61

CHAPTER 3

SPECTROPHOTOMETRIC DETERMINATION OF HYPOCHLORITE IN

ENVIRONMENTAL SAMPLES

3.1 INTRODUCTION

3.2 ANALYTICAL CHEMISTRY

3.3 APPARATUS

3.4 REAGENTS AND SOLUTIONS

3.5 PROCEDURE

3.6 RESULTS AND DISCUSSION

3.7 APPLICATIONS

3.8 CONCLUSIONS

3.9 REFERENCES

62

3.1 INTRODUCTION

Hypochlorite refers to the salts of hypochlorous acid (HOCl). Hypochlorite is

inherently an unstable compound. It can decompose over the time and results in the

formation of crystalline salts and oxygen gas. Hypochlorite is widely used as

bleaching agent [1], disinfectant in fabrics, wood pulp and food industries. It has

been extensively used for hygienic chemical studies on tap water for treating skin

cancers [2], disinfectant in milk industry [3] and for the determination of blood urea.

It is also used for the preparation of surgically active anticeptic compounds for

controlling and preventing infection in wounds. Sodium hypochlorite is used as a

screening agent for the identification of cocane [4]. The lethal action of calcium

hypochlorite on Bacillus anthracoides spores [5] resulted from the alteration of

structural organization of spores which lead to disturbance of normal permeability

barrier with loss of life – sustaining components and unbalance of metabolic process.

Biological toxins can be extremely hazardous even in minute quantities.

Investigators must ensure that appropriate equipment and safety procedures are in

place for the specific toxin used and the type of experiments performed in their

laboratory. Some toxins are inactivated by autoclaving for one hour at 121°C, while

others are inactivated by exposure to sodium hypochlorite, sodium hypochlorate and

sodium hydroxide [6].

The common method for making sodium hypochlorite is to react chlorine with

a solution of caustic soda. The final concentration of the sodium hypochlorite solution

depends on the initial concentration of the starting caustic soda solution. The

following equation gives the chemical reaction involved, regardless of concentration:

Cl2 + 2NaOH �����������������2O

A more active but less stable sodium hypochlorite can be produced by chlorinating a

solution of soda ash according to the following equation:

Cl2 + 2Na2CO3 + H2O ������������������������3

On further chlorination, hypochlorous acid will be produced:

Cl2 + Na2CO3 + H2O ���������������������3

Most of the commercial production processes involve the reaction of chlorine with caustic soda as mentioned in the above equation.

63

Sodium hypochlorite (bleach) manufacturers are now frequently required to

provide high quality sodium hypochlorite with limits on chlorate ion and transition

metal ions. Sodium hypochlorite "decomposes" by two mechanisms. The first is the

2nd order process that forms chlorate ion

3OCl-�����3- + 2 Cl-

In the presence of transition metal ions, decomposing bleach forms oxygen whether

transition metal ion acts as catalyst.

2OCl- ���2 + 2Cl-

Majority of the municipalities require the delivered sodium hypochlorite (9 to 16 wt%

NaOCl) should contain 0.1-0.4 wt% excess caustic, <1,500 mgL-1 ClO3-, <0.5

mgL-1 iron and <0.05 mgL-1 nickel and copper. Important considerations for

minimizing ClO3- formation include: high pH (i.e. excess caustic), dilution

(decomposition is 2nd order with respect to OCl-) and temperature control.

The active ingredient in most of the chlorine bleaches is sodium

hypochlorite(NaOCl). The oxidizing action of hypochlorite ion (OCl-) kills germs and

also decolorizes many stains and dyes. The quantity of hypochlorite ion in a sample of

bleach can be determined by finding out how much iodine (I2) it can produce by

oxidizing an iodide ion (I-). The quantity of iodine produced is estimated by titrating

it with sodium thiosulfate, which converts the colored iodine back to colorless iodide

ion.

The equations are:

Oxidation of iodide ion to iodine with bleach:

2H+ + OCl- + 2I- → I2 + Cl- + H2O

Titrating iodine with thiosulfate:

I2 + 2S2O32- → 2 I-+ S4O6

2-

Sodium hypochlorite is recommended and used by the majority of dentists

because this solution presents several important properties: antimicrobial effect [7,8],

tissue dissolution capacity and acceptable biologic compatibility of less concentrated

solutions. In relation to antimicrobial effect, the studies have shown that sodium

hypochlorite decreases microorganism number during the treatment of teeth with

apical periodontitis.

