158

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

159

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

160

arsenopyrite (FeAsS), loellingite (FeAs2), enargite (CuS.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.

The maximum permissible limit for arsenic in water is 0.05 mgL-1 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

161

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], inductively 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

162

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-���������

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-���������������

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.

Kellen and Jaselskis 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-��� ��� ��� ������� ��� �� ��� ��� ��������� 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

163

diethyldithiocarbamate spectrophotometric method on hair and urine samples taken

from smelter workers and individuals residing in two polluted areas and three 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 HCl solution (> 2 M), which was extracted into chloroform.

Arsenic was determined by measuring the absorbance of the CHCl3 extracted at

335 nm. Beer's law was obeyed in the range of 0-��� ��� ��� ������� ��� ����������

molar absorptivity value of the system was 1.62×104 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 formed a red-colored radical instantaneously in 0.5��� M sulfuric acid or

0.5��������������������������������������������������� �������������� �� �����

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

�������� �������������� �����������!���������� �������"�����-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

164

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

As-PBHA extract was 1.1×104 Lmol-1cm-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 µg 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].

165

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

arsenic using 2-mercaptoethanol 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

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 1.13×105 Lmol�cm� at 640 nm. Beer's law was obeyed

for 0–�������� ������������ ��!��� ������ ��� ��������ation (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 4.8×104 Lmol-1cm-1. Beer's law was

obeyed in the range 0.3-����������������������������������������������!���������

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 µg and the

coefficient of variation was 5.1% at the 0.1 µg 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(III) and Sb(III) did not

166

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 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 3.05×104

Lmol-1cm-1. Beer’s law was obeyed in the concentration range of 0–30 µg 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 KBrO3 in the presence of KBr and methyl orange was bleached by the

excessive KBrO3, 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 Lmol-1cm-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 HCl 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

167

2.64×104 Lmol-1cm-1. Beer’s law was valid over the concentration range 0-1.20

mgmL-1 of arsenic.

Shao-Min 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

and 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,

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� of arsenic. The molar absorptivity was found to be

3.24×105 Lmol�cm�. 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 Lmol-1cm-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 + HCl). 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 µgmL-1

of As(V) with lower detection limits 0.15 µgmL-1. The molar absorptivity and

Sandell’s sensitivity were found to be 0.5×104 Lmol-1cm-1 and 0.0149 µgcm-2

respectively. The relative standard deviation for five replicate analyses of 4 µgmL-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.

168

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

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

SnI4 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 we�������������#����������������$���������������%�����

the 0.05 g of Fe.

Cherian and Narayana reported a spectrophotometric method for the

determination of arsenic in environmental and biological samples using azure B as a

chromogenic reagent [50]. The method was based on the reaction of arsenic(III) with

169

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 μgmL-1 of As(III). The molar

absorptivity, Sandell’s sensitivity, detection limit and quantitation limit of the method

were found to be 1.12×104 Lmol-1cm-1, 6.71×10-3 μgcm-2, 0.02 μgmL-1 and 0.08

μgmL-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

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 μgmL-1 of As(III) in a final volume of 10 mL. The molar

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

1.43×104 Lmol-1cm-1 and 5.26×10-2 μg 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

170

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

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

µgL-1. The coefficient of variation for the spectrophotometry at 50 µgL-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

171

permanganate absorbance at 524.2 nm was monitored for arsenic determination. The

acetic acid/sodium acetate and HCl 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 ������

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.

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.

172

6.3 APPARATUS

A Systronics 2201 UV-Visible Double Beam 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 μgmL-1) was prepared by dissolving 0.1734 g of NaAsO2 in 100 mL of water.

Working standard was prepared by appropriate dilution of stock. Toluidine blue

(0.01 %), safranine 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 μgmL-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 μgmL-1 of arsenic(III) were

transferred in to a series of 10 mL calibrated flasks. Potassium iodate (2 %, 1 mL)

173

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 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 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 [44].

