 1 mL of 0.05 % 2-(4-aminophenyl)

benzimidazole and 1 mL of 2N hydrochloric acid. The contents were mixed well and allowed

to stand for 2 min. Then 1 mL of 0.05 % N-(1-naphthyl) ethylenediamine dihydrochloride

was added and diluted to 25 mL with distilled water. The solutions were then transferred into

60 mL separating funnels and treated with 1 mL of 5N sodium hydroxide and 5 mL of

isoamyl alcohol as organic solvent. The solutions were equilibrated for one minute and the

organic phase was collected into 5 mL volumetric flask. Then it is diluted up to the mark

with methanolic hydrochloric acid and the absorbance values were measured at 565 nm

against reagent blank.

3.3 Results and Discussion

This method involves diazotization of 2-(4-aminophenyl) benzimidazole (APB) as amine

with fixed nitrite in acidic medium and coupling with N-(1-naphthyl) ethylenediamine

dihydrochloride (NEDA) in aqueous medium. The preliminary studies have been carried out

by using various aromatic amines for diazotization process and phenols/naphthols as

coupling agents for the determination of nitrite by diazocoupling reaction. Initial studies were

carried out using 10 mL aliquots of sodium arsenite containing 6 μg of nitrite. This solution

was introduced into a 25 mL calibrated flask containing 2 mL of amine in acidic condition

Chapter 3 __________________________________________________________________________

56

after allowing 2 min. for diazotization and 2 mL coupling agent in 2N sodium hydroxide.

The solutions were diluted up to the mark with distilled water and the reagent blanks were

also prepared simultaneously for each combination of amine and coupling agent. The

absorption spectrum of the blank and sample was then recorded over the wavelength range

400-700 nm. Based on these observations the combination of 2-(4-aminophenyl)

benzimidazole (APB) as amine and N-(1-naphthyl) ethylenediamine dihydrochloride

(NEDA) as coupling agent has resulted an azo dye with λmax at 555 nm. The combination of

these reagents gave high sample absorbance and very low blank absorbance in acidic

medium for the determination of nitrite through the diazo-coupling reaction.

3.3.1 Optimization study

3.3.1.1 Effect of amine

The effect of amine concentration was varied in order to establish the optimum quantity of

amine required for maximum absorbance by varying its concentration in the range 0.1 - 5 mL

using 0.05 % APB. Different volumes of amine were taken in a series of 25 mL volumetric

flasks containing 2 mL of 2N hydrochloric acid and these solutions were treated with 10 mL

aliquots of sodium arsenite containing 6 µg of nitrite. The solutions were allowed to stand for

five minutes and treated with 2 mL of 0.05 % coupling agent. These studies revealed that 0.2

mL of amine is sufficient enough to give maximum absorbance to the sample. Higher

concentrations of amine did not enhance the sample absorbance values; hence 1 mL of 0.05

% of amine has been fixed as optimum concentration in all further studies (Fig. 3.1).

Chapter 3 __________________________________________________________________________

57

Fig. 3.1 Effect of amine

3.3.1.2 Effect of acidity

Further the effect of acidity during diazotization process was examined in order to establish

the optimum acidity for maximum color development. In these experiments 10 mL aliquots

of alkaline sodium arsenite containing 6 µg of nitrite were added into series of 25 mL

calibrated flasks containing 1 mL of 0.05 % amine and various volumes of 2N hydrochloric

acid (0.1-5.0 mL). These solutions were allowed to stand for two minutes and treated with 5

mL of coupling agent. The absorbance values were measured at 555 nm. It is evident from

the graph that the overall acidity in the range 0.3 - 0.7 gave maximum absorbance. Hence the

required acidity was provided by the addition of 1 mL of 2N hydrochloric acid during

diazotization process (Fig. 3.2).

