CHAPTER 6

SPECTROPHOTOMETRIC DETERMINATION OF ARSENIC IN

ENVIRONMENTAL AND BIOLOGICAL SAMPLES

6.1 INTRODUCTION

6.2 ANALYTICAL CHEMISTRY

6.3 APPARATUS

6.4 REAGENTS AND SOLUTIONS

6.5 PROCEDURES

6.6 RESULTS AND DISCUSSION

6.7 APPLICATIONS

6.8 CONCLUSIONS

6.9 REFERENCES
6.1 INTRODUCTION

Arsenic is a naturally occurring dissolved element in ground and surface

waters throughout the world. Arsenic is an element classed as a semi-metal or
metalloid. This means it has some properties of metal and some properties of non-
metal. Arsenic occurs in two distinct solid forms. One is a brittle gray metal, while the
other is a yellow, non-metallic form, rarely seen outside the laboratory. Arsenic and
its compounds often have a garlic-like odor when crushed or when scratched with a
hard object. Elemental arsenic has very few uses. Nearly all the applications are as
salts or oxides of arsenic. Arsenic compounds can be very toxic and their uses are
strictly controlled by health and environmental regulations.

Arsenic has been found in nature since antiquity. Aristotle made reference to
sandarach (arsenic trisulfide) in the 4th century B.C. In the 1st century A.D., Pliny

stated that sandarach is found in gold and silver mines and arsenic (arsenic trioxide) is
composed of the same matter as sandarach. By the 11th century three species of

arsenic were known, white, yellow and red - since then recognized as arsenic trioxide,
arsenic trisulfide (orpiment) and arsenic disulfide (realgar) respectively [1].

The name arsenic comes from the Greek word arsenikon, which means
orpiment. Orpiment is a bright yellow mineral composed of arsenic sulfide (AS2S3),
and is the most highly visible common arsenic mineral. Historians say that arsenic
was discovered in 1250 A. D. by Albertus Magnus, a German monk who spent his life
studying and classifying natural materials. It is believed that he heated soap and
orpiment together and isolated elemental arsenic. Arsenopyrite (FeAsS) is the most
common mineral from which, the arsenic sublimes leaving ferrous sulfide on heating.

The terrestrial abundance of arsenic is about 5 g/ton being found widely

dispersed in nature. Some samples of arsenic have been found which vary in purity
from about 90 to 98 %. The commonly associated impurities encountered in these
samples are antimony, bismuth, iron, nickel and sulfur. Normally arsenic is found in
nature, combined as sulfides, arsenides, sulfoarsenides, arsenites and occasionally as
oxide and oxychloride. The most commonly encountered minerals of arsenic are

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arsenopyrite (FeAsS), loellingite (FeAs2), enargite (C11S.AS2S5), orpiments (AS2S3)
and realgar (AS2S3).

Arsenic occurs in nature in inorganic as well as organic forms. Erosion of

arsenic containing surface rocks probably accounts for a significant amount of arsenic
in water supplies [2]. The other major sources of environmental arsenic are the
smelting of nonferrous metal ores, especially copper. Arsenic is an essential nutrient
and is a constituent of many food such as meat, fish, poultry grains and cereals [2]. In
excessive amounts, arsenic causes gastrointestinal damage and cardiac damage.
Chronic doses can cause vascular disorders such as blackfoot disease [2]. Arsenic and
its compound are reported to be carcinogenic, mutagenic and teratogenic in nature.
1

The maximum permissible limit for arsenic in water is 0.05 mgL‘ as recommended
by WHO [2]. The threshold limit proposed by ACGIH for arsenic in air is
0.5 mgm*3 [3].

The most infamous use of arsenic is as a poison. However, arsenic can now
be detected during autopsy, so this use of the element has become a legend of the
past. These days the most important use of arsenic is in the preservation of wood. It
is used in the form of a compound called chromated copper arsenate (CCA) and is
added to wood used to build houses and other wooden structures. CCA prevents
organisms from growing in the wood and causing it to rot. Arsenic is also used as a
weed killer and rat poison. Arsenic has been used to improve the roundness of lead
shot. Trace amounts of arsenic are alloyed with lead in storage batteries. Arsenic is
also used in the manufacture of high efficiency solar cells. Alloys of gallium, arsenic
and phosphorous are used in the semiconductor industry for the production of light-
emitting diodes (LEDs) in watches, clocks, calculators and numerous other instrument
displays.

Arsenic has significant medicinal properties and it has been used as a

therapeutic agent for more than 2,400 years [4]. In the 15th century, William
Withering, who discovered digitalis, was a strong proponent of arsenic-based
therapies. He argued, "Poisons in small doses are the best medicines and the best
medicines in too large doses are poisonous" [5]. In the 18th century, Thomas Fowler
compounded a potassium bicarbonate based solution of arsenic trioxide (AS2O3) that

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would bear his name. Fowler's solution was used empirically to treat a variety of
diseases during the 18th, 19th and early 20th centuries [6]. Pharmacology texts of the
1880’s describe the use of arsenical pastes for cancers of the skin and breast and
arsenous acid was used to treat hypertension, bleeding gastric ulcers, heartburn and
chronic rheumatism [5]. Arsenic's reputation as a therapeutic agent was enhanced in
1910 when Nobel laureate Paul Ehrlich developed salvarsan, an organic arsenial for
treating syphilis and trypanosomiasis. However, as medicine evolved in the
20th century, enthusiasm for medicinal arsenic waned rapidly [5].

