chapter 5 - shodhganga

125

CHAPTER 5

NEW REAGENTS FOR THE SPECTROPHOTOMETRIC

DETERMINATION OF HYPOCHLORITE

5.1 INTRODUCTION

5.2 ANALYTICAL CHEMISTRY

5.3 APPARATUS

5.4 REAGENTS AND SOLUTIONS

5.5 PROCEDURES

5.6 RESULTS AND DISCUSSION

5.7 APPLICATIONS

5.8 CONCLUSIONS

5.9 REFERENCES

126

5.1 INTRODUCTION

In nature, chlorine exists as various species at different oxidation states,

such as chloride (Cl-

), hypochlorite (OCl-

), chlorite (ClO2

-

), chlorate (ClO3

-

) and

perchlorate (ClO4

-

), etc. In the pharmaceutical industry, NaOCl can be used as a

key raw material. The released Cl2 from NaOCl in acidic solution could be

hazardous [1,2]. Hypochlorite (OCl-

) is commonly used in bleaches and

disinfectants. In human tissues, OCl-

or its conjugate acid, hypochlorous acid

(HOCl), may be formed from hydrogen peroxide and chloride by the catalytic

action of myeloperoxidase, a heme containing enzyme released by activated

monocytes and neutrophils [3-5]. Biological toxins can be extremely hazardous

even in minute quantities. Some toxins are inactivated by autoclaving for one hour

at 121°C, while others are inactivated by exposure to sodium hypochlorite or

sodium hypochlorite and sodium hydroxide [6]. Many solutions have been tested

with the intent of encountering an irrigating solution which permits the substitution

of sodium hypochlorite because of its toxicity. The antibacterial action of

hypochlorite acid occurs by the oxidation of bacterial enzymes which lead to the

disorganization of their metabolism [7].

Hypochlorite was first produced in 1789 in Javelle, France, by passing

chlorine gas through a solution of sodium carbonate. The resulting liquid, known

as "Eau de Javelle" was a weak solution of sodium hypochlorite. However, this

process was not very efficient and alternate production methods were sought. One

such method involved the extraction of chlorinated lime (known as bleaching

powder) with sodium carbonate to yield low levels of available chlorine. This

method was commonly used to produce hypochlorite solutions for use as a hospital

antiseptic which was sold under the trade names "Eusol" and "Dakin's solution."

Near the end of the nineteenth century, E. S. Smith patented a method of

hypochlorite production involving hydrolysis of brine to produce caustic soda and

chlorine gas which then mix to form hypochlorite. Both electric power and brine

solution were in cheap supply at this time and various enterprising marketers took

advantage of this situation to satisfy the market's demand for hypochlorite. Bottled

solutions of hypochlorite were sold under numerous trade names; one such early

brand produced by this method was called Parozone. Today, an improved version

127

of this method, known as the Hooker process, is the only large scale industrial

method of sodium hypochlorite production.

Sodium hypochlorite is now used in endodontics during root canal

treatments. It is the medicament of choice due to its efficacy against pathogenic

organisms and pulp digestion. Historically, Henry Drysdale Dakin's solution (0.5

%) had been used. Its concentration for use in endodontics today varies from 0.5 %

to 5.25 %. At low concentrations it will dissolve mainly necrotic tissue; whereas at

higher concentrations tissue dissolution is better but it also dissolves vital tissue, a

generally undesirable effect. It has been shown clinical effectiveness does not

increase conclusively for concentrations higher than 1% [8]. US Government

regulations (21 CFR Part 178) allow food processing equipment and food contact

surfaces to be sanitized with solutions containing bleach provided the solution is

allowed to drain adequately before contact with food, and the solutions do not

exceed 200 parts per million (ppm) available chlorine (for example, one tablespoon

of typical household bleach containing 5.25 % sodium hypochlorite, per gallon of

water). If higher concentrations are used, the surface must be rinsed with potable

water after sanitizing, 1 in 5 dilution of household bleach with water (1 part bleach

to 4 parts water) is effective against many bacteria and some viruses, and is often

the disinfectant of choice in cleaning surfaces in hospitals (Primarily in the United

States). The solution is corrosive, and needs to be thoroughly removed afterwards,

so the bleach disinfection is sometimes followed by an ethanol disinfection. Even

"scientific grade", commercially produced disinfection solutions such as Virocidin-

X usually have sodium hypochlorite as their sole active ingredient, though they

also contain surfactants (to prevent beading) and fragrances (to conceal the bleach

smell).

Household bleach sold for use in laundering clothes is a 3-6% solution of

sodium hypochlorite at the time of manufacture. Strength varies from one

formulation to another and gradually decreases with long storage. A 12% solution

is widely used in waterworks for the chlorination of water and a 15% solution is

more commonly [9] used for disinfection of waste water in treatment plants. The

crystalline salt is also sold for the same use; this salt usually contains less than 50%

of calcium hypochlorite. However, the level of "active chlorine" may be much

128

higher. It can also be found on store shelves in "Daily Sanitizing Sprays", as the

sole active ingredient at 0.0095%.