0 1 2 3 4 50.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Sample

Reagent blank

Ab

so

rba

nc

e

Amine(mL)

Chapter 3 __________________________________________________________________________

58

Fig. 3.2 Effect of acidity

3.3.1.3 Effect of diazotization time

The optimum time period required for diazotization of amine with nitrite was examined by

treating 10 mL aliquots of alkaline sodium arsenite solution containing 6 μg of nitrite in a

series of 25 mL calibrated flasks containing 1 mL of 0.05 % amine and 1 mL of 2N

hydrochloric acid. These flasks were allowed to stand for different time intervals and were

treated with 1 mL of 0.05 % coupling agent and the absorbance values were measured at 555

nm. It is evident from the graph that the time required for maximum absorbance is in the

range of 60 -180 sec. Hence one minute time period is allowed in all further studies for

complete diazotization before the addition of coupling agent for maximum sample

absorbance (Fig. 3.3).

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.90.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Reagent blank

Sample

Ab

so

rban

ce

Concentration of acid(N)

Chapter 3 __________________________________________________________________________

59

Fig.3.3 Effect of diazotization time

3.3.1.4 Effect of coupling agent concentration

The effect of coupling agent concentration was studied by varying its volume in the range

0.1- 5 mL. In these experiments 10 mL aliquots of alkaline sodium arsenite containing 6 µg

of nitrite were added into a series of 25 mL calibrated flasks containing 1 mL of 0.05 %

amine and 1 mL of 2N hydrochloric acid. Varying volumes of coupling agent (0.5 %) was

added and a reagent blank was also prepared simultaneously for each concentration of the

coupling agent. The absorbance measurements were made at 555 nm against water and

respective reagent blanks. The maximum absorbance value was obtained in the volume range

(1 - 3 mL). Hence 1 mL of 0.05 % NEDA is sufficient enough to produce maximum sample

absorbance which has been incorporated in the working procedure (Fig.3.4).

0 100 200 300 400 5000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Reagent blank

Sample

Ab

so

rba

nc

e

Diazotisation time(Sec)

Chapter 3 __________________________________________________________________________

60

Fig. 3.4 Effect of coupling agent

Attempts have been made to extract the formed azo dye into organic solvent to lower the

detection limits so that the developed method can be extended to measure the trace levels of

nitrogen dioxide present in the atmospheric air as well as in industrial flue gases.

3.3.1.5 Effect of extraction pH

In order to establish the most suitable pH range for the quantitative extraction of the azo dye

into organic solvent was next investigated. In these experiments, 10 mL aliquots of alkaline

sodium arsenite solution containing 2 µg of nitrite were added to a series of 25 mL calibrated

flasks containing 1 mL of 0.05 % amine and 1 mL of 2N hydrochloric acid. After standing

time of 2 min. the solutions were treated with 1 mL of 0.05 % coupling agent. These

solutions were transferred into 60 mL separating funnels and treated with 5 mL of various

buffer solutions in the pH range 2 - 14. The solutions were extracted with 5 mL of isoamyl

alcohol and the organic extracts were collected into 5 mL calibrated flasks. These extracts

0 1 2 3 4 50.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Reagent blank

Sample

Ab

so

rba

nc

e

NEDA(mL)

Chapter 3 __________________________________________________________________________

61

have been diluted to the mark with methanolic hydrochloric acid to restore the original color

and the absorbance values were measured. It has been found that λmax has been shifted from

555 nm in aqueous phase to 565 nm in organic phase. These studies have been revealed that

the extraction is quantitative in the pH range 9 - 12. Hence the required pH range during

extraction was maintained by the addition of 5 mL of 1N NaOH into separating funnels

before extracting the dye into organic solvent as shown (Fig.3.5).