The concentration of arsenic in plants can vary depending on factors like the
species of plant, the type of arsenic in the soil, and the location of a given species.
Arsenic can exist in a variety of oxidation states in inorganic and organic forms in
many environmental matrices such as natural water and soils [7]. Therefore precise
knowledge of the arsenic compounds present in a system is required for an accurate
assessment of the environmental and biological impact of arsenic, which has resulted
in an increasing need of analytical method for their determination at micro trace or
even ultra trace levels.

6.2 ANALYTICAL CHEMISTRY

Various methods for the analysis of arsenic have been reported in the
literature. Many analytical techniques based on flow injection analysis with hydride
generation [8], atomic absorption spectroscopy [9], gas fluorometry-atomic absorption
spectroscopy [10], induced coupled plasma-atomic absorption spectroscopy [11],
neutron activation analysis [12] and fluorescence spectroscopy [13] are used for the
arsenic determination.

A literature survey revealed that a large number of reagents are suitable for the
spectrophotometric determination of arsenic. Cristau reported a spectrophotometric
determination of arsenic using hypophosphorus acid [14]. The method was influenced
of the polyvinylpyrrolidone and stannous chloride. Powers et al. reported silver
diethylthiocarbamate as a spectrophotometric reagent for the determination of arsenic
[15]. This method was based on the reduction of arsenic by zinc and generated arsine
was absorbed in silver diethylthiocarbamate. Molybdenum blue was also used as a

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spectrophotometric reagent for the determination of arsenic in tungstun-free
steels [16].

Takashi and Kazuo reported quercetin as a reagent for the spectrophotometric

determination of arsenic in alloys [17]. Beer's law was obeyed in the range 0-20 pg of
arsenic in 50 mL and exhibited an absorption maximum at 398 nm.

Steckel and Hall described silver diethyldithiocarbamate as a reagent for the

determination of trace quantities of arsenic [18]. Generated hydride reacted with
As(III) formed a stable red As(III)-silver diethyldithiocarbamate complex and the
absorbance was measured at 540 nm. Beer's law was obeyed for 0-18 pg of arsenic.
Takashi and Kazuo used rutin as a reagent for the spectrophotometric determination
of arsenic [19] and Kazuo et al. reported a spectrophotometric determination of
arsenic with morin [20].

Pakalns reported a method for the spectrophotometric determination of arsenic

in a wide variety of salts, white metals, Cu alloys and all types of steels [21]. The
method involved the extraction of the yellow molybdoarsenic acid with BuOH and
subsequent reduction to the blue colored complex. Stara and Stary described
8-mercaptoquinoline as a reagent for the determination of arsenic [22].
8-Mercaptoquinoline reacted with arsenic formed a complex exhibited an absorption
maximum at 380 nm. Only Sn(II) interfered with the determination.

Gerald and Bruno described a method for the determination of submicro

amounts of arsenic [23]. The method was based on the reduction of silver and
iron(III) ions by arsine in the presence of ferrozine. Beer’s law was obeyed in range
0.1-1.5 pg of arsenic in 5 mL of solution. Haywood and Riley described a
spectrophotometric determination of arsenic in sea water, potable water and effluents
[24].

Silvia and Victoria described the main sources of arsenic emission in Romania
are ore smelters and refineries [25]. Arsenic determinations were carried out by the
silver diethyldithiocarbamate spectrophotometric method on hair and urine samples
taken from smelter workers and individuals residing in two polluted areas and three

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areas not polluted by arsenic. Arsenic in hair was found to be a more reliable
biological test than tests on urine, obviously reflecting the differences in arsenic
concentrations in workroom air. Arsenic analysis of hair on people living in two
locations near an ore smelter and a refinery indicated high levels compared to those of
individuals residing in nonpolluted areas. Epidemiological studies were necessary in
order to ascertain effects of heavy arsenic exposure in relation with concurrent
exposures to respiratory irritants and metals.

Takatomi et al. reported bismuthiol-II as a reagent for the spectrophotometric

determination of arsenic [26]. Arsenic(V) reacted with bismuthiol-II which formed a
light yellow precipitate in HC1 solution (> 2 M), which was extracted into chloroform.
Arsenic was determined by measuring the absorbance of the CHCI3 extracted at
335 nm. Beer's law was obeyed in the range of 0-50 pg of arsenic in 10 mL. The
molar absorptivity value of the system was 1.62x104 Lmolcm'1.

Gowda and Thimmaiah reported promazine hydrochloride as a reagent for the

spectrophotometric determination of arsenic(III), cerium(IV) and nitrite [27]. The
reagent fonned a red-colored radical instantaneously in 0.5-2 M sulfuric acid or
0.5-2.5 M phosphoric acid solution. The red radical exhibited maximum absorbance
at 505 nm. Beer's law was valid over the concentration range of 0.5-15 ppm in
sulfuric acid and 0.5-21 ppm in phosphoric acid. The sensitivities of the reaction in
sulfuric and phosphoric acid media were 0.022 and 0.019 pgcm'2 respectively. The
effects of acidity, time, temperature, reagent concentration, and diverse ions were
reported. Arsenic(III) and nitrite were indirectly determined. The proposed method
offered the advantages of good sensitivity, simplicity, rapidity, selectivity and a wider
range of determination without the need for extraction. Hiroshi and Kamihiko
described determination of arsenic using cinchonidine by spectrophotometric method
[28].