6.5.4 Determination of Arsenic in Soil SamplesA 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 HCl. 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 [44].

6.5.5 Determination of Arsenic in Plant MaterialA 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 [44]. The results are listed in

Table 6A2.

174

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 μgmL-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 HCl 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 HCl 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 could

175

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 μgmL–1 of arsenic. The molar absorptivity and Sandell’s sensitivity of the

system is found to be 1.076×104 Lmol-1cm-1 and 9.66×10-3 μgcm-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 σ/s) and quantitation limit

(QL=10 σ/s) [where σ 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 μgmL-1

and 0.934 μgmL-1 respectively. Adherence to Beer’s law graph for the determination

of arsenic using toluidine blue is presented in Figure VIA2.

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 μgmL-1 of arsenic. The molar absorptivity and Sandell’s sensitivity of the

system is found to be 1.388×104 Lmol-1cm-1, 7.490×10-3 μgcm-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 σ/s) and quantitation limit (QL=10 σ/s)

[where σ 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 μgmL-1 and

0.759 μgmL-1 respectively. Adherence to Beer’s law graph for the determination of

arsenic using safranine O is presented in Figure VIA3.

176

6.6.4 Effect of Divers IonsThe 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

interfering species were established at those concentrations that do not cause more

th���&���'������� ���� �� �����(����� ����������)***+� ���������-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 [44].

The precision and accuracy of the proposed is evaluated by replicate analysis of

samples containing arsenic at four 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.

177

FIGURE VIA1ABSORPTION SPECTRA OF COLORED SPECIES OF TOLUIDINE BLUE (a)

AND SAFRANINE O (b)

Wavelength (nm)

200 300 400 500 600 700 800 900

Abs

orba

nce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

a

b

FIGURE VIA2ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF ARSENIC

USING TOLUIDINE BLUE AS A REAGENT

C oncentra tion o f arsen ic (µgm L-1)

0 2 4 6 8 10 12 14

Ab

sorb

an

ce

0 .0

0 .2

0.4

0.6

0.8

1.0

1.2

178

FIGURE VIA3ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF ARSENIC

USING SAFRANINE O AS A REAGENT

Concentration of arsenic (µgmL-1)

0 2 4 6 8 10 12 14

Abs

orba

nce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

FIGURE VIA4EFFECT OF pH ON COLOR INTENSITY

pH

0 1 2 3 4 5 6 7 8

Abs

orba

nce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Toluidine blueSafranine O

179

SCHEME VI

SCHEME OF THE REACTIONS

2 AsO2 - + 2 IO3 - + 8 H+ I2 + 2 AsO2 - + 4 H2O

N

S+

CH3

NH2(CH3)2N

NH

S

CH3

NH2(CH3)2N

I2 , H+

Toluidine Blue (Colored) Toluidine Blue (Colorless)

N

N+

CH3

NH2NH2

CH3 NH

N

CH3

NH2NH2

CH3 I2 , H+

Safranine O (Colored) Safranine O (Colorless)

180

TABLE 6A1

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF ARSENIC

(2 μgmL-1)

$������������������������������)����-1+����$������������������������������)����-1)

Toluidine blue Safranine O Toluidine blue Safranine O

Fe3+ 150 100 V5+ 125 100

Ni2+ 75 100 Zn2+ 1000 1000

Cd2+ 500 600 PO43- 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

181

TABLE 6A2DETERMINATION OF ARSENIC IN ENVIRONMENTAL SAMPLES USING

TOLUIDINE BLUE AND SAFRANINE O AS REAGENTS

Toluidine blue Safranine O

Samples As3+ added As3+ found Recovery RSD As3+ found Recovery RSD (µgmL-1) (µgmL-1) (%) (%) (µgmL-1) (%) (%)

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

a Tap 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 1.25a 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 1.34trial 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 0.97aPlant 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.