Fig.3.5 Effect of extraction pH

3.3.1.6 Effect of Variation of solvents during extraction

Several polar solvents like 1-butanol, isoamylalcohol, isoamyl acetate and non polar solvents

like benzene, toluene were used for extracting the azo dye. Among several solvents isoamyl

alcohol gave the lower blank absorbance and higher sample absorbance values. In these

experiments 10 mL aliquots of sodium arsenite solution containing 0-2 µg nitrite were added

to series of 25 mL standard flasks containing 1 mL of 0.05 % amine and 1 mL of 2N

hydrochloric acid. The contents were mixed well and allowed to stand for 2 min. Then 1 mL

of 0.05 % N-(1-naphthyl) ethylenediamine dihydrochloride was added and diluted to 25 mL

with distilled water (Table 3.1).

0 2 4 6 8 10 12 1 40 .0 0

0 .0 5

0 .1 0

0 .1 5

0 .2 0

0 .2 5

0 .3 0

0 .3 5

0 .4 0

0 .4 5

0 .5 0

0 .5 5

R e ag en t b lan k

S a m p le

Ab

so

rba

nc

e

p H

Chapter 3 __________________________________________________________________________

62

Table 3.1 The effect of solvents

Sl No Solventa (mL) Absorbance

Blank vs. Solvent Sample vs. Blank

1) 1-Butanol (10) 0.0243 0.4986

2) Isoamyl acetate (5) 0.0271 0.4376

3) Isoamyl alcohol (5) 0.0015 0.5401

4) Isoamyl alcohol 0.0335 0.4162

+

Isoamyl acetate (5)*

5) Benzene (5) 0.0526 0.3953

6) Toluene (5) 0.0431 0.2934

aBased on the solubility of solvents in aqueous phase, different volumes have been used. In

all these cases the extract was collected into 5 mL standard flask and made up to mark with

methanolic HCl to restore the original color of the dye.

* 1:1 ratio V/V

3.3.1.7 Effect of equilibration time during extraction

The time required for the quantitative extraction of the azo dye into isoamyl alcohol was then

investigated. Ten mL aliquots of alkaline sodium arsenite solution containing 2 µg of nitrite

were added to a series of 25 mL calibrated flasks containing 1 mL of 0.05 % amine and 1 mL

of 2N hydrochloric acid. After standing time of 1 min. the solutions were treated with 1 mL

of 0.05 % coupling agent and diluted to 25 mL with water. After color development, the

solutions were transferred into 60 mL separating funnels and equilibrated with 5 mL isoamyl

alcohol for varying lengths of time from 30 - 240 seconds. The absorbance of the extract

after diluting to 5 mL with methanolic hydrochloric acid was measured against reagent blank

at 565 nm. These studies have revealed that about one minute equilibration time is adequate

for the complete extraction of the dye into organic solvent (Fig.3.6).

Chapter 3 __________________________________________________________________________

63

Fig. 3.6 Effect of equilibration time

3.3.1.8 Species responsible for color

2-(4-aminophenyl) benzimidazole on treatment with nitrite undergoes diazotization in acidic

medium to form corresponding diazonium ion which couples with N-(1-

naphthyl)ethylenediamine dihydrochloride instantaneously in aqueous media to form an pink

colored azo dye 2-(4-diazophenyl)benzimidazole-N-(1-naphthyl)ethylenediamine

dihydrochloride, which has λmax at 555 nm. The dye has been extracted quantitatively into

organic solvent in alkaline condition to lower the detection limit. The dye has λmax at 565 nm

in organic phase. The extracted organic phase was collected in 5 mL volumetric flasks and

made up to the mark with methanolic hydrochloric acid to restore the original color. The

species responsible for color is shown in the scheme 3.1.

0 50 100 150 200 2500.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

R eagent b lank

Sam ple

Ab

so

rban

ce

Equ ilibration tim e(Sec)

Chapter 3 __________________________________________________________________________

64

Scheme 3.1 Species responsible for color

3.3.1.9 Calibration procedure

10 mL aliquots of sodium arsenite solution containing 0 - 10 µg nitrite were added to series

of 25 mL standard flasks containing 1 mL of 0.05 % 2-(4-aminophenyl) benzimidazole and

1mL of 2N HCl. The contents were mixed well and allowed to stand for 2 min. Then 1 mL of

0.05 % N-(1-naphthyl) ethylenediamine dihydrochloride was added and diluted to 25 mL

with distilled water and the absorbance values were measured at 555 nm using 1cm quartz

cuvette (Fig.3.7 and 3.8).