Agrawal and Patke reported a simple, sensitive and selective method for the
determination of microamounts of arsenic(III) in the environment [29]. Arsenic
formed a yellow colored complex with N-phenylbenzo-hydroxamic acid (PBHA) at
pH 4.5-5.2 which was extracted into chloroform. The effective molar absorptivity of

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As-PBHA extract was 1.1 * 104 LmoF’cm'1 at 410 nm. Many common ions associated
with arsenic did not interfere. The effect of pH, reagent concentration and solvent was
described. The arsenic in trace quantities were estimated in the industrial effluents,
soil and grass samples.

Merry and Zarcinas reported sodium tetrahydroborate(III) reagent for the

hydride generation in the spectrophotometric determination of arsenic and antimony
by the silver diethyldithiocarbamate method [30]. The use of oxidising acids for the
digestion of sediment, soil and plant material for arsenic and other metals was not
suitable for antimony. This was overcome by the addition of a reducing agent in the
later stages of digestion which allowed the determination of both arsenic and
antimony simultaneously.

Howard and Arbab-Zavar described the silver diethyldithiocarbamate

spectrophotometric procedure for the determination of arsenic to perform the
differential determination of inorganic arsenic(III) and arsenic(V) species [31]. The
method was based on pH control of the reduction characteristics of the borohydride
ion. Arsine was generated at pH 6 from arsenic(III) following acidification, arsine
was then generated and trapped from arsenic(V) species. The procedure was
appropriate to the determination of 2-40 pg of each arsenic species. Antimony(III),
bismuth(III), chromium(VI), copper(II), gold(III), nickel(II), platinum(IV), silver(I),
tellurium(IV) and tin(II) interfered in the determination of arsenic(III) and
methylarsenic and dimethylarsenic species, platinum(IV) and silver(I) interfered in
the determination of arsenic(V). Interference effected due to platinum was masked by
the addition of 1,10-phenanthroline to the reductant mixture. Interference due to
bismuth, copper, gold, nickel, silver, tellurium and tin was overcome by the
preliminary extraction of the interferents from the sample as their dithizonates.
Elzbieta and Wieckowska used rhodamine-6G as a reagent for the spectrophotometric
determination of arsenic [32].

Ali Naki et al. described a method for the spectrophotometric determination of

arsenic using 2-mereaptoethanol as a reagent [33]. Qian-Feng and Peng-Fei reported a
highly sensitive spectrophotometric method for determination of arsenic based on the
formation of an ion-association complex between arsenoantimonomolybdenum blue

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and malachite green [34]. The ion-association complex was soluble in the presence of
triton X-305 and arsenic was determined directly in aqueous solution. The molar
absorptivity was found to be 1.13xl05 Lmor'cm”1 at 640 nm. Beer's law was obeyed

for 0-5 pg of arsenic. The lower limit of determination (absorbance = 0.01) was 4
ngmL'1 in the final solution.

Tianze and Ming described a simple and sensitive procedure for the
spectrophotometric determination of traces of arsenic [35]. In this method arsine
generated at pH 5.3 reacted with silver acetate in the aqueous solution in the presence
of tween-80, which formed a yellow silver sol with an absorption maximum at 420
nm. The molar absorptivity was found to be 4.8x104 Lmof’cm"1. Beer's law was

obeyed in the range 0.3-5.0 pg of arsenic in 20 mL solution. This method was used in
the determination of arsenic in water and waste water samples.

Maher presented a procedure for the spectrophotometric determination of

arsenic in environmental extracts [36]. In this method arsenic was converted into
arsine using a zinc reductor column, the evolved arsine trapped in a potassium iodide
- iodine solution and the arsenic determined spectrophotometrically as an
arsenomolybdenum blue colored complex. The detection limit was 0.024 pg and the
coefficient of variation was 5.1% at the 0.1 pg level. The method was free from
interferences by other elements at levels normally found in environmental samples.

Kavlentis developed a spectrophotometric determination of arsenic(III) and

antimony(III) using isonicotinoylhydrazones of 4-dimethylaminobezaldehyde
(4-DBIH) and 2-hydroxynaphthaldehyde (2-HNIH) [37]. In this method 4-DBIH and
2-HNIH reacted with As(III) and Sb(III) respectively in CH3COOH medium which
formed colored complexes stable in presence of EDTA. As(IlI) and Sb(III) did not
react with 2-HNIH and 4-DBIH. The Sb(III)-2-HNIH complex was extracted into
isoamyl alcohol.

Lata et al. reported a reducing agent succinyldihydroxamic acid for the

extractive spectrophotometric determination of arsenic in polluted water and
environmental samples [38]. This method was based on the reduction of yellow
molybdoarsenic heteropoly acid with succinyldihydroxamic acid into molybdoarsenic

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blue. The blue colored dye exhibited an absorption maximum at 780 nm in n-butanol.
Beer's law was obeyed in the range of 0.02-0.14 ppm of arsenic.