182

TABLE 6A3 COMPARISON OF THE METHOD REPORTED WITH EARLIER METHODS

ε = Molar absorptivity, ss = Sandell’s sensitivity

Reagent Method Beer’s law)����-1)

ε (Lmol-1cm-1)

�)����-2)

λmax(nm)

Ref. No.

Tween-80 Spectrophotometry 0.3-������ ε = 4.80×104---------

420 35

Rhodamine B Spectrophotometry 0.04-0.4 mgL-1

ε = 3.24×105---------

553 44

Oleic acid Spectrophotometry 0.25-20 ε = 0.50×104ss = 1.49×10-2

435 46

Azure B Spectrophotometry 0.2-10 ε = 1.12×104ss = 6.71×10-3

644 50

Variamine blue Spectrophotometry 0.2-14 ε = 1.43×104ss = 5.26×10-2

556 52

Proposed MethodToluidine blue

Safranine OSpectrophotometry

Spectrophotometry

1.2-10.5

0.4-11.5

ε = 1.076×104ss = 9.66×10-3

ε = 1.388×104ss = 7.490×10-3

628

532

183

6.9 REFERENCES

1. J. C. Deilar, H. J. Emeleus, R. Nyholm and A. F. T. Dickenson, Comprehensive

Inorganic Chemistry, 2 (1975) 547.

2. “Water Quality and Treatment”, American Water Work Association, (1992) 83.

3. N. I. Sax, “Cancer Causing Chemicals”, Van Nostrand Reinholds, New York,

(1981) 290

4. C. D. Klaassen, Heavy Metals and Heavy-Metal Antagonists. In: J. G. Hardman,

A. G. Gilman, L. E. Limbird, eds. Goodman & Gilman's the Pharmacological

Basis of Therapeutics. New York: Mc Graw-Hill, (1996) 1649-1672.

5. S. M. Aronson, R. I. Med., 77 (1994) 233.

6. Y. L. Kwong and D. Todd, Blood, 89 (1997) 3487.

7. E. G. Soto, E. A. Rodriguez, D. P. Rodriguez and E. F. Fernandez, Anal. Lett., 29

(1996) 2701.

8. G. Samanta and D. Chakraborthi, Fresenius’ J. Anal. Chem., 357 (1997) 827.

9. N. Ybanez, M. L. Cervera and R. Montoro, Anal. Chim. Acta, 258 (1992) 61.

10. G. Samanta, A. Chatterjee, P. P. Chowdhary, C. R. Chanda and D. Chakraborthi,

Environ. Tech., 16 (1995) 223.

11. D. Das, A. Chatterjee, G. Samanta and D. Chakraborthi, Chem. Environ. Res.,

13 (1992) 279.