Violet dye

2-(4-diazophenyl) benzimidazole-N-(1-naphthyl) ethylenediamine dihydrochloride

- -

NH-(CH2)2-NH2.2HClNH-(CH2)2-NH2.2HCl

2N HClNO2

NN

N

N

N

N

N

NH2

N

H

HH

Cl

H

N

N+

+

+

+

Chapter 3 __________________________________________________________________________

65

Fig.3.7 Calibration plot

Fig. 3.8 Absorption spectra

0 2 4 6 8 10

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Absorb

an

ce

Amount of nitrite(g)

400 450 500 550 600 650 700

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Wavelength/nm

F 10 g ,,

E 8g ,,

D 6 g ,,

C 4g ,,

B 2 g nitrite

A reagent blankF

E

D

C

B

A

Ab

sorb

an

ce

Chapter 3 __________________________________________________________________________

66

3.3.1.10 Interference study

In order to evaluate the suitability of the proposed method for the determination of nitrogen

dioxide in air and nitrite/nitrate in water and soil samples, the effect of interference of several

ions in the determination was examined. Initially the effect of common atmospheric air

pollutants like sulphur dioxide, hydrogen sulphide and formaldehyde in the determination of

nitrogen dioxide was studied. These species were introduced in the form of their respective

anions. Formaldehyde did not interfere up to 2×104 µg in the proposed method. Sulphite at

concentrations above 5×102 µg interfered causing decrease in absorbance value. However

higher concentrations (up to 1×103 µg) of sulphite can be overcome by the addition of 1mL

of 0.05 % formaldehyde solution which reacts with the suphite to form a stable adduct prior

to nitrite determination. Sulfide, up to 4 µg did not interfere but at higher concentrations it

interfered by decreasing the absorbance values. Up to 50 µg of sulfide interference was

overcome by precipitating as lead sulphide by the addition of 1 mL of 0.01 % lead acetate

before nitrite estimation. The interference of other several anions and cations were evaluated

to check the suitability of the method for the determination of nitrite and nitrate in water and

soil samples. Anions like oxalate, carbonate, sulphate, citrate, phosphate, bicarbonate and

nitrate did not interfere upto 1×104 µg levels. Cations like Fe2+, Cu2+, Ni2+, Hg2+, Co2+, Fe3+,

Ba2+, Mo2+, Mg2+ and Zn2+ up to the 1×104 µg level did not interfere. However copper (II)

interfered at 2×103 µg level by decreasing the absorbance and this was overcome by adding 2

mL of 0.05 M EDTA solution before the addition of coupling agent. Iron (III) gives negative

interference at 2×102 µg which was overcome by precipitating it as hydroxide and removing

through centrifugation (Table 3.2).

Chapter 3 __________________________________________________________________________

67

Table 3.2 Interference studies

atreated with 2 mL of 0.05 % formaldehyde solution before the addition of coupling agent.

btreated with 1mL of 0.01 % lead acetate solution centrifuged and washed the residue, the

centrifugate and washings were mixed and used for color development.

ctreated with 1 mL of 1N NaOH solution centrifuged and washed the residue, the centrifugate

and washings were mixed and used for color development.

dtreated with 2 mL of 0.05 M EDTA solution.