Palanivelu et al. reported a chemical enhancement method for the

spectrophotometric determination of trace amounts of arsenic [39]. Jiayu et al.
reported a spectrophotometric method for the determination of arsenic using
dithioantipyrylmethane (DTPM) as a reagent [40], The method was based on the
reaction of arsenic with DTPM, which formed 1:2 arsenic-DTPM complex in acidic
medium and the complex was measured at 336 nm with molar absorptivity 3.05x104
Lmof’em"1. Beer’s law was obeyed in the concentration range of 0-30 pg of arsenic.

Dianwen and Jianping reported methyl orange as a spectrophotometric reagent

for the determination of arsenic [41]. In 0.18-1.08 M H2SO4 medium, As(III) was
oxidized by KBr03 in the presence of KBr and methyl orange was bleached by the
excessive KBrC>3, and the decrease in color was inversely proportional to the amounts
of arsenic and was used for the determination of arsenic in sludge. The absorption
maximum of methyl orange was 510 nm. The molar absorptivity value of the system
was 4.81><104 Lmof’cm'1.

Yubiao reported gibberellin as a reagent for the spectrophotometric

determination of arsenic [42]. The method was systematically studied on arsenium
molybdenum acid reduction in 0.84 M HC1 medium, the As-Mo heteropoly acid was
reduced into very stable As-Mo heteropoly blue by gibberellin. The maximum
absorption wavelength was 838 nm. The molar absorptivity of the system was
2.64xl04 Lmof’cm’. Beer’s law was valid over the concentration range 0-1.20
mgmL'1 of arsenic.

Liu et al. used chlorpromazine reagent for the sensitive spectrophotometric

determination of arsenic [43] The method was based on the formation of heteropoly
arsenomolybdic chlorpromazine complex in aqueous phase exhibited maximum
absorption at 320 and 350 nm respectively.

Pillai et al. described a sensitive method for the determination of traces of

arsenic(III) [44]. The method involved bleaching of the pinkish red colored dye,

155
rhodamine B, by the action of iodine which was released by the reaction between
potassium iodate and arsenic in slightly acidic medium. The color of the dye was
measured at 553 nm. Beer’s law was obeyed in the concentration range of
0.04-0.4 mgL"1 of arsenic. The molar absorptivity was found to be
3.24*105 LmoF'cm-1. The proposed method was successfully applied for the
determination of arsenic in environmental and biological samples. Gudzenko et al.
described a spectrophotometric determination of trace amounts of arsenic using
michler's thioketone as a reagent [45]. The molar absorptivity value was
1.02*105 Lmof'cm'1 at 640 nm. Beer's law was obeyed in the concentration range
0.008-0.12 mgL"1 of arsenic.

Abd El-Hafeez and El-Syed described a simple, rapid and selective procedure
for the indirect spectrophotometric determination of arsenic(V) [46]. The method was
based on the reduction of As(V) to As(III) with hydroiodic acid (KI + HC1). The
liberated iodine, equivalent to each analyte which was quantitatively extracted with
oleic acid surfactant. The iodine-HOL system exhibited maximum absorbance at
435 nm. The calibration graphs were found to be linear over the range 0.25-20 pgmL'1
of As(V) with lower detection limits 0.15 pgmL"1. The molar absorptivity and
Sandell’s sensitivity were found to be 0.5X104 Lmof’cm-1 and 0.0149 pgem'2
respectively. The relative standard deviation for five replicate analyses of 4 pgmL'1 of
arsenic was 0.9%. The proposed procedure in the presence of EDTA as a masking
agent for foreign ions was successfully applied to the determination of arsenic in
copper metal, in addition to their determination in spiked and polluted water samples.

Kundu et al. reported a spectrophotometric method for the determination of

arsenic in ppm level. The method was based on the color bleaching of methylene blue
in anionic micellar medium [47]. Arsine gas was formed by borohydride reduction of
arsenite/arsenate. Arsine generation and color bleaching (quantification of arsenic)
was done in one-pot. The presence of silver or gold nanoparticles made the
determination faster. Different calibration graphs at the three different ranges of
arsenic concentration such as 0-8.63, 0-1.11 and 0-0.11 ppm were constructed and
limit of detection (LODs) were found to be 1.3, 0.53 and 0.03 ppm respectively. The
method was simple, rapid, reproducible (relative standard deviations lies within ±5%)
and eco-friendly. The method was free from phosphate and silicate interferences and

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applied for real sample analysis. Sano et al. reported ammonium
pyrrolidinedithiocarbamate as spectrophotometric reagent for the determination of
arsenic [48].

Taniai et al. described an automated on-line solvent extraction system for the
determination of arsenic or tin in steel by electrothermal atomic absorption
spectrometry (ET-AAS) [49]. The method was based on the formation of ASI3 and
SnLj in concentrated hydrochloric acid and sulfuric acid media respectively. They are
extracted into benzene and back extracted into water and 0.25 M sulfuric acid,
respectively. An improved gravity phase separator was developed for the recycling of
organic solvent used in the automated on-line solvent extraction system. Using the
proposed system, arsenic or tin contained in the acid dissolved steel sample solution
was automatically extracted and back-extracted. Then, the back-extracted solutions
were used for the determination of arsenic or tin by ET-AAS. In the determination of
arsenic, 800 mgL'1 of cobalt solution had to be used as the matrix modifier to exclude
the effect of coexisting substances such as iodide ion. In the determination of tin,
1000 mgL'1 of palladium solution had to be used in the same manner. By this method,
detection limits of As and Sn were 0.2 jig of As in the 0.1 g of Fe and 0.1 jig of Sn in
the 0.05 gofFe.