64

Sodium hypochlorite neutralizes amino acids forming water and salt. With the

reduction in concentration of hydroxyl ions, there is a reduction of pH. Hypochlorous

acid, a substance present in sodium hypochlorite solution, when in contact with

organic tissue acts as solvent, releases chlorine that combined with the protein amino

group and forms chloramines. Hypochlorous acid (HOCl) and hypochlorite ions

(OCl-) lead to amino acid degradation and hydrolysis.

The antimicrobial effectiveness of sodium hypochlorite based on its high pH

(hydroxyl ions action) is similar to the mechanism of action of calcium hydroxide.

The high pH of sodium hypochlorite interferes in the cytoplasmic membrane integrity

with an irreversible enzymatic inhibition, biosynthetic alterations in cellular

metabolism and phospholipid degradation.

Sodium hypochlorite in higher concentrations is more aggressive while in

lower concentrations (0.5% to 1%), it is biocompatible. For a substance to be

biocompatible, it must present a discrete tissue reaction at all periods and moderate or

intense tissue reaction at 7 days which decreases in intensity with time until reaching

a non-significant tissue reaction [9].

3.2 ANALYTICAL CHEMISTRY

The determination of hypochlorite in environmental and biological samples such as natural water and tap water can be of interest in biochemical research. Hence there is a need for a rapid and sensitive method for the determination of hypochlorite. Iodometric [10], coulometric [11], polarographic [12], chemiluminescence[13-15], radiolytically-induced redox [16], normal pulse voltametry [17,18], colorimetric [19] and potentiometric [20] methods are most commonly used. However, colorimetric methods are often preferred as they involve less expensive instrumentation and provide better sensitivity when appropriate chromogenic reagents are available.

Anwar et al. reported two simple spectrophotometric procedures for the

quantitative estimation of hypochlorite [21]. One of the method was based directly on

the absorbance of OCl- in alkaline aqueous media. The other method took the

advantage of the quantitative reaction of OCl- with NH3 in alkaline solution to form

65

chloramine, which has a higher molar absorptivity. Results of the spectrophotometric

assays are compared with results obtained by the titrimetric procedure of NF XII

(National formulary, 1965).

Bunikiene and Ramanauskas presented an indirect spectrophotometric

detection of trace amounts of OCl- was based on oxidation of OCl- with I- [22].

Subsequent reaction of the oxidized product with brilliant green and measured of

change in the absorbance of brilliant green solution. The detection was conducted in

acid medium (7M HCl) with NaOAc addition or in universal buffer medium and the

absorbance was measured at 628 or 684 nm respectively. The absorbance was

proportional to OCl- concentration in the range 0.04-1.60 μgmL-1. The sensitivity of

the method was 0.02-0.05 μgmL-1 of OCl-.

Fleet and Ho presented a approach to the automated determination of sodium

hypochlorite and hydrogen peroxide [23]. This method was based on the use of a

porous catalytic silver electrode. The principle of both methods involved the

quantitative liberation of oxygen, which was measured colorimetrically by the

electrode.

Isacsson and Wettermark reported a sensitive method for the determination of hypochlorite in aqueous solution which involved the measurement of the chemiluminescence, produced during alkaline oxidation of luminol in presence of hydrogen peroxide. Micromolar and submicromolar quantities could be detected by this method [24].

Tarasankar et al. described a method for the spectrophotometric determination

of hypochlorite [25]. In this method, AgNO3 was mixed with 0.5 % gelatin at a pH 8,

Ag+ reduced by CO to form a Ag sol solution. Aliquots of the sol solution acidified to

pH <7, were added to water samples containing OCl- and measured at 415 nm. The

method was used for OCl- concentrations of 0.04-1.0 mgL-1.