Interferent

Tolerance limit (µg)

Formaldehyde

Sulphite

aSulphite

Sulphide

bSulphide

CO32-, C2O4

2-, Citrate, NO3-, tartarate, Fe2+,

Hg2+, Ni2+, Co2+, Zn2+, Ba2+, Mg2+.

cFe2+

Fe3+

cFe3+

Cu2+

dCu2+

2 × 104

5 × 102

1 × 103

4

50

1 × 104

1 × 104

2 × 102

1 × 103

2 × 103

5 × 103

Chapter 3 __________________________________________________________________________

68

3.4 Application study

The proposed method has been applied to determine nitrite/nitrate in environmental samples

like air, water and soil. In order to check the validation of the proposed method, the samples

were simultaneously determined by using Griess - Ilosvey reaction as standard method. The

results obtained by the proposed method are in good agreement with those obtained by the

standard method.

3.4.1 Determination of nitrogen dioxide in air

Air samples were drawn through 10 mL of sodium arsenite absorber solution taken in an

impinger at a flow rate of 0.3 Lmin-1. The sampled solution was made up to 25 mL with

sodium arsenite absorber solution. 10 mL of made up solution was taken into 25 mL

calibrated flask containing 1 mL of 0.05 % APB and 1 mL of 2N hydrochloric acid. The

contents were mixed well and allowed to stand for 2 min.The coupling agent (1 mL of 0.05

% NEDA) was added and diluted to 25 mL with distilled water and the absorbance values

were measured at 555 nm as shown in the table 3.3. The extraction procedure was adopted

when the nitrite levels are well below the detection limit.

Table 3.3 Determination of nitrogen dioxide in atmospheric air

Trapping solution: 10 mL of alkaline sodium arsenite.

Sampling rate: 0.3 Lmin-1

The volume of solution taken for analysis was 5 mL from 25 mL made up solution

Sl.No. Volume of air proposed method standard method

sampleda (L) NO2-(µg) NO2(ppb)* NO2

-(µg) NO2(ppb)*

1) 45 0.801 59 0.786 58.07

2) 36 0.713 52 0.690 51.00

aAir was sampled on different days

*V

gNOppbNOofionConcentrat

82.0

5325)()( 2

2

Chapter 3 __________________________________________________________________________

69

Where V is the volume of air sampled, 0.82 is the factor of absorption efficiency for sodium

arsenite as trapping medium, 532 is the conversion factor to convert µgL-1 of nitrite to ppb

of nitrogen dioxide at 298 K and 101.3 kpa.

3.4.2 Determination of nitrite and nitrate in water samples

10 mL of the water sample was treated with 1 mL of 1N sodium hydroxide and centrifuged.

The centrifugate has been collected and the residue was washed with 5 mL portions of water

and centrifuged again. All the centrifugates were mixed well and made up to 25 mL in a

calibrated flask.

Nitrite determination 10 mL of the made up solution was transferred to a 25 mL calibrated

flask containing 1 mL of 0.05 % APB and 1 mL of 2N hydrochloric acid. The contents were

mixed well and allowed to stand for 2 min. Then 1 mL of 0.05 % NEDA solution was added

and diluted to 25 mL with distilled water. The absorbance was measured at 555 nm. If the

color intensity is very low the extraction procedure can be followed and absorbance values

can be measured at 565 nm against reagent blank.

Nitrate determination 10 mL of made up solution was taken and treated with 5 mL of NH3-

NH4Cl buffer solution (pH = 8.5) and passed through copperized cadmium reductor column

at a flow rate of 1 mLmin-1.The column was washed with five 3 mL portions of water and the

eluents were collected in a 25 mL standard flask and diluted to the mark with water, 5 mL of

the made up solution was taken and analyzed for total nitrite content. The nitrate content can

be calculated by the difference between total nitrite and original nitrite content (Table 3.4).

Chapter 3 __________________________________________________________________________

70

Table 3.4 Determination of nitrite/nitrate in water samples

Sample (mL) Nitrite present originally (µg) Total nitrite found in 10 mL (µg)

proposed standard proposed standard

method method method method

Ground water

(Bore well)a 6.94 7.23 52.02 53.81

Ground water

(Bore well)b 26.02 23.87 193.18 177.26

)()(

)(gnitrateofreduction

thebyformednitrite

gpresent

originallynitritegnitriteTotal

46

62

10

)()()( 221

3

gpresentorginallyNOgNOtotalgmLNO

aSamples collected from Kadugodi, Bangalore.

bSamples have been collected from T.Dasarahalli area, Bangalore.