Cherian and Narayana reported a spectrophotometric method for the

determination of arsenic in environmental and biological samples using azur B as a
chromogenic reagent [50], The method was based on the reaction of arsenic(III) with
potassium iodate in acid medium to liberate iodine. Bleaching of the violet color of
azur B by the liberated iodine was the basis of the determination and was measured at
644 nm. Beer’s law was obeyed in the range 0.2-10 pgmL'1 of As(III). The molar
absorptivity, Sandell’s sensitivity, detection limit and quantitation limit of the method
were found to be 1.12xl04 Lmol ’cm'1, 6.71xl0'3 jigcm'2, 0.02 figmL'1 and 0.08
jigmL'1 respectively.

Behpour et al. described a simple, sensitive, rapid and reliable

preconcentration method for the spectrophotometric determination of trace amounts
of arsenic [51]. Arsenic was retained on a minicolumn of adsorbent naphthalene, as an

157
ion associate of arsenomolybdate and methyltrioctylammonium ions. The contents of
column was dissolved in a small volume of N,N-dimethylformamide having stannous
chloride as a solvent. The absorbance was measured at 715 nm at room temperature.
The method allowed determination of arsenic in the range of 1-8 ngmL'1 in the initial
solution with r=0.999 (n=6). The relative standard deviation for 15 replicate
measurements of 6.0 ngmL'1 of arsenic was 1.3 % and the detection limit was 0.067
ngmL'1. The preconcentration factors of 100 and 167 could be achieved when using a
5 and 3 mL DMF for dissolving adsorbent respectively. The optimized method was
successfully applied to determination of arsenic in natural water, synthetic sample and
fish.

Narayana et al. described a spectrophotometric determination of arsenic using

variamine blue reagent [52]. The method was based on the reaction of arsenic(III)
with potassium iodate in acid medium which liberated iodine, oxidized variamine blue
to a violet colored species having an absorption maximum at 556 nm. Beer’s law was
obeyed in the range 0.2-14 jxgmL'1 of As(III) in a final volume of 10 mL. The molar
absorptivity and Sanded’s sensitivity for the colored system were found to be
1.43xl04 Lmof'cm"1 and 5.26x1 O'2 pg cm'2 respectively.

Graham described a spectrophotometric determination of arsenic in solutions

containing nitric acid necessitates the removal of nitrate ions without the loss of
arsenic [53], A convenient and effective method for its removal was achieved by
treatment with formic acid.

Kunihiro et al. reported a spectrophotometric determination of arsenic in steels

by flow injection analysis using a teflon (PTFE) filter tube concentration method [54].
In this method arsenic coprecipitated with beryllium hydroxide at pH 10, which was
collected with a filter tube for 5 minutes. The precipitate was eluted with 1 M nitric
acid at 0.6 mL per minute and the eluate was reacted with ammonium molybdate.
The molybdoarsenate complex was reduced with ascorbic acid. The obtained
molybdenum blue complex was determined by spectrophotometry at 840 nm. The
calibration curve for arsenic was linear over the range of 0 to 100 ppb. The limits of
detection and determination for arsenic were 0.7 and 2 ppb respectively. The relative

158
standard deviation for 20 ppb of arsenic was 1.3% (n=8). The iron as a matrix did not
interfere with the determination of arsenic up to a million-fold to arsenic amount.

Keisuke and Emiko described a simple and sensitive spectrophotometric

method for the determination of arsenic in water samples [55]. The method was
based on the formation of micro particles of ethyl violet and molybdoarsenate, which
gave an apparently homogeneous blue color to the solution. The absorption of the
excess dye gradually decreased due to its conversion to a colorless carbinol species
under strongly acidic conditions. Consequently, the sufficiently low reagent blank
enables the spectrophotometric determination of arsenic with the detection limit of 4
pgL'1. The coefficient of variation for the spectrophotometry at 50 pgL'1 was
3.5% (n = 5).

Revanasiddappa et al. developed a sensitive spectrophotometric method for

the determination of arsenic in environmental samples [56]. Hashemi and Modasser
described a simple spectrophotometric method for the sequential determination of
inorganic arsenic species in a sample [57]. The method was based on the sequential
arsine generation from As(III) and As(V) using selective medium reactions, collection
of the arsine generated in an absorbing solution containing permanganate and ethanol
at 5°C and subsequent reduction of permanganate by arsine. The decrease in
permanganate absorbance at 524.2 nm was monitored for arsenic determination. The
acetic acid/sodium acetate and HC1 media were used for selective arsine generation
from As(III) and remaining As(V) in one solution, respectively. The effect of
interferences and their possible mechanisms were discussed. Interferences from
transition metal ions were removed by using a Chelex 100 resin. Under optimized
conditions, the established method was applicable to the determination of 3-30 pg of
each arsenic species. Good recoveries (96-102%) of spiked artificial sea water, tap
water and standard mixtures of As(III) and As(V) were also found. However, most of
these methods suffer from certain limitations such as; interference by a large number
of ions, low sensitivity and need extraction into organic solvents or heating. Thus
there is need to develop an entirely new method, which would overcome the existing
inadequacies in the determination of arsenic.

