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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
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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
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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]
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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
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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
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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
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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.
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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%),