Bamnolker et al. described a spectrophotometric method for the detection of

hypochlorite traces in solutions containing 4M NaOH based on the reaction of the

66

reagent 3,3'-dimethylnaphthidine (DMN) with chlorine [26]. The reagent containing

DMN, hydrochloric acid, and DMF was used. This common reagent enables the

liberation and detection of chlorine in situ in acidic solution. The effect of factors

such as acidity, temperature, oxidizing agents and metallic impurities on the

absorption spectrum was studied. The chlorine DMN complex has an absorption

maximum at 550 nm and obeys Beer's law for solutions containing up to 3 mgL-1 of

OCl-.

Gonzalez-Robledo et al. described the reaction of hypochlorite with luminol by stopped-flow chemiluminescence spectrometry method [27]. The emission was observed by using a conventional fluorescence detector at 425 nm. The method yields linear response over three orders of magnitudes with an RSD of about 1 %. The method was highly selective and rapid (80 samples per hour) and was applied to the routine determination of hypochlorite in different water samples.

Watanabe et al. presented the simultaneous determination of chlorine dioxide

and hypochlorite by high-performance liquid chromatography [28]. The chromogenic

substance formed by the oxidative condensation reaction using 4-aminoantipyrine

(4-AA) and phenol was determined at 503 nm by a post-column reaction system.

Chlorine dioxide and hypochlorite were separated within 3 minutes by the applied

elution solution (1.7 mM sodium carbonate–1.8 mM sodium carbonate) at 0.5 mL per

minute into a laboratory-prepared PTFE tube column (13 cm×1.0 mm I.D.×2 mm

O.D.) packed with Waters Accell QMA as the anion-exchange material. A linear

correlation between the peak height and concentration was obtained within the range

of 1–20 �gmL-1 for chlorine dioxide and 47–200 �gmL-1 for hypochlorite with good

reproducibility (relative standard deviations of 4.0 and 2.2%, respectively). The limits

of detection of chlorine dioxide and hypochlorite were approximately 0.2 and

������-1 respectively.

Han et al. developed a simple spectrophotometric method for quantitative

detection of hypochlorite (OCl-) or hypochlorous acid (HOCl) [29]. The OCl- or

HOCl sample was first incubated with an excess amount of tris(2-

carboxyethyl)phosphine(TCEP). The concentration of the residual TCEP was then

measured as the amount of 2-nitro-5-thiobenzoate produced after reaction with

67

5,5'-dithiobis(2-nitrobenzoic acid). The concentration of OCl- or HOCl was equivalent

to the amount of decrease in the concentration of TCEP because one mole of TCEP

was rapidly and irreversibly oxidized to TCEP oxide by one mole of OCl- or HOCl.

This method was more sensitive and convenient than the standard procedure of

NaOCl assay, which involves reaction with KI, followed by titration of the liberated

triiodide with thiosulfate.

Chiswell and O'Halloran reported acid yellow 17 as a spectrophotometric

reagent for the determination of low concentrations of residual free chlorine [30]. The

detection limit was 50 ngmL� for free chlorine, and the calibration graph was linear

��������������� �������� . Cyclic voltammetry was used to explain the findings of the

spectrophotometric work.

Tian and Dasgupta described a flow injection method for the simultaneous

determination of hydroxide, chloride, hypochlorite and chlorate ions present in

chloralkali cell effluents in concentrations ranging from sub-millimolar to

severaLmolar [31]. For the measurement of hypochlorite and chlorate, colorimetric

iodometry was used.

Icardo et al. reported a flow injection analytical (FIA) procedure for the

detection of free chlorine in industrial formulations and H2O samples [32]. The

manifold was provided with a gas-diffusion unit, which permits the removal of

interfering species and also the preconcentration of chlorine. The detection of chlorine

was performed from the oxidation by o-dianisidine as a chromogenic reagent to a

colored product, which was monitored at 445 nm. The method was linear over the

range 0.04-1.00 mgL-1 of chlorine. The limit of detection was 0.04 mgL-1, the

reproducibility of the procedure (as relative standard deviation of the slope) was 3.7

% for four independent calibrations, the precision (as relative standard deviation of 30

continuous FIA peaks of 0.56 mgL-1 of chlorine) was 1.4 % and the sample

throughout was 40 per hour.

Narayana et al. presented azure B as a reagent for the facile

spectrophotometric determination of hypochlorite [33]. The method was based on the

reaction of hypochlorite with potassium iodide in an acidic medium to liberate iodine.