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 The aim of the present work described in this chapter is to provide a simple
and sensitive method for the determination of arsenic using toluidine blue and
safranine O as new reagents. The proposed method has been successfully applied for
the determination of arsenic in various environmental and biological samples.

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6.3 APPARATUS

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

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

6.4 REAGENTS AND SOLUTIONS

All chemicals were of analytical reagent grade or chemically pure grade and
distilled water was used throughout the study. Arsenic(III) stock solution
(1000 pgmL'1) was prepared by dissolving 0.1734 g of NaAs02 in 100 mL of water.
Working standard was prepared by appropriate dilution of stock. Toluidine blue
(0.01 %), saffanine O (0.02 %), hydrochloric acid (1 M), potassium iodate (2 %) and
sodium acetate (1 M) were used.

6.5 PROCEDURES

6.5.1 Using Toluidine Blue as a Reagent

Aliquots of sample solution containing 1.2-10.5 pgmL'1 of arsenic(III) were
transferred in to a series of 10 mL calibrated flasks. Potassium iodate (2 %, 1 mL)
then hydrochloric acid (1 M, 1 mL) were added and mixture was gently shaken until
the appearance of yellow color indicating the liberation of iodine. This was followed
by addition of toluidine blue (0.01 %, 0.5 mL) and 2 mL of sodium acetate solution.
The solution was kept for 2 minutes and made up to the mark with distilled water. The
absorbance was measured at 628 nm against the corresponding reagent blank. Reagent
blank was prepared by replacing the analyte (arsenic) solution with distilled water.
The absorbance corresponding to the bleached color, which in turn corresponds to the
analyte (arsenic) concentration, was obtained by subtracting the absorbance of the
blank solution from that of the test solution. The amount of the arsenic present in the
volume taken was computed from the calibration graph (Figure VIA2).

6.5.2 Using Safranine O as a Reagent

Aliquots of sample solution containing 0.4-11.5 pgmL'1 of arsenic(III) were
transferred in to a series of 10 mL calibrated flasks. Potassium iodate (2 %, 1 mL)

161
then hydrochloric acid (1 M, 1 mL) were added and mixture was gently shaken until
the appearance of yellow color indicating the liberation of iodine. This was followed
by addition of safranine O (0.02 %, 0.5 mL) and 2 mL of sodium acetate solution.
The solution was kept for 2 minutes and made up to the mark with distilled water. The
absorbance was measured at 532 ran against the corresponding reagent blank. Reagent
blank was prepared by replacing the analyte (arsenic) solution with distilled water.
The absorbance corresponding to the bleached color, which in turn corresponds to the
analyte (arsenic) concentration, was obtained by subtracting the absorbance of the
blank solution from that of the test solution. The amount of the arsenic present in the
volume taken was computed from the calibration graph (Figure VIA3).

6.5.3 Determination of Arsenic in Polluted Water Samples

Water samples from a river receiving effluent of steel plant and fertilizer
factory were collected in polyethylene bottles, which was filtered through whatman
41 filter paper. A few drops of 10 % KI was added to convert any arsenic(V) to
arsenic(III). Arsenic content was determined directly according to the proposed
method and also by the reference method [42].

6.5.4 Determination of Arsenic in Soil Samples

A known weight (1.0012 g) of a soil sludge sample was placed in a 50 mL
beaker and extracted 4 times with a 5 mL portion of concentrated HC1. The extract
was boiled for about 30 minutes. Arsenic(V) if any was reduced to As(III) by the
process described above. The solution was cooled and diluted to 25 mL volumetric
flask with distilled water. Suitable aliquot of the sample was analyzed by the proposed
method and also by the reference method [42].

6.5.5 Determination of Arsenic in Plant Material

A sample of plant material (grass-5g) was digested with 10 mL of HNO3 for
about 25 minutes. After cooling, 1 mL of perchloric acid was added and heating was
continued for about another 10 minutes. arsenic(V) if any was reduced to As(III) by
the process described. The solution was transferred to a 25 mL volumetric flask and
diluted to volume with water. Suitable aliquot of the sample was analyzed by the
proposed method and also by the reference method [42]. The results are listed in
Table 6A2.

162
6.6 RESULTS AND DISCUSSION

6.6.1 Absorption Spectra

6.6.1.1 Using toluidine blue as a reagent
This method involves the liberation of iodine by the reaction of arsenic(III)
with potassium iodate in an acidic medium. The liberated iodine bleaches the blue
color of toluidine blue and measured at 628 nm. This decrease in absorbance is
directly proportional to the As(III) concentration. The absorption spectra of colored
species of toluidine blue are presented in Figure VIA1 and reaction system is
presented in Scheme VI.