The common method for masking 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 NaOCl + NaCl + H

2O [1]

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

chlorinating a solution of soda ash according to the following equation:

Cl2 + 2Na

2CO

3

+ H2O NaOCl + NaCl + 2 NaHCO

3[2]

On further chlorination, hypochlorous acid will be produced:

Cl2 + Na

2CO

3 + H

2O HOCl + NaCl + NaHCO

3[3]

Most of the commercial production processes involve the reaction of

chlorine with caustic soda as mentioned in the above equation.

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

-

ClO3

-

+ 2 Cl

-

[4]

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

whether transition metal ion acts as catalyst.

The active ingredient in most of the chlorine bleaches is sodium

hypochlorite. The oxidizing action of hypochlorite ion 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 it can produce by

oxidizing an iodide ion. The quantity of iodine produced is estimated by titrating it

2OCl

-

O2 + 2Cl

-

[5]

129

with sodium thiosulphate, 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 [6]

Titrating iodine with thiosulfate:

I2 + 2S

2O

3

2-

2I

-

+ S4O

6

2-

[7]

Sodium hypochlorite has been used for the disinfection of drinking water.

A concentration equivalent to about 1 liter of household bleach per 4000 liters of

water is used. The exact amount required depends on the water chemistry,

temperature, contact time, and presence or absence of sediment. In large-scale

applications, residual chlorine is measured to titrate the proper dosing rate. For

emergency disinfection, the United States Environmental Protection Agency

recommends the use of 2 drops of 5%ac household bleach per quart of water. If the

treated water doesn't smell of bleach, 2 more drops are to be added. The use of

chlorine-based disinfectants in domestic water, although widespread, has led to

some controversy due to the formation of small quantities of harmful byproducts

such as chloroform.

Sodium hypochlorite is recommended and used by the majority of dentists

because this solution presents several important properties: antimicrobial effect

[10, 11], tissue dissolution capacity and acceptable biologic compatibility in less

concentrated solution. Sodium hypochlorite neutralizes amino acids forming water

and salt. With the exit 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 a solvent releases chlorine that combines with protein amino

group to form chloramines. Hypochlorous acid and hypochlorite ions lead to amino

acid degradation and hydrolysis.

Sodium hypochlorite in lower concentration is biocompatible. For a

substance to be biocompatible, it must present a discrete tissue reaction at all

130

periods and moderate or intense tissue reaction at 7 days which decrease in

intensity with time until reaching a non-significant tissue reaction [12].

5.2. 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 the determination of

hypochlorite. Several methods have been reported for the determination of

hypochlorite. These include colorimetric, chemiluminescent, potentiometric,

amperometric, titrimetric, spectrophotometric, Iodometric, polarographic and

radiolytically-induced redox methods [13-22].

Soto et al. described an environmental friendly method for the automatic

determination of hypochlorite in commercial products using multisyringe flow

injection analysis [23]. The methodology was based on the selective decomposition

of hypochlorite by a cobalt oxide catalyst giving chloride and oxygen. The

difference of the absorbance of the sample before and after its pass through a

cobalt oxide column was selected as analytical signal. As no further reagent was

required this work can be considered as a contribution to environmental friendly

analytical chemistry. The dynamic concentration range was 0.04–0.78 g L1

(relative standard deviation lower than 3%), where the extension of the

hypochlorite decomposition was of 90 ± 4%. The accuracy of the method was

established by iodometric titration.

Evaluation of the interaction between sodium hypochlorite and

chlorhexidine gluconate and its effect on root dentin was described [24]. Forty-four

extracted single-rooted human teeth were instrumented and irrigated with both

sodium hypochlorite and chlorhexidine to produce a precipitate. Root canal

surfaces were analyzed with the environmental scanning electron microscope

(ESEM). The amount of remaining debris and number of patent tubules were

determined. There were no significant differences in remaining debris between the

negative control group and the experimental groups. There were significantly

fewer patent tubules in the experimental groups when compared with the negative

control group. The sodium hypochlorite/ chlorhexidine precipitate tends to occlude

131

the dentinal tubules. Until this precipitate was studied further, caution should be

exercised when irrigating with sodium hypochlorite and chlorhexidine.

March and Simonet reported a green method for the determination of

hypochlorite in bleaching products [25]. The method, based on a flow injection

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

allows the determination of hypochlorite in the range 0.07–0.42 g Cl2 L-1

. In order

to achieve high selectivity a mini-column containing cobalt oxide, which

effectively catalyses hypochlorite decomposition to chloride and oxygen, was

inserted in the flow system. The difference in absorbance of samples no circulated

and circulated through the mini-column was selected as analytical signal; thus, the

method only requires 20 mg of solid, reusable catalyst, and a NaOH solution of pH

10.4; providing a sample throughput of 12 samples h1

in triplicate injection. Its

usefulness for analysis of bleaching products was demonstrated. Determination of

hypochlorite in water using a chemiluminescent test strip was described. The test

strip responded linearly to hypochlorite over two linear ranges, the first 2.0–

10.3 mg L1

and the second 10.3–51.4 mg L1

, with a detection limit of 0.4 mg L1

.

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.