68

Bleaching of the blue color of azure B by the liberated iodine was the basis of the

determination and exhibited an absorption maximum at 644 nm. Beer’s law was

obeyed in the range 0.2-1.0 μgmL-1 of hypochlorite in a final volume of 10 mL. The

molar absorptivity and Sandell’s sensitivity for the colored system were found to be

1.49×104 Lmol-1cm-1 and 3.25×10-4 μgcm-2 respectively.

Kodera et al. reported a electroanalytical method which was based on an

anodic reaction for the hypochlorite ion [34]. The determination was carried out by

linear sweep-voltammetry at a Pt electrode. The peak current of observed oxidation

wave was proportional to the amount of hypochlorite ion. The influences of various

physical and chemical factors (repeatability, supporting electrolyte, pH, sweep rate,

temperature, metal ion, and dissolved oxygen) were investigated. A fairly good

correlation (r=0.987) between this method and iodometric titration was achieved for

standard samples (n=5). This method was useful to analyze the hypochlorite ion.

Ballesta et al. developed a selective and reusable chemiluminescent test strip

to determine hypochlorite [35]. The hypochlorite sensitive test strip contains a 10

mm×9 mm piece of anionic cellulose paper in fluoresceinate cycle, glued to a 10

mm×4cm×0.5 mm polyester strip. The measurement of the chemiluminescence in a

luminometer when 1 mL of sample was injected into a conventional cell containing

the strip makes it possible to determine hypochlorite. The composition of the

membrane and reaction conditions was adjusted to obtain adequate sensitivity and

selectivity. The test strip responded linearly to hypochlorite over two linear ranges,

the first 2.0-10.3 mgL-1 and the second 10.3-51.4 mgL-1 with a detection limit of 0.4

mgL-1. The reproducibility using the same disposable test strip at a medium level of

the range was 6.6%, as relative standard deviation (R.S.D.), and 12.3% using different

test strips. The procedure was applied to the determination of hypochlorite in different

types of waters.

Narayana et al. described an easy spectrophotometric method for the

determination of hypochlorite using thionin [36]. The method was based on the

reaction of hypochlorite with potassium iodide in acidic medium to liberate iodine.

Bleaching of the violet color of thionin by the liberated iodine was the basis of the

69

determination and was measured at 600 nm. The decrease in absorbance was directly

proportional to the concentration of hypochlorite. Beer’s law was obeyed in the range

0.2–1.2 µgmL-1 of hypochlorite. The molar absorptivity, Sandell’s sensitivity,

detection limit, and quantitation limit are found to be 1.489×104 Lmol-1cm-1,

3.25×10�� µgcm��, 0.1026 µgmL-1 and 0.3112 µgmL-1 respectively.

Antonio et al. described the determination of hypochlorite in bleaching

products with flower extracts to demonstrate the principles of flow injection analysis

[37]. The use of crude flower extracts to the principle of analytical chemistry

automation with the flow injection analysis (FIA) procedure was developed to

determine hypochlorite in household bleaching products. The FIA comprises a group

of techniques based on injection of a liquid sample into a moving, nonsegmented

carrier stream of a suitable fluid.

March and Simonet developed a green method for the determination of

hypochloride in bleaching products [38]. The method was based on a flow injection

system and measurement of the native absorbance of hypochlorite at 292 nm for the

determination of hypochlorite in the range 0.07–0.42 gL� �of chlorine. A mini-column

containing cobalt oxide was inserted in the flow system catalysed the hypochlorite

decomposition to chloride and oxygen. The method required 20 mg of solid, reusable

catalyst and a NaOH solution of pH 10.4, provided a sample throughput of 12

samples per hour in triplicate injection. The usefulness of the analysis of bleaching

products was demonstrated. Colorimetric methods are often preferred, however, as

they involve less expensive instruments and provide better sensitivity when the

appropriate chromogenic reagents are available.

In this chapter, rhodamine B and methylene blue have been used as rapid,

sensitive and selective reagents for the determination of hypochlorite. The developed

method has been successfully employed for the determination of hypochlorite in tap

water, natural water and milk samples.