12. M. Shull and J. D. Winefordner, Anal. Chem., 43 (1971) 799.

13. Y. Madrid, D. Chakraborti and C. Camara, Mikrochim. Acta, 120 (1995) 63.

14. B. Cristau, Ann. Pharm. Fr., 16 (1958) 26.

15. G. W. Powers, R. L. Martin, F. J. Piehl and J. M. Griffin, Anal. Chem.,

31(1959)1589

16. M. Elisabeth and M. Jean, Chim. Anal., (Paris), 43 (1961) 276.

17. T. Takashi and H. Kazuo, Bunseki Kagaku, 11 (1962) 1180.

18. L. M. Steckel and J. R. Hall, U.S. At. Energy Comm., Y-1406 (1962) 18.

19. T. Takashi and H. Kazuo, Bunseki Kagaku, 12 (1963) 914.

20. H. Kazuo, T. Takashi and W. Shizuko, Bunseki Kagaku, 12 (1963) 918.

21. P. Pakalns, Anal. Chim. Acta, 47 (1969) 225.

22. V. Stara and J. Stary, Talanta, 17 (1970) 341.

23. G. J. Kellen and B. Jaselskis, Anal. Chem., 48 (1976) 1538.

24. M. G. Haywood and J. P. Riley, Anal. Chim. Acta, 85 (1976) 219.

184

25. G. Silvia and C. Victoria, Environ. Health Pers., 19 (1977) 107.

26. M. Takatomi, Y. Yoshikazu and U. Shunzo, Bunseki Kagaku, 27 (1978) 419.

27. S. H. Gowda and K. N. Thimmaiah, Microchem. J., 23 (1978) 291.

28. F. Hiroshi and I. Kamihiko, Bunseki Kagaku, 28 (1979) 627.

29. Y. K. Agrawal and S. K. Patke, Intern. J. Environ. Anal. Chem., 8 (1980) 157.

30. R. H. Merry and B. A. Zarcinas, Analyst, 105 (1980) 558.

31. G. Howard and M. H. Arbab-Zavar, Analyst, 105 (1980) 338.

32. W. Elzbieta and E.Wieckowska, Biul. Wojsk. Akad. Tech., 30 (1981) 103.

33. C. Ali Naki, A. Huseyin and B. Fikret, Chim. Acta Turc., 9 (1981) 109.

34. W. Qian-Feng and L. Peng-Fei, Talanta, 30 (1983) 275.

35. Z. Tianze and W. Ming, Intern. J. Environ. Anal. Chem., 15 (1983) 1.

36. W. A. Maher, Analyst, 108 (1983) 939.

37. E. Kavlentis, Anal. Lett., 20 (1987) 2043.

38. C. Lata, J. Raju and V. K. Gupta, J. Indian Chem. Soc., 67 (1990) 500.

39. K. Palanivelu, N. Balasubramanian and T.V. Ramakrishna, Talanta, 39 (1991)

555.

40. W. Jiayu, S. Jiayun, C. Haofei and J. Qimin, Huaxue Fence, 29 (1993) 351.

41. H. Dianwen and L. Jianping, Guangdong Gongxueyuan Xuebao, 13 (1996) 84.

42. W. Yubiao, Yingyang Xuebao, 19 (1997) 453.

43. L. Shao-Min, X. Yue-Qin and W. Yu-Biao, Chinese Chem. Lett., 10 (1999) 155.

44. A. Pillai, G. Sunita and V. K. Gupta, Anal. Chim. Acta, 408 (2000) 111.

45. L. V. Gudzenko, R. P. Pantaler and A. B. Blank, J. Anal. Chem., 56 (2001) 721.

46. G. M. Abd El-Hafeez and S. G. El-Syed, Anal. Sci., 17 (2001) 1189.

47. S. Kundu, S. K. Ghosh, M. Mandal, T. Pal and A. Pal, Talanta, 58 (2002) 935.

48. Y. Sano, T. Kato, I. Nukatsuka and K. Ohzeki, Bunseki Kagaku, 52 (2003) 1153.

49. T. Taniai, A. Sakuragawa and A. Uzawa, ISIJ Int., 44 (2004) 1852.

50. T. Cherian and B. Narayana, Anal. Lett., 38 (2005) 2207.

51. M. Behpour, S. M. Ghoreishi and S. Salehi, Acta Chim. Slov., 52 (2005) 323.

52. B. Narayana, T. Cherian, M. Mathew and C. Pasha, Indian J. Chem. Tech., 13

(2006) 36.

53. F. C. Graham, J. Sci. Food Agric., 24 (2006) 1115.

185

54. W. Kunihiro, O. Takashi, I. Junichi and I. Masayuki, Bunseki Kagaku, 55

(2006) 251.

55. M. Keisuke and K. Emiko, Anal. Sci., 22 (2006) 1085.

56. H. D. Revanasiddappa, B. P. Dayananda and T. Kumar, Environ. Chem. Lett.,

5 (2007) 151.

57. M. Hashemi and P. Modasser, Talanta, 73 (2007) 166.