Jackson et al. described the indirect detection of bleach (sodium

hypochlorite) in beverages as evidence of product tampering [26]. In this work,

household bleach was added to 23 different beverages at each of three levels. The

impact of sodium hypochlorite on these beverages over a 13-day study period was

evaluated using the following techniques: diphenylamine spot test for oxidizing

agents, potassium iodide-starch test paper for oxidizing agents, pH, iodometric

titration for quantitating hypochlorite, ion chromatography for chloride and

chlorate quantitation, automated headspace sampling with gas chromatography-

flame ionization detection (GC-FID) for determination of chloroform, and visual

and organoleptic observations. This study has shown that hypochlorite was fragile

when added to most common beverages and typically breaks down either partially

or completely over time. In cases where a beverage was suspected of being

132

adulterated with bleach but tests for hypochlorite were negative, it was still

possible to characterize the product to demonstrate that the results were consistent

with the addition of bleach. An adulterated product will give a positive test for

oxidizing agents using the diphenylamine spot test. It was likely that the pH of the

adulterated product will be higher than a control of that product. Ion

chromatographic analysis showed elevated chloride and chlorate as compared with

a control. And, chloroform may also be detected by GC-FID especially if the

beverage that was adulterated contains citric acid.

Pasha and Narayana reported a method for the spectrophotometric

determination of hypochlorite using rhodamine B [27]. The proposed method

reports the reaction of hypochlorite with potassium iodide in an acid medium with

iodine liberation. The liberated iodine bleaches the pinkish red color of the

rhodamine B and can be measured at 553 nm. This decrease in absorbance was

directly proportional to the hypochlorite concentration and obeyed Beer's law in

the range of 0.1- 4.0 µg mL-1

of hypochlorite. The molar absorptivity, Sandell's

sensitivity, detection limit and quantitation limit of the method were found to be

2.57×105

L mol-1

cm–1

, 2.01×10-3

µg cm-2

, 0.070 µg mL-1

and 0.212 µg mL-1

respectively. The optimum reaction conditions and other analytical parameters

were evaluated. The effect of interfering ions on the determination was also

described. Antonio et al. described the determination of hypochlorite in bleaching

products with flower extracts to demonstrate the principles of flow injection

analysis [28]. The use of crude flower extracts to the principle of analytical

chemistry automation with the flow injection analysis was developed to determine

hypochlorite in household bleaching products.

Narayana et al. described an easy spectrophotometric method for the

determination of hypochlorite [29]. 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

determination and was measured at 600 nm. Beer’s law was obeyed in the range

0.2-- 1

of hypochlorite. The molar absorptivity, Sandell’s sensitivity,

detection limit, quantitation limit were found to be 1.48×104

Lmol-1

cm-1

, 3.25×10-3

-2 -1 -1

respectively.

133

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

determination of hypochlorite [30]. The method was based on the reaction of

hypochlorite with potassium iodide in an acidic medium to liberate iodine. Beer’s

law was obeyed in the range of 0.2--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

L mol-1

cm-1

and 3.25×10-4 -2

respectively. GC–

MS comparison of the behavior of chlorine and sodium hypochlorite towards

organic compounds dissolved in water was described [31]. Chlorine and sodium

hypochlorite were used as commonly employed disinfecting agents. Comparison of

the chlorinating agents was performed in terms of the assortment and relative

amounts of reaction products. Quantum chemical calculations were applied to

propose structures of the reacting particles and a numerical parameter to estimate

an extent of conversion of aromatic substrates during chlorination.

Harriram et al. reported oxidative degradation of indigocarmine by

hypochlorite--a tool for determination of hypochlorite in commercial samples [32].

Hypochlorite dissociates indigocarmine to produce isatin-5-monosulphonic acid,

with a stoichiometry of 2:1. The suitability of reaction between hypochlorite and

indigocarmine as an indicator reaction for the determination of high levels of

hypochlorite in synthetic and commercial samples was investigated. Michael

Goldsmith et al. described the effect of sodium hypochlorite irrigant concentration

on tooth surface strain [33]. The effect of root-canal irrigation with different

concentrations of sodium hypochlorite (3%, 5.1%, 7.3% NaOCl) on the

mechanical properties of teeth was investigated in vitro. Root canals of 13

extracted, human premolars, denuded of enamel, were prepared with nickel-

titanium rotary instruments (Quantec™) to a standard size by using saline

irrigation. An electrical strain gauge was bonded to the cervical aspect of each

tooth. The 10 experimental teeth were subjected to 5 successive, 30-minute periods

of irrigation. The irrigants were used in the following order: (a) saline; (b) 3.0%

NaOCl; (c) 5.1% NaOCl; (d) 7.3% NaOCl; (e) saline. Three control teeth were

irrigated with saline only for all five periods. After each irrigation, the teeth were

cyclically loaded to 110 N while the surface strain was measured. Changes in

strain of the test teeth after each irrigation regimen followed broadly similar

134

patterns that were different from the control teeth. There was no difference,

however, in the strain recorded after irrigation by the different irrigants within the

experimental group.

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 several

molar [34]. The hydroxide concentration was determined by the heat of

neutralization of the injected sample into an acidic carrier stream and the chloride

concentration was calculated from the measured conductance data. For the

measurement of hypochlorite and chlorate, colorimetric iodometry was used.