70

3.3 APPARATUS

A Secomam Anthelie NUA 022 UV-Visible spectrophotometer with 1 cm

quartz cell was used. A WTW pH 330-pH meter was used.

3.4 REAGENTS AND SOLUTIONS

All chemicals were of analytical reagent grade or chemically pure grade.

Standard hypochlorite solution (1000 µgmL-1) was prepared by dissolving 1.4446 g of

sodium hypochlorite in 1000 mL water and standardized by the iodometric method

[10]. Rhodamine B (0.05 %), methylene blue (0.05 %), sodium acetate (1 M),

hydrochloric acid (2 M) and potassium iodide (2 %) solutions were used.

3.5 PROCEDURES

3.5.1 Using Rhodamine B as a Reagent

Aliquots of sample solution containing 0.1–4.0 μgmL-1 of hypochlorite were

transferred into a series of 10 mL calibrated flasks, 1 mL of 2 M hydrochloric acid and

1 mL of 2% potassium iodide were added and the mixture was gently shaken until the

appearance of yellow color, indicating the liberation of iodine. A 0.5 mL of 0.05 %

rhodamine B solution was then added to it followed by the addition of 2 mL of 1 M

sodium acetate and the reaction mixture was shaken for 2 minutes and the contents

were diluted to 10 mL with distilled water and mixed well. The absorbance of the

resulting solutions were measured at 553 nm against distilled water. A blank was

prepared by replacing the analyte (hypochlorite) solution with distilled water. The

absorbance corresponding to the bleached color which in turn corresponds to the

analyte (hypochlorite) concentration was obtained by subtracting the absorbance of

the blank solution from that of test solution. The amount of the hypochlorite present

in the volume taken was computed from the calibration graph (Figure IIIB1).

3.5.2 Using Methylene Blue as a Reagent

Aliquots of sample solution containing 0.1–6.0 μgmL-1 of hypochlorite were

transferred into a series of 10 mL calibrated flasks, 1 mL of 2 M hydrochloric acid and

71

1 mL of 2% potassium iodide were added and the mixture was gently shaken until the

appearance of yellow color, indicating the liberation of iodine. A 0.5 mL of 0.05 %

methylene blue solution was then added to it followed by the addition of 2 mL of 1 M

sodium acetate and the reaction mixture was shaken for 2 minutes, the contents were

diluted to 10 mL with distilled water and mixed well. The absorbance of the resulting

solutions were measured at 665.6 nm against distilled water. A blank was prepared

by replacing the analyte(hypochlorite) solution with distilled water. The absorbance

corresponding to the bleached color which in turn corresponds to the

analyte(hypochlorite) concentration was obtained by subtracting the absorbance of

the blank solution from that of test solution. The amount of the hypochlorite present

in the volume taken was computed from the calibration graph (Figure IIIB2).

3.5.3 Determination of Hypochlorite in Natural / Tap Water Samples

Aliquots of natural / tap water sample containing not more than 6.0 μgmL-1 of

hypochlorite were treated with 0.5 mL of 1M NaOH and 0.5mL of 0.2M EDTA. The

solution was mixed and centrifuged to remove any precipitate formed. The

centrifugate was transferred to a 10 mL calibrated flask and its hypochlorite content

was determined directly according to the general procedure for the determination of

the hypochlorite (Table 3A1 and Table 3A2).

3.5.4 Determination of Hypochlorite in Milk

A known volume of milk (20 mL) was placed in a 50 mL beaker and

coagulated with 8–10 mL of 1 M citric acid. The solution was centrifuged to remove

the precipitate. The centrifugate was transferred to a 100 mL calibrated flask. A

sample solution of suitable aliquot was determined according to the general procedure

for the determination of hypochlorite (Table 3A1 and Table 3A2).

3.6 RESULTS AND DISCUSSION

3.6.1 Absorption Spectra

This method involves the liberation of iodine by the reaction of hypochlorite

with potassium iodide in an acidic medium. The liberated iodine selectively bleaches

the color of rhodamine B and is measured at 553 nm. This decrease in absorbance is

72

directly proportional to the hypochlorite concentration and obeys Beer’s law in the

range of 0.1–4.0 μgmL-1 of hypochlorite. The absorption spectrum of rhodamine B

is presented in Figure IIIA1, and the reaction system is represented in Scheme III.