3.4.3 Determination of nitrite and nitrate in soil samples

A known weight (0.5 g) of soil sample was taken in a 50 mL beaker and extracted with three

5 mL portions of 0.5 % sodium carbonate solution and centrifuged. The clear centrifugate

solutions were collected in 25 mL calibrated flask and diluted to the mark. The nitrite and

nitrate contents can be measured by following the procedure described under water samples

(Table 3.5).

Chapter 3 __________________________________________________________________________

71

Table 3.5 Determination of nitrate in soil samples

Weight of soila total nitriteb found in 25 mL nitrate in soil samplec (µgg-1)

taken (g) extract (µg)

proposed standard proposed standard

method method method method

0.50d 6.208 6.25 16.72 16.83

0.50e 4.63 4.01 12.47 10.76

aNitrite was not detected in these soil samples

)()(

)(gnitrateofreduction

thebyformednitrite

gpresent

originallynitritegnitriteTotal

c

46

62

)(

)()()( 221

3

gsoilofweight

gpresentorginallyNOgNOtotalggsoilinNO

dSoil samples have been collected from Banashankari area Bangalore

eSoil samples have been collected from Sunkanapalya Kengeri area Bangalore.

3.5 Conclusion

The proposed method based on the diazo-coupling reaction between

aminophenylbenzimidazole and naphthylethylenediamine dihydrochloride is sensitive and

simple for the estimation of nitrogen dioxide/nitrite/nitrate at trace level. The reaction

conditions have been optimized and the method obeys Beer’s law over the concentration

range 0 - 10 µg in aqueous phase and 0 - 2 µg in organic phase. The proposed method has

been applied to determine nitrogen dioxide levels of ambient air after fixing it as nitrite using

sodium arsenite trapping solution. The results obtained by this method are in good agreement

with standard method [23]. It has been applied to measure nitrite and nitrate levels in bore

well water, soil samples and the results are in quite agreement with the standard method. .

The application of this method for routine monitoring of nitrite/nitrate levels of industrial

Chapter 3 __________________________________________________________________________

72

effluents at trace level will be a useful analytical procedure and it serves as an alternative to

other existing procedures.

3.6 References

[1] Legge A.H and Krupa S.V, Air quality of an area proximal to anthropogenic

emissions, acidic deposition: sulphur and nitrogen oxides, Lewis Publishers, Chelsla,

Michigan, 249 - 345 (1990).

[2] Aswathanarayana U, Geoenvironment: An Introduction, edited by Rotterdam, Brookfield

V.T and Balkema A. A, 270 (1995).

[3] Baird C, Toxic organic chemicals, In Environmental Chemistry, W.H. Freeman and

Company. New York, NY, 215 - 286 (1995).

[4] Peirce J.J; Weiner R.F and Vesilind P.A, Environmental pollution and control. 4th Edn.

Butterworth - Heinemann, Boston (1998).

[5] Kazemzadeh A and Ensafi A.A, Sequential flow injection spectrophotometric

determination of nitrite and nitrate in various samples, Analytica Chimica Acta, 442, 319 -

326 (2001).

[6] Buckman H.O and Brady N.C, The nature and properties of soils, 7th Edn. Macmillan

Company, New York (1969).

[7] Puckett L.J, Identifying the major sources of nutrient water pollution, Environmental

Science and Technology, 29, 408A - 414A (1995).

[8] Habibe O; Fait P and Alaaddin C, Spectrophotometric determination of nitrite in water

samples with 4-(1-methyl-1-mesitylcyclobutane-3-yl)-2-aminothiazole, Analytical Letters,

39, 823 - 833 (2006).