Watanabe et al. presented simultaneous determination of chlorine dioxide

and hypochlorite in water by high-performance liquid chromatography [35]. A

linear correlation between the peak height and concentration was obtained within

the range of 1.0–20.0 g mL-1

for chlorine dioxide and 47.0–200.0 g mL-1

for

hypochlorite, with good reproducibility (relative standard deviations of 4.0 and

2.2%, respectively, replicated determination). The limits of detection of chlorine

dioxide and hypochlorite were approximately -1

(S/N=3),

respectively.

Han et al. presented a simple spectrophotometric method for the

quantitative determination of hypochlorite (OCl ) or hypochlorous acid (HOCl)

[36]. The OCl or HOCl sample was first incubated with an excess amount of

tris(2-carboxyethyl)phosphine. The concentration of the residual tris(2-

carboxyethyl)phosphine was then measured as the amount of 2-nitro-5-

thiobenzoate produced after reaction with 5,5 -dithiobis(2-nitrobenzoic acid). The

concentration of OCl or HOCl was equivalent to the amount of decrease in the

concentration of tris(2-carboxyethyl)phosphine because one molecule of tris(2-

carboxyethyl)phosphine was rapidly and irreversibly oxidized to tris(2-

carboxyethyl)phosphine oxide by one molecule of OCl or HOCl. This method was

more sensitive and convenient than the standard procedure of NaOCl assay which

involves reaction with potassium iodide followed by titration of the liberated

triiodide with thiosulfate.

135

Gonzalez-Robledo et al. reported the determination of hypochlorite in

waters by stopped-flow chemiluminescence spectrometry [37]. The emission was

observed by using a conventional fluorescence detector with its shutter off and set

at 425 nm. Methods based on direct rate measurements on the formation and decay

steps of the chemiluminescence process were used in addition to those involving

conventional peak-height or peak-area measurements, which were evaluated

comparatively. The methods yield linear responses over three orders of magnitudes

with an RSD of about 1%. The proposed method, which was highly selective and

rapid (80 samples h-1

), was applied to the routine determination of hypochlorite in

waters. The results were in good agreement with those obtained by the classical

N,N-diethyl-p-phenylendiamine spectrophotometric method. Bamnolker et al.

reported a method for the spectrophotometric determination of hypochlorite traces

in solutions containing 4 M NaOH based on the reaction of the reagent 3,3 -

dimethylnaphtidine with chlorine [38]. Use was made in common reagent

containing 3,3 -dimethylnaphtidine, hydrochloric acid and dimethylformamide.

This common reagent enables the liberation and determination of chlorine in situ in

strongly acidic solutions. The effect of factors such as acidity, temperature,

presence of other solvents, oxidation agents and metallic impurities on the

absorption spectrum were studied. The factors affecting the chemical stability of

the common reagent were also discussed.

Tarasankar et al. described a method for the spectrophotometric

determination of hypochlorite [39]. 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 mg

L-1

. Vieira et al. reported the effect of sodium hypochlorite and citric acid solutions

on healing of periodontal pockets [40]. 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 luminal in presence of hydrogen peroxide [41]. Micromolar and

submicromolar quantities could be detected by this method.

136

Fleet and Ho presented a fully automated procedure for the determination

of sodium hypochlorite and hydrogen peroxide, based on the use of a porous

catalytic silver electrode [42]. The principle of both methods involved the

quantitative liberation of oxygen, which was measured coulometrically by the

electrode. The procedures were suitable for the continuous monitoring of the

contents of bleaching baths. Anwar et al. reported two simple spectrophotometric

procedures for the quantitative estimation of hypochlorite [43]. 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 chloramine, which had a higher molar absorptivity.

Bunikiene and Ramanauskas reported an indirect spectrophotometric

detection of trace amounts of OCl-

, was based on oxidation of OCl-

with I-

[44].

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

change in the absorbance of brilliant green solution. The diction 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

propotional to OCl-

concentration in the range 0.04-1.60 -1

. Hussain et al.

presented quantitative spectrophotometric methods for determination of sodium

hypochlorite in aqueous solutions [45]. Erdey and Vigh described indirect

determination of hypochlorite and hypobromite by ascorbinometric titration of

thallium(III) ions [46]. To the hypochlorite or hypobromite solution an excess of

thallium(I) sulphate solution was added, and thallium(III) ions, formed in

equivalent amount, can be titrated with ascorbic acid in the presence of variamine

blue as an indicator.

The present work describes a simple, rapid and sensitive method for the

spectrophotometric determination of hypochlorite using crystal violet, xylene

cyanol FF and malachite green as reagents. The developed method has been

successfully employed for the determination of hypochlorite in water and milk

samples. Comparison of spectrophotometric method with the earlier methods are

given in table 5.

137

5.3 APPARATUS

5.3.1 Spectrophotometer

A SHIMADZU (Model No: UV-2550) UV-Visible spectrophotometer with

1 cm matching quartz cells were used for the absorbance measurements.

5.4 REAGENTS AND SOLUTIONS

All reagents used were of analytical reagent grade and distilled water was

-1

)

was prepared and the stock solution was diluted as needed. Potassium iodide (2%),

hydrochloric acid (2M), acetate buffer solutions (1M), crystal violet (CV) (0.05%),

xylene cyanol FF (XFF) (0.05%), malachite green (MLG) (0.03%) were also used.