6.6.1.2 Using safranine O as a reagent

In this method is also involves the liberation of iodine by the reaction of
arsenic(III) with potassium iodate in an acidic medium. The liberated iodine bleaches
the pinkish red color of safranine O and measured at 532 nm. This decrease in
absorbance is directly proportional to the As(III) concentration. The absorption
spectra of colored species of safranine O are presented in Figure VIA1 and reaction
system is presented in Scheme VI.

6.6.2 Effect of Iodide Concentration and Acidity

The effect of iodide concentration and acidity on the decolorization is studied
with 2 pgmL'1 of arsenic solution. The oxidation of iodate to iodine was effective in
the pH range 1.0 to 1.5, which could be maintained by adding 1 mL of 1 M HC1 in a
final volume of 10 mL. The liberation of iodine from potassium iodate in an acidic
medium is quantitative. The appearance of yellow color indicates the liberation of
iodine. Although any excess of iodate in the solution will not interfere. It is found that
1 mL of 2 % potassium iodate and 1 mL of 1 M HC1 are sufficient for the liberation of
iodine from iodate by arsenic and 0.5 mL of 0.01 % toluidine blue or 0.02 % safranine
O was used for subsequent decolorization.

Constant and maximum absorbance values are obtained in the pH=4±0.2.

Hence the pH of the reaction system is maintained at 4±0.2 throughout the study. This

163
could be achieved by the addition of 2 mL of 1 M sodium acetate solution in a total
volume of 10 mL. Effect of pH on color stability is presented in Figure VIA4.

6.6.3 Analytical Data

6.6.3.1 Using toluidine blue as a reagent
The adherence to Beer’s law is studied by measuring the absorbance values of
solutions varying arsenic concentration. A straight line graph is obtained by plotting
absorbance against concentration of arsenic. Beer’s law is obeyed in the range of
1.2-10.5 pgmL"1 of arsenic. The molar obsorptivity and Sandell’s sensitivity of the
system is found to be 1.076xl04 Lmof’cmf1 and 9.66xl0'3 pgcm'2 respectively.
Correlation coefficient (n = 10) and slope of the calibration curve are 0.9996 and
0.107 respectively. The detection limit (Dl=3.3 cr/s) and quantitation limit
(Ql=10 a/s) [where a is the standard deviation of the reagent blank (n=5) and s is the
slope of the calibration curve] of arsenic determination are found to be 0.308 pgmL1
and 0.934 pgmL1 respectively. Adherence to Beer’s law graph for the determination
of arsenic using toluidine blue is presented in Figure VIA1.

6.6.3.2 Using safranine O as a reagent

The adherence to Beer’s law is studied by measuring the absorbance values of
solutions varying arsenic concentration. A straight line graph is obtained by plotting
/ *
absorbance against concentration of arsenic. Beer’s law is obeyed in the range of
0.4-11.5 pgmL'1 of arsenic. The molar obsorptivity and Sandell’s sensitivity of the
system is found to be 1.388xl04 Lmof’cm'1, 7.490xl0'3 pgcm*2 respectively.
Correlation coefficient (n=10) and slope of the calibration curve are 0.9998 and 0.132
respectively. The detection limit (DL=3.3 a/s) and quantitation limit (QL=10 a/s)
[where a is the standard deviation of the reagent blank (n=5) and s is the slope of the
calibration- curve] for arsenic determination are found to be 0.250 pgmL'1 and
0.759 pgmL'1 respectively. Adherence to Beer’s law graph for the determination of
arsenic using safranine O is presented in Figure VIA2.

6.6.4 Effect of Divers Ions

The effect of various foreign ions at microgram levels on the determination of
arsenic using toluidine blue and safranine O is studied. The tolerance limits of

164
interfering species were established at those concentrations that do not cause more
than ±2.0% error in absorbance values for arsenic(III) at 2 pgmL'1, The tolerance
limits of foreign ions are listed in Table 6A1. The results indicated that most of the
common ions did not interfere. Urea, uric acid, glucose, citrate, tartarate and anions
like sulphate and phosphate did not interfere.

6.7 APPLICATIONS

The proposed method is applied to the quantitative determination of arsenic in

various environmental and biological samples. The results of the analysis are
presented in Table 6A2, compare favorably with those from a reference method [42].
The precision and accuracy of the proposed is evaluated by replicate analysis of
samples containing arsenic at three different concentrations.

6.8 CONCLUSIONS

1. The new reagents provide a simple, rapid, sensitive and highly specific method for
the spectrophotometric determination of arsenic.
2. The reagents have the advantage of high sensitivity and selectivity.
3. Proposed method has less interference from the common metal ions and anions.
4. The developed method does not involve any stringent reaction conditions and
offers the advantages of high stability of the reaction system (more than 8 hours).
5. The proposed method has been successfully applied for the determination of
arsenic in various environmental and biological samples. A comparison of the
method reported is made with earlier methods and is given in Table 6A3.