[9] Bruning-Fann C.S and Kaneene J.B, The effects of nitrate, nitrite and n-nitroso

compounds on human health: a review, Veterinary and human toxicology, 35, 521 - 538

(1993).

[10] Gapper L.W; Fong B.Y; Otter D.E; Indyk H.E and Woollard D.C, Determination of

nitrite and nitrate in dairy products by ion exchange LC with Spectrophotometric detection,

International Diary Journal, 14, 881 - 887 (2004).

[11] Favell D.J, A comparison of the vitamin C content of fresh and frozen vegetables, Food

Chemistry, 62, 59 - 64 (1998).

Chapter 3 __________________________________________________________________________

73

[12] Kidmose U; Knuthsen P; Edelenbos M; Justesen U and Hegelund E, Carotenoids and

flavonoids in organically grown spinach (spinacia oleracea L) genotypes after deep frozen

storage, Journal of the Science of Food and Agriculture, 81, 918 - 923 (2001).

[13] Harrison M.N and Davies A, Nitrate and nitrite in foods and the diet, Food Additives

and Contaminants, 11, 519 - 532 (1994).

[14] Huang K.J; Wang H; Guo Y.H; Fan R.L and Zhang H.S, Spectrofluorimetric

determination of trace nitrite in food products with a new fluorescent probe 1, 3, 5, 7-

tetramethyl-2,6-dicarbethoxy-8-(3,4-diaminophenyl)-difluoroboradiaza-s-indacene

Talanta, 69, 73 - 78 (2006).

[15] Hurst J.K and Lumar S.V, Toxicity of peroxynitrite and reactive nitrogen species toward

Escherichia coli, Chemical Research in Toxicology, 10, 802 - 810 (1997).

[16] Reszka K.J; Massac Z and Chignell C.F, Lactoperoxidase - catalyzed oxidation of the

anticancer agent mitoxantrone by nitrogen dioxide radicals, Chemical Research in

Toxicology, 10, 1325 - 1330 (1997).

[17] Wang C.J; Huang H.P; Tseng T.H; Lin Y.L and Shiow S.J, N-Nitroso -N-(3-keto-1, 2-

butandiol)-3’-nitrotyramine: a new genotoxic agent derived from the reaction of tyrosine

and glucose in the presence of sodium nitrite, Archives of Toxicology, 70, 10 - 15 (1995).

[18] Zuo Y; Wang C and Van T, Simultaneous determination of nitrite and nitrate, in rain,

snow and lake water samples by ion-pair high-performance liquid chromatography,Talanta,

70, 281 - 285 (2006).

[19] Moorcroft M.J; Davis J and Compton R. G, Detection and determination of nitrate and

nitrite: a review, Talanta, 54,785 - 803 (2001).

[20] Burakhama R; Oshimab M; Grudpan K and Motomizu Sh, Simple flow-injection system

for the simultaneous determination of nitrite and nitrate in water samples, Talanta, 64, 1259 -

1265 (2004).

[21] Aydin A; Ercan O and Tascioglu S, A novel method for the Spectrophotometric

determination of nitrite in water, Talanta, 66, 1181 - 1186 (2005).

[22] Yue X.F; Zhang Zh.Q and Yan H.T, Flow injection catalytic spectrophotometric

simultaneous determination of nitrite and nitrate, Talanta, 62, 97 - 101 (2004).

[23] APHA, Standard methods for the examination of water and wastewater analysis, 19th

Edn. Washington D.C. 4 - 83 - 4 - 88 (1995).

Chapter 3 __________________________________________________________________________

74

[24] Pratt P.F; Nithipatikom K and Campbell W.B, Simultaneous determination of nitrate

and nitrite in biological samples by multichannel flow injection analysis, Analytical

Biochemistry, 231, 383 - 386 (1995).

[25] Wu Q.F and Liu P.F, Spectrophotometric determination of micro amounts of nitrite in

water and soil, Talanta, 30, 374 - 376 (1983).