5.5 PROCEDURE

5.5.1 Using Crystal Violet as a Reagent

Aliquots of sample solution containing 0.06 - 0.40 µg mL-1

of hypochlorite

was transferred in to a series of 10 mL calibrated flasks, 1 mL of hydrochloric acid

and 1 mL of potassium iodide were added. The mixture was gently shaken until the

appearance of yellow color indicating the liberation of iodine. To this solution, 0.5

mL of crystal violet was added followed by the addition of 2 mL of 1M sodium

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

diluted to 10 mL with distilled water. The absorbances of the resulting solution

were measured at 582 nm against distilled water. The absorbance corresponding to

the bleached color which in turn corresponds to the analyte concentration was

obtained by subtracting the absorbance of the blank solution from that of the test

solution.

5.5.2. Using Xylene Cyanol FF as a Reagent

Aliquots of sample solution containing 1.00-3.00 µg mL-1

of hypochlorite


and 1 mL of potassium iodide were added. The mixture was gently shaken until

the appearance of yellow color indicating the liberation of iodine. To this solution,

0.5 mL of xylene cyanol FF was added followed by the addition of 2 mL of sodium




138



solution.

5.5.3. Using Malachite Green as a Reagent

Aliquots of sample solution containing 1.00-2.00 µg mL-1

of hypochlorite


and 1 mL of potassium iodide were added. The mixture was gently shaken until the

appearance of yellow color indicating the liberation of iodine. To this solution, 0.5

mL of malachite green was added followed by the addition of 2 mL of sodium






solution.

5.5.4. Determination of Hypochlorite in Water Samples

Each filtered environmental water samples were analyzed for hypochlorite.

All the tested samples gave negative results. To these samples known amounts (not

more than 0.06- 0.40 µg mL-1

of hypochlorite fo hypochlorite-crystal violet system,

1.00-3.00 µg mL-1

of hypochlorite for hypochlorite-xylene cyanol FF system ,

1.00- 2.00 µg mL-1

of hypochlorite for hypochlorite-malachite green system) were

spiked and analyzed for chromium by the proposed methods and also by the

reference methods [29]. The results are summarized in table 5.1A, 5.1B and 5.1C.

5.5.5. Determination of Hypochlorite in Milk Samples

A known volume of commercial milk sample was placed in a 50 mL beaker

and coagulated with suitable volume of 1 mL citric acid. The solution was

centrifuged to remove the precipitate. All the tested samples gave negative results.

To these samples a known amount of hypochlorite was added. The centrifugate

was transferred to a 100 mL calibrated flask. A suitable aliquot of the sample

solution was determined directly, according to the procedure described above. The

results are summarized in table 5.1A, 5.1B and 5.1C.

139

5.6. RESULTS AND DISCUSSION

5.6.1. Absorption Spectra

5.6.1.1 Using Crystal Violet as a Reagent

This method involves the liberation of iodine by the reaction of

hypochlorite with potassium iodide in an acid medium. The liberated iodine

selectively bleaches the color of crystal violet and is measured at 582 nm. This

decrease in absorbance is directly proportional to the hypochlorite concentration

and obeys Beer’s law in the range of 0.06- 0.40 µg mL-1

of hypochlorite. The

absorption spectrum of crystal violet is presented in fig.VA and the reaction system

is presented in scheme 5.3A.

5.6.1.2 Using Xylene Cyanol FF as a Reagent

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

hypochlorite with potassium iodide in an acid medium. The liberated iodine

selectively bleaches the color of xylene cyanol FF and is measured at 612 nm. This

decrease in absorbance is directly proportional to the hypochlorite concentration

and obeys Beer’s law in the range of 1.00-3.00 µg mL-1

of hypochlorite. The

absorption spectrum of xylene cyanol FF is presented in fig.VB and the reaction

system is presented in scheme 5.3A.

5.6.1.3 Using Malachite Green as a Reagent

Similarly the liberated iodine selectively bleaches the color of malachite

green and is measured at 615 nm. This decrease in absorbance is directly

propotional to the hypochlorite concentration and obeys Beer’s law in the range of

1.00-2.00 µg mL-1

of hypochlorite. The absorption spectrum of malachite green is

presented in fig.VC and the reaction system is presented in scheme 5.3A.

5.6.2 Effect of Reagent Concentration and Acidity

In the present method all parameters influencing the color development are

investigated and the optimum values obtained are incorporated in the

recommended procedure. The reaction of hypochlorite with potassium iodide in

acidic medium results in the liberation of iodine, and the liberated iodine bleaches

the color of crystal violet or xylene cyanol FF or malachite green to colorless

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

140

quantitative and the appearance of yellow color indicates the liberation of iodine.

The optimum acidity to bleach the color of reaction system is fixed to be 2M HCl

and the bleached reaction systems formed are found to be stable for more than 2

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

range 1.0-1.2 ranges, which could be maintained by adding 1 mL of 2M HCl in a

final volume of 10 mL. The absorbance corresponding to the bleached color, which

in turn corresponds to the hypochlorite concentration, is obtained by subtracting

the absorbance of the blank solution from that of the test solution.