165
FIGURE VIA1
ABSORPTION SPECTRA OF COLORED SPECIES OF TOLUIDINE BLUE (a)
AND SAFRANINE O (b)
Absorbance
o
CO
o
CD

0.4 -

0.2 -

0.0 —I—

200 300 400 500 600 700 800 900

Wavelength (nm)

FIGURE VIA2
ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF ARSENIC
USING TOLUIDINE BLUE AS A REAGENT
00
o
Absorbance

o>
o
1
o

0.0 --
10 12 14
1
Concentration of arsenic (pgmL )
- \

166
FIGURE VIA3
ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF ARSENIC
USING SAFRANINE O AS A REAGENT
— —
CM

5
Absorbance
CO

Concentration of arsenic (pgmL'1)

FIGURE VIA4
EFFECT OF pH ON COLOR INTENSITY

1.4

1.2

1.0 -
Absorbance

oo
O
— 1------------------ !—
©
co
d
o
CM
d
o

h
*
Q,

167
SCHEME VI

SCHEME OF THE REACTIONS

2 AsO" + 2 IO~3 + 2 H ---------- l2+ 2 AsO'3 + 4 H20

N^r/<^CH3 '2■ H+

(CH3)2N" (ch3)2n

Toluidine Blue (Colored) Toluidine Blue (Colorless)

h3c 3
<2 • H

h2n nh2 nh2

ri r^i

Safranine O (Colored) Safranine O (Colorless)

168
TABLE 6A1
EFFECT OF DIVERSE IONS ON THE DETERMINATION OF ARSENIC
(2 ngmL'1)

Foreign ions Tolerance limit (figmL'1) Foreign ions Tolerance limit (jj-gmL'1)
Toluidine blue Safranine 0 Toluidine blue Safranine O
Fe3+ 150 100 V5" 125 100
Ni2+ 75 100 Zn2+ 1000 1000
Cd2+ 500 600 P043' 1000 1000
Ba2+ 1000 750 Tartarate 1250 1000
Bi3+ 1000 1250 Oxalate 1000 1250
Al3+ 500 400 Sulfate 750 1000
Ca2+ 400 300 Nitrate 750 1000
Co2+ 175 150 Glucose 1000 1200

169
TABLE 6A2
DETERMINATION OF ARSENIC IN ENVIRONMENTAL SAMPLES USING
TOLUIDINE BLUE AND SAFRANINE O AS REAGENTS

Toluidine blue Saffanine O

Samples As3+ added As3+ found Recovery RSD As 3+ found Recovery RSD
(pgmL') (pgmL’) (%) (%) (pgmL ) (%) (%)

a Ground Water 2.00 2.01 100.50 1.99 1.98 99.00 2.02

Samples 4.00 3.96 99.00 3.03 3.98 99.50 1.51
6.00 5.99 99.83 0.84 5.96 99.33 0.67
8.00 7.92 99.00 0.25 7.95 99.38 0.88

aTap Water 2.00 1.94 97.00 1.03 1.96 98.00 3.57

Samples 4.00 3.98 99.50 3.50 3.97 99.25 1.51
6.00 5.94 99.00 0.50 5.96 99.33 0.16
8.00 7.96 99.50 0.75 7.98 99.75 1.25
a Industrial Water 2.00 1.99 99.50 1.51 1.96 98.00 0.76
Samples 4.00 3.97 99.25 1.51 3.95 98.75 0.51
(Collected from the Indus- 6.00 5.96 99.33 1.34 5.94 99.00 1.34
trial zone of Mangalore city) 8.00 7.95 99.37 1.76 7.97 99.63 0.75

aRiver Water 2.00 1.96 98.00 1.67 2.00 100.00 2.62

Samples 4.00 3.95 98.75 1.40 3.95 98.75 1.66
6.00 5.86 98.00 0.72 5.94 99.00 0.57
8.00 7.89 98.62 0.89 7.96 98.50 0.66

aSoil Samples 2.00 1.98 99.00 1.20 2.00 100.00 1.81

4.00 4.01 100.25 1.31 3.96 99.00 0.37
6.00 5.94 99.00 0.27 5.99 99.83 0.96
8.00 7.96 99.50 0.32 7.95 99.37 0.97
aPlant Material 2.00 2.01 100.50 1.32 1.98 99.00 1.00
(Grass Samples) 4.00 3.96 99.00 0.60 3.96 99.00 0.80
6.00 6.01 100.17 0.36 5.95 100.17 0.86
8.00 7.96 99.50 0.55 7.92 99.00 0.89

a. Arsenic was not detected.

170
TABLE 6A3
COMPARISON OF THE METHOD REPORTED WITH EARLIER METHODS

Reagent Method Beer’s law s (Lmor’cnT1) ^-max Ref.

(pgmL'1) ss (pgcm'2) (nm) No.
Tween-80 Spectrophotometry 0.3-5.0 pg e = 4.80x104 420 35

Rhodamine B Spectrophotometry 0.04-0.4 e = 3.24xl05 553 44

mgL'1
Oleic acid Spectrophotometry 0.25-20 8 = 0.50x104 435 46
ss= 1.49x1 O'2
Azur B Spectrophotometry 0.2-10 E= 1.12xl04 644 50
ss = 6.71 xlO'3
Variamine blue Spectrophotometry 0.2-14 £ = 1.43 x 104 556 52
ss - 5.26xl0'2
Proposed Method

Toluidine blue Spectrophotometry 1.2-10.5 s = 1.076xl04 628

ss = 9.66x10'3
Safranine 0 Spectrophotometry 0.4-11.5 s= 1.388xl04 532
ss = 7.490x10'3

s = Molar absorptivity, ss = Sandell’s sensitivity

171
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