Similarly the liberated iodine selectively bleaches the blue color of methylene

blue and is measured at 665.6 nm. This decrease in absorbance is directly proportional

to the hypochlorite concentration and obeys Beer’s law in the range of 0.1–6.0

μgmL-1 of hypochlorite. The absorption spectrum of methylene blue is presented in

Figure IIIA2, and the reaction system is represented in Scheme III.

3.6.2 Effect of Iodide Concentration and Acidity

The oxidation of iodide to iodine by hypochlorite is effective in the pH range

1.0-1.5, which could be maintained by adding 1 mL of 2M HCl in the final volume of

10 mL. The liberation of iodine from potassium iodide in an acidic medium is

quantitative. The appearance of yellow color indicated the liberation of iodine.

However, concentration of hydrochloric acid should be maintained at the 1.0-1.2 mL

ranges. It is found that 1 mL each of 2 % potassium iodide and 2 M HCl are

sufficient for the liberation of iodine from iodide by hypochlorite and 0.5 mL of each

0.05 % rhodamine B and methylene blue is used for subsequent decolorization. The

bleached reaction system is found to be stable for 3 hours for each rhodamine B and

methylene blue reagents. Effect of pH on color stability is presented in Figure IIIC1

and IIIC2.

3.6.3 Analytical Data

3.6.3.1 Using rhodamine B as a reagent

The adherence to Beer’s law is studied by measuring the absorbance values of

solutions varying hypochlorite concentration. A straight line graph is obtained by

plotting absorbance against concentration of hypochlorite. Beer’s law obeyed in

the range of 0.1–4.0 μgmL-1 of hypochlorite (Figure IIIB1). The molar absorptivity

and Sandell’s sensitivity for colored system is found to be 2.57×105 Lmol-1cm-1,

2.01×10-3 μgcm-2 respectively. Correlation coefficient(n = 10) and slope of the

calibration curve are 0.995 and 0.471 respectively. The detection limit (DL=3.3σ/s)

and quantitaion limit (QL=10σ/s) [where σ is the standard deviation of the reagent

73

blank (n=5) and s is the slope of the calibration-curve] for hypochlorite determination

were found to be 0.070 μgmL-1 and 0.212 μgmL-1 respectively.

3.6.3.2 Using methylene blue as a reagent

The adherence to Beer’s law is studied by measuring the absorbance values of

solutions varying hypochlorite concentration. A straight line graph is obtained by

plotting absorbance against concentration of hypochlorite. Beer’s law obeyed in the

range of 0.1–6.0 μgmL-1 of hypochlorite (Figure IIIB2). The molar absorptivity and

Sandell’s sensitivity for colored system is found to be 1.12×104 Lmol-1 cm-1,

4.61×10-3 μgcm-2 respectively. The detection limit (DL=3.3σ/s) and quantitaion limit

(QL=10σ/s) where σ is the standard deviation of the reagent blank (n=5) and s is the

slope of the calibration- curve, for hypochlorite determination were found to be 0.031

μgmL-1 and 0.093 μgmL-1 respectively.

3.6.4 Effect of Diverse Ions

The effect of diverse ions on the determination of hypochlorite by the

proposed procedure is examined. The tolerance limits of interfering species are

established at the concentration required to cause not more than a ± 2% error in the

recovery of hypochlorite at 1.00 μgmL-1. The tolerance limits of diverse ions are

summarized in Table 3A1. The interference of iron(III) can be masked by the

addition of 1 mL of 2% sodium fluoride while cupric ions were masked by the

addition of 1 mL of 1 % EDTA.

3.7 APPLICATIONS

The proposed method is capable of determining with a high degree of

precision the amount of hypochlorite in samples of tap water, natural water and milk.

The results of analysis of the above samples (Table 3A2 and Table 3A3) compared

favorably with those from a reference method [19,36]. Statistical analysis of the

results by the use of t and F–tests showed that there was no significant difference

between the accuracy and precision of the proposed and reference methods. The

74

precision of the proposed method was evaluated by replicate analysis of samples

containing hypochlorite at different concentrations.