5.7 Analytical Data

5.7.1. Using Crystal Violet as a Reagent

Adherence to Beer’s law is studied by measuring the absorbance value of

the solution varying hypochlorite concentration. Beer’

s law is obeyed in the range

of 0.06-0.40 µg mL-1

of hypochlorite. The molar absorptivity, Sandell’s sensitivity

detection limit, quantitation limit are found to be 5.73× 104

L mol-1

cm-1

, 8.97×10-4

µg cm-2

, 0.109, 0.015 L-1

, 0.045 L-1

respectively. The precision and

accuracy of the method is studied by analyzing the coupling solution containing

known amounts of the cited reagents within Beer’s law limit. The low values of the

standard deviation in percentages and the error indicates the high accuracy of the

two methods. Adherences to Beer’s law for the determination of above methods

are shown in figure 5A.

5.7.2 Using Xylene Cyanol FF as a Reagent




of 1.00-3.00 µg mL-1

of hypochlorite. The molar absorptivity, Sandell’s sensitivity

detection limit, quantitation limit are found to be 0.38× 104

L mol-1

cm-1

, 0.013 µg

cm-2

, 0.052 L-1

, 0.156 L-1

respectively. The precision and accuracy of

the method is studied by analyzing the coupling solution containing known

amounts of the cited reagents within Beer’s law limit. The low values of the



are shown in figure 5B.

141

5.7.2 Using Malachite Green as a Reagent




of 1.00-2.00 µg mL-1

of hypochlorite. The molar absorptivity, Sandell’s

sensitivity detection limit, quantitation limit are found to be 2.39×104

L mol-1

cm-1

,

2.16×10-3

µg cm-2

, 0.010 L-1

, 0.030 L-1

respectively. The precision and

accuracy of the method is studied by analyzing the coupling solution containing

known amounts of the cited reagents within Beer’s law limit. The low values of the



are shown in figure 5C.

5.7.3 Effect of Diverse Ions.

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

proposed procedure is examined. An error of ±2% is considered tolerable. The

interference of Fe(III) can be masked by the addition of sodium fluoride. The

tolerance limits of the foreign ions are in shown 5.2A and 5.2B.

5.8 APPLICATIONS

A new reaction scheme for the spectrophotometric determination of

hypochlorite is developed. The feasibility of the technique for the determination of

hypochlorite in water and milk samples are examined. The results are shown in

table 5.1A, 5.1B and 5.1C. Statistical analysis of the results by t and F test show

that there is no significant difference between accuracy and precision of the

proposed and published method. The precision of the proposed method is

evaluated by replicate analysis of samples containing hypochlorite at different

concentrations.

5.9 CONCLUSIONS

The reagents provide a simple, rapid and sensitive method for the

spectrophotometric determination of hypochlorite. Introduction of crystal violet,

xylene cyanol FF and malachite green as new reagents make the method versatile.

Reliability, excellent reproducibility and independence from temperature make the

method versatile. The proposed method has been successfully applied to the

determination of traces of hypochlorite in water and milk samples.

142

TABLE 5: COMPARISON OF THE SPECTROPHOTOMETRIC METHOD

WITH EARLIER METHODS

Reagent

Molar

absorptivity

(L mol-1

cm-1

)

Beer’s law

(µg mL-1

)

Ref

Azure B 644 1.49×104

0.20-1.00 [29]

Thionin 600 1.48×104

0.20-1.20 [30]

Rhodamine B 553 2.57×105

0.10-4.00 [27]

Proposed method

Using CV 582 5.73×104

0.06-0.40

Using XFF 610 0.38×104

1.00-3.00

Using MLG 615 2.39×104

1.00-2.00

TABLE 5.1A: DETERMINATION OF HYPOCHLORITE IN WATER AND

MILK SAMPLES (USING CV AS A REAGENT)

Standard method Proposed methodsamples Hypochlorite

added

µg mL-1

a

Hypochlorite

found

µg mL-1

Recovery

(%)

a

Hypochlorite

found

µg mL-1

Recovery

(%)

b

t-testc

F-test

Water

sample

0.20

0.40

0.198±0.01

0.388±0.008

99.00

97.00

0.210±0.02

0.386±0.01

105.00

96.50

1.12

3.13

4.00

1.56

Milk

sample

0.20

0.40

0.197±0.01

0.396±0.006

98.50

99.00

0.196±0.01

0.399±0.003

98.00

99.80

0.89

1.12

1.00

4.00

a

Average of 5 determinations.

b

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

c

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

143

TABLE 5.1B: DETERMINATION OF HYPOCHLORITE IN WATER AND

MILK SAMPLES (USING XFF AS A REAGENT)

Standard method Proposed methodb

t-testc

F-testsamples Hypochlorite

added

µg mL-1

a

Hypochlorite

found

µg mL-1

Recovery

(%)

a

Hypochlorite

found

µg mL-1

Recovery

(%)

Water

sample

1.00

2.00

1.02±0.01

2.06±0.03

102.00

103.00

0.98±0.02

1.96±0.05

98.00

98.00

2.24

1.79

4.00

2.78

Milk

sample

1.00

2.00

1.02±0.03

2.05±0.01

102.00

102.50

1.02±0.03

1.98±0.02

102.00

99.00

1.49

2.24

1.00

4.00

a


b


c

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

TABLE 5.1C: DETERMINATION OF HYPOCHLORITE IN WATER AND

MILK SAMPLES (USING MLG AS A REAGENT)

Standard method Proposed methodb

t-testc

F-test

samples

Hypochlorite

added

µg mL-1

a

Hypochlorite

found

µg mL-1

Recovery

(%)

a

Hypochlorite

found

µg mL-1

Recovery

(%)

Water

sample

1.00

2.00

1.02±0.01

2.06±0.03

102.00

103.00

0.99±0.03

2.02±0.02

99.00

101.00

0.75

2.24

2.25

2.25

Milk

sample

1.00

2.00

1.02±0.03

2.05±0.01

102.00

102.50

1.01±0.03

1.98±0.01

101.00

99.00

1.12

1.79

1.00

1.00

a


b


c

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

144

TABLE 5.2A: EFFECT OF DIVERSE IONS ON THE DETERMINATION OF

HYPOCHLORITE USING CV AND XFF AS REAGENTS.

Foreign Ion Tolerance Limit -1

)

Chloride, Phosphate Acetate, Borate,

Sulphate

>800

Fe 3+

* 50

Cd2+

, Zn2+

, Ni2+

, Co2+

>500

Mg2+

, Al3+

, Ti4+

, U6+

, In3+

300

Ba2+

, Ca2+

600

Mo6+

, W6+

100

* Masked by masking agents.

TABLE 5.2B: EFFECT OF DIVERSE IONS ON THE DETERMINATION OF

HYPOCHLORITE USING MLG AS A REAGENT.

Foreign Ion-1

)

Chloride, Phosphate, Acetate, Borate,

Sulphate

1500

Fe 3+

* 50

Cd2+

, Zn2+

, Ni2+

, Co2+

>1000

Mg2+

, Al3+

, Ti4+

, U6+

, In3+

500

Ba2+

, Ca2+

1300

Mo6+*

, W6+

80

* Masked by masking agents.

145

FIGURE 5A: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION

OF HYPOCHLORITE USING CV AS A REAGENT.

Concentra tion (µg/ml)

0.0 0.1 0.2 0.3 0.4 0.5

Ab

so

rb

an

ce

0.0

0.1

0.2

0.3

0.4

0.5

FIGURE 5B: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION

OF HYPOCHLORITE USINF XFF AS A REAGENT.

Concentration (µg/ml)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Ab

sorb

ance

0.00

0.05

0.10

0.15

0.20

0.25

146

FIGURE 5C: ADHERANCE TO BEER’S LAW FOR THE DETERMINATION

OF HYPOCHLORITE USING MLG AS A REAGENT

Conce ntra tion (µg/ml)

0.0 0.5 1.0 1.5 2.0 2.5

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

FIGURE VA: ABSORPTION SPECTRA OF COLORED SPECIES OF CV

Wavelength (nm)

350 400 450 500 550 600 650 700 750

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

147

FIGURE VB: ABSORPTION SPECTRA OF COLORED SPECIES OF XFF.

Wavelength (nm)

350 400 450 500 550 600 650 700 750

Absorbance

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

FIGURE VC: ABSORPTION SPECTRA OF COLORED SPECIES OF MLG

Wavelength (nm)

350 400 450 500 550 600 650 700

Ab

so

rb

an

ce

0

1

2

3

4

5

148

REACTION SCHEMES 5.3A:

NaOCl HCl NaCl HOCl

KI+ HClHI+ KCl

HOCl+ 2HI I2+ HCl+ H

2O

+ +

N

+CH

3CH

3

NN

CH3

CH3

CH3

CH3

Cl

-

I2

H

+

Crystal Violet (Colored) Crystal Violet (Leuco form)

N

CH3

CH3

N

CH3

CH3

N

CH3

CH3

so3

-

CH3

NH

so3

-

CH3

N

+

CH3

H

CH3

I2/H

+

N

+

so3

-

so3

-

H

CH3

HNH

CH3

CH3

CH3

Xylene cyanol FF (Colored) Xylene cyanol FF (Leucoform)

N

+

CH3

CH3

N

CH3

CH3

I2/H

+

N

CH3

CH3

N

CH3

CH3

H

Cl

-

Malachite green (Colored) Malachite green (Leucoform)

149

5.9. REFERENCES

1. J. Wang, C. Winskog, E. Edston & S. M. Walther, Acta Anaesthesiol. Scand., 49

(2005) 183.

2. J. Wang. L. Zhang & S. M. Walther, J. Trauma., 56 (2004) 850.

3. S. J. Weiss, M. B. Lampert & S. T. Test, Science, 222 (1983) 625.

4. C. S. Foot, T. E. Goyne & R. I. Lehrer, Nature, 301 (1983) 715.

5. M. Wasil, B. Halliwell, D. C. Hutchison & H. Baum, Biochem. J., 243 (1987)

219.

6. R. W. Wannemacher, Procedures for Inactivation and Safety Containment of

Toxins. Proc. Symposium on Agents of Biological Origin, U. S. Army Research,

Development and Engineering Center, Aberdeen proving Ground, MD. pp.115

(1989).

7. E. V. F. Fachin, L. Hahn & A. L. F. Palmini, Rev. Bras. Odontol., LI (1994) 14.

8. M. Zehnder, D. Kosicki, H. Luder, B. Sener & T. Waltimo, Oral Surg. Oral

Med. Oral Pathol. Oral Radio Endodon., 94 (2002) 756.