3.8 CONCLUSIONS

1. The reagents provide a simple and rapid method for the spectrophotometric

determination of hypochlorite.

2. The reagents have the advantage of sensitivity and wide range of determinations

without the need for extraction or heating.

3. The method does not involve any stringent reaction conditions and the accuracy of

the method is comparable with most methods reported in the literature.

4. The proposed method has been successfully applied to the determination of traces

of hypochlorite in samples of tap water, natural water and milk. A comparison of

the method reported is made with earlier methods and is given in Table 3A4.

75

FIGURE IIIA1

ABSORPTION SPECTRA OF RHODAMINE B Vs REAGENT BLANK

Wavelength (nm)

0 200 400 600 800 1000

Abs

orba

nce

0.0

0.5

1.0

1.5

2.0

2.5

FIGURE IIIA2

ABSORPTION SPECTRA OF METHYLENE BLUE Vs REAGENT BLANK

Wavelength (nm)

400 500 600 700 800 900 1000

Abs

orba

nce

0.0

0.1

0.2

0.3

0.4

76

FIGURE IIIB1

ADHERENCE TO BEER’S LAW FOR HYPOCHLRITE USING RHODAMINE B

AS A REAGENT

H ypochlorite concentration (µgm L -1)

0 1 2 3 4 5 6 7

Abs

orba

nce

0 .0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

FIGURE IIIB2

ADHERENCE TO BEER’S LAW FOR HYPOCHLRITE USING METHYLENE

BLUE AS A REAGENT

Hypochlorite concentration (µgm L -1)

0 2 4 6 8 10

Abs

orba

nce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

77

FIGURE IIIC1EFFECT OF pH ON COLOR INTENSITY USING RHODAMINE B AS A

REAGENT

pH

1 2 3 4 5 6 7 8

Abso

rban

ce

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

FIGURE IIIC2

EFFECT OF pH ON COLOR INTENSITY USING METHYLENE BLUE

AS A REAGENT

p H

1 2 3 4 5 6 7 8

Ab

sorb

an

ce

0 .06

0 .08

0 .10

0 .12

0 .14

0 .16

0 .18

0 .20

0 .22

0 .24

78

SCHEME III

NaOCl + HCl → NaCl + HOCl

KI + HCl → HI + KCl

HOCl + 2HI → I2 + HCl + H2O

(H3CH2C)2N N(CH2CH3)2O

COOH

Cl

I2

(H3CH2C)2N N(CH2CH3)2O

COOH

Rhodamine B (colored) Rhodamine B (colorless)

(CH3)2N

N

S+ N(CH3)2

Cl-

I2 , H+

N(CH3)2(CH3)2N

NH

S

Methylene blue (colored) Methylene blue (colorless)

79

TABLE 3A1

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF HYPOCHLORITE

USING RHODAMINE B AND METHYLENE BLUE AS REAGENTS (1.0 μgmL-1)

Foreign ion Tolerance limit

(μg)

Foreign ion Tolerance limit

(μg)

Cd(II)

Zn(II)

Ni(II)

Co(II)

Mg(II)

Ba(II)

Cu(II)*

Fe(III)*

In(III)

V(IV)

Ti(IV)

Al(III)

50

50

75

50

75

25

25

75

50

100

50

50

Mo(VI)

W(VI)

U(VI)

Chloride

Phosphate

Bromide

Nitrate

Acetate

Sulphate

Borate

50

50

100

150

150

250

250

125

100

100

* Masked by masking agents.

80

TABLE 3A2

DETERMINATION OF HYPOCHLORITE IN ENVIRONMENTAL SAMPLES

USING RHODAMINE B AS A REAGENT

Proposed method Reference method [19,36]

SamplesHypo-chlorite added(μgmL-1)

Hypo-chloritefounda

(μgmL-1)

Relativeerror

Hypo-chloritefounda

(μgmL-1)