9. Metcalf & Eddy, Inc. Wastewater Engineering: Treatment, Disposal &

Reuse, 3rd

Edn, pg 497 (1991).

10. A. Bystron & G. Sundqvist, J. Dent. Res., 89 (1981) 321.

11. A. Bystron & G. Sundqvist, Oral Surg. Oral Med. Oral Pathol., 55(1983) 307.

12. R. Holland, I. J. Soares & I. M. Soares, Endod. Dent. Traumatol., 8 (1992) 223.

13. D. Leggett, N. Chen & D. Mahadevappa, The Analyst, 107 (1982) 433.

14. L. Moberg & B. Karlberg, Anal. Chim. Acta, 407 (2000) 127.

15. J. Ballesta Claver, M. C. Valencia Miron & L. F. Capitan- Vallvey, Anal. Chim.

Acta, 522 (2004) 267.

16. E. Pobozy, K. Pyrzynska & K. Szostek, Microchem. J., 51 (1995) 379.

17. A. P. Soldatkin, D. V. Gorchkov, C. Martelet & N. Jaffrezic-Renault,

SensorActuat. B., 43 (1997) 99.

18. Standard Methods for the Examination of Water and Wastewater, 15th

Edn.,

American Public Health Association, Washington, DC (1980).

19. C. Madec, F. Quentel, J. Courtot-Coupez & M. Dore, Analusis, 15 (1987) 69.

20. APHA, AWWA, WPCF, Metodos Normalizados Para el Analisis de Aguas

Potables Y Residuals, Ed. Diaz de Santos, S. A. Madrid, Espana (1992).

150

21. H. P. Paviet, H. Jacek. D. Thomas, M. Stanislaw, L. Ning ping, W. Mark,

R. Andrzej & Z. Zbigniew, WM’02 Conference February, 24 (2002).

22. H. Iketake & A. Yanada, Bunseki Kagaku, 49 (2000) 977.

23. N. O. Soto, B. Horstkotte, J. G. March, P. L. Lopez de Alba, L. Lopez Martinez

& V. Cerda Martin, Anal. Chim. Acta, 611 (2008) 182.

24. T. B. Bui, J. Craig Baumgartner & J. C. Mitchell, J. Endod., 34 (2008) 181.

25. J. G. March & B. M. Simonet, Talanta, 73 (2007) 232.

26. D. S. Jackson, D. E. Crockett & K. A. Wolnik, J. Forensic Sci., 51 (2006) 827.

27. C. Pasha & B. Narayana, J. Braz. Chem. Soc., 18 (2007) 167.

28. R. L. Antonio, P. K. Roberta, C. E. Tadeu Gomes & C. C. Cristina Schmitt, J.

Chem. Educ., 82 (2005) 1815.

29. B. Narayana, M. Mathew, K. Vipin, N. V. Sreekumar & T. Cherian, J. Anal.

Chem., 60 (2005) 706.

30. B. Narayana, K. Vipin, M. Mathew & N. V. Sreekumar, Indian J. Chem., 43A

(2004) 573.

31. A. T. Lebedev, G. M. Shaydullina, N. A. Sinikova & N. V. Harchevnikova,

Water Res., 38 (2004) 3713.

32. A. Harriram, V. Govender & S. B. Jonnalagadda, J. Environ. Sci. Health, Pt. A:

Toxic/Hazard. Subst. Environ. Eng., 38 (2003) 1055.

33. M. Goldsmith, K. Gulabivala & J. C. Knowles, J. Endod., 28 (2002) 575.

34. K. Tian & P. K. Dasgupta, Talanta, 52 (2000) 623.

35. T. Watanabe, T. Idehara, Y. Yoshimura & H. Nakazawa, J. Chromatogr. A.,

796 (1998) 397.

36. J. Han, T. Ching Chu, G. Han, J. Browne, I. Brown & P. Han, Microchem. J.,

58 (1998) 218.

37. D. Gonzalez-Robledo, M. Silva & D. Perez-Bendito, Anal. Chim. Acta, 228

(1990) 123.

38. H. Bamnolker, J. Lapid, Y. Givra, Y. Sorek & Z. Gavra, Microchem. J., 40

(1989) 246.

39. P. Tarasankar, G. Ashes & M. S. Durga, Chem. Anal. (Warsaw)., 33 (1988) 703.

40. E. M. Vieira, T. J. O'Leary & A. H. Kafrawy, Periodontol, 53 (1982) 71.

41. U. Isacsson & G. Wettermark, Anal. Chim. Acta, 83 (1976) 227.

42. B. Fleet & A. Y. W. Ho, Talanta, 19 (1972) 317.

151

43. H. Anwar, P. Trudell & R. Arnold, J. Pharm. Sci., 59 (1970) 1168.

44. L. Bunikiene & E. Ramanauskas, Chem. Tech., 12 (1970) 57.

45. A. Hussain, P. Trudell & A. J. Repta, J. Pharm. Sci., 59 (1970) 1168.

46. L. Erdey & K. Vigh, Talanta, 10 (1963) 439.

chapter 5 - shodhganga

Documents