Relativeerror t-

testb

F-testc

Tap water-1

1.002.003.00

0.98 ± 0.04

2.04 ± 0.02

3.05 ± 0.02

-0.02+0.02+0.02

1.04 ± 0.06

2.08 ± 0.02

3.07 ± 0.03

+0.04+0.02+0.02

0.673.010.50

2.251.002.25

Tap water-2

1.002.003.00

1.02 ± 0.01

2.06 ± 0.03

3.07 ± 0.10

+0.02+0.03+0.02

1.06 ± 0.01

2.10 ± 0.05

3.09 ± 0.02

+0.06+0.05+0.03

4.001.330.40

1.002.724.00

Natural water d

1.002.003.00

0.97 ± 0.04

1.98 ± 0.06

3.01 ± 0.02

-0.03-0.01+0.01

1.06 ± 0.05

2.01 ± 0.10

3.04 ± 0.04

+0.06+0.01+0.01

3.000.601.50

1.562.724.00

Milk d 1.002.003.00

1.02 ± 0.03

2.05 ± 0.01

3.04 ± 0.02

+0.02+0.03+0.01

1.05 ± 0.04

2.09 ± 0.01

3.06 ± 0.02

+0.05+0.05+0.02

1.504.002.00

1.711.001.00

a. Hypochlorite concentration (μgmL-1) ± Standard deviation (n=5)

b. Tabulated t- value for (4,4) degrees of freedom at 95% probability level is 2.306

c. Tabulated F- value for (4,4) degree of freedom at 95% probability level is 6.39

d. Tested and shown to be free from hypochlorite

81

TABLE 3A3DETERMINATION OF HYPOCHLORITE IN ENVIRONMENTAL SAMPLES

USING METHYLENE BLUE AS A REAGENT

Proposed method Reference method [19,36]

SamplesHypo-chlorite added(μgmL-1)

Hypochloritefounda

(μgmL-1)

Relativeerror

Hypochloritefounda

(μgmL-1)

Relativeerror

t-testb F-testc

Tap

water-1

1.00

3.00

5.00

0.994 ± 0.01

3.012 ± 0.01

4.984 ± 0.03

-0.006

+0.004

-0.003

1.014 ± 0.02

3.036 ± 0.01

5.017 ± 0.04

+0.014

+0.012

+0.003

2.00

4.00

1.65

4.00

1.00

1.78

Tap

water-2

1.00

3.00

5.00

1.058 ± 0.02

2.972 ± 0.01

5.028 ± 0.03

+0.018

-0.009

+0.005

1.096 ± 0.02

3.012 ± 0.02

5.032 ± 0.02

+0.096

+0.004

+0.006

2.90

4.00

0.25

1.00

4.00

2.25

Natural

water d

1.00

3.00

5.00

1.022 ± 0.01

3.042 ± 0.02

5.032 ± 0.02

+0.022

+0.014

+0.006

0.986 ± 0.02

3.014 ± 0.015

5.054 ± 0.03

-0.014

+0.005

+0.011

3.60

2.80

1.37

4.00

1.78

2.25

Milk d 1.00

3.00

5.00

1.038 ± 0.02

3.101 ± 0.02

5.028 ± 0.04

+0.038

+0.034

+0.005

1.077 ± 0.03

3.082 ± 0.02

5.032 ± 0.01

+0.077

+0.027

+0.006

2.44

1.46

0.13

2.25

1.00

1.56

a. Hypochlorite concentration (μgmL-1) ± Standard deviation (n=5)

b. Tabulated t- value for (4,4) degrees of freedom at 95% probability level is 2.306

c. Tabulated F- value for (4,4) degree of freedom at 95% probability level is 6.39

d. Tested and shown to be free from hypochlorite

82

TABLE 3A4 COMPARISON OF THE METHOD REPORTED WITH EARLIER METHODS

Reagent Method Beer’s law�����-1)

ε (Lmol-1cm-1)���������-2)

λmax(nm)

Ref. No.

Azure B Spectrophotometry 0.2-1.0 ε = 1.49×104

ss = 3.25×10-4644 33

Thionin Spectrophotometry 0.2-1.2 ε = 1.489×104

ss = 3.25×10-3600 36

Proposed Method

Rhodamine B

Methylene blue

spectrophotometry

spectrophotometry

0.1–4.0

0.1-0.6

ε = 2.57×105

ss = 2.01×10-3

ε = 1.12×104

ss = 4.61×10-3

553

665.6

ε = Molar absorptivity, ss = Sandell’s sensitivity

83

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