C H A P T E R 5

Phenoxazines as new spectrophotometric reagent for the determination of chromium(VI) in

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Chapter V Phenoxazines chromium(VI)

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Chapter V Phenoxazines chromium(VI)

y.l. Introduction

Chromium(VI) as toxicant enters the environment from industrial effluent or

waste disposal sources, such as waste from steel works, electroplating and tanning

industries [1]. Traces of chromium may also enter drinking water supply system.

chromiimi(III) and (VI) are two common forms of inorganic chromium in the

environment. There are many differences in their biochemical properties and toxicity.

For example, a trace amount of Cr(III) is essential to human beings. While, Cr(VI) is

harmfiil to human health, especially because it is known to be a strong oxidizing

agent, posing a high risk to humans and animals due to its carcinogenic and

mutagenic properties [2]. Thus, the determination of chromium in environmental and

biological samples is of great interest.

The methods proposed for the determination of metals in soil and industrial

effluent matrices include electroanalytical [3], radioanalytical [4] and

chromatography [5]. However, these methods have proved to be deficient with

respect to specificity, sensitivity, simplicity and analysis time. Various optical

methods such as inductively coupled plasma atomic emission spectrometry,

electrothermal atomization atomic absorption spectrometry (ETAAS) and atomic

absorption spectrometry (AAS) have been used for detection, quantitation and

characterization of these metals.

Amongst the optical methods visible spectrophotometry seems to be the most

appropriate for the determination of toxic metals, as it provides sensitive, precise and

accurate measurements of suitable analytes and offers practical and economical

advantages over other methods. Besides, visible spectrophotometric detection is much

more viable as a usefiil technique to develop a portable on-line or at-line system.

We propose an analytical procedure for determination of chromium(VI) in

environmental and biological sample by spectrophotometry, using phenoxazines as

chromogenic agent in presence of sulfanilamide (SAA), sulfadoxine (SDX),

sulfamethoxazole (SMX) or sulfadazine (SDZ). Different experimental conditions

like wavelength, the effect of acid, the amount of chromogenic agent, the effect of

coexistence ions, limit of detection and the ranges of applicability of Beer's law on

the determination of chromium(VI) have been studied

73

Chapter V Phenoxazines chromiunt(VI)

V.2. Experimental

V.2.L Apparatus

Specord 50 UV-Vis spectrophotometer with 1.0 cm silica quartz matched cell

was used for measuring the absorbance.

V.2.2. Reagents

Phenoxazines (Aldrich, India), sulphanilamide (SAA), sulphadoxine (SDX),

sulphamethaxazole (SMX) and sulfadazine (SDZ) (Smithkline Beecham, India) were

used as received. Distilled water was used to prepare all solutions. Distilled ethyl

alcohol was used to dissolve PNZ and a minimum amount of IM hydrochloric acid

was used to increase solubility of SDX and SMX.

Stock solutions of chromium(VI) (1000 ^g ml'') was prepared by dissolving

known amount of potassiimi chromate in 100 ml of distilled water. Solutions of

required strength were prepared by diluting the stock solution with distilled water.

Fresh solutions of phenoxazine (PNZ), chlorophenoxazine (CPZ) and

triflouromethylphenoxazine (TMP) (0.025%, w/v) were prepared by dissolving 25 mg

each of the sample in 100 ml distilled ethyl alcohol. Aqueous solutions of SAA, SDX,

SMX and SDZ (0.05%, w/v) were prepared by dissolving 50 mg each and diluting

quantitatively to 100 ml with distilled water. Two ml of IM hydrochloric acid was

added during the preparation of SDX and SMX to enhance the solubility. Solutions of

diverse ions were prepared by dissolving their corresponding salts. All reagents used

were of analytical grade and were used as received.

V.2.3. Analytical Procedure

To a series of 25 ml standard flasks, 2 ml of SAA/SDX/SMX/SDZ (0.05 %,

w/v), 1 ml of 2 M HCl, 2 ml of PNZ/CPZ/TPM (0.025%, w/v) and different aliquots

of standard solutions of chromium(VI) were added. The solutions were mixed well

and allowed to stand for 5 min at room temperature. The solutions were made up to

the mark with distilled water. The absorbance was measured at 540 nm for PNZ and

510 nm for CPZ/ TMP method against the corresponding reagent blank prepared

under identical conditions but without chromium.

74

Chapter V Phenoxazines chromium(VI)

The calibration graph was constructed. Concentration of chromium in test

solution was calculated from the regression equation computed from the Beer's law

data as a reference. The optical characteristics for the determination of chromium

with PNZ/CPZ/TMP using SAA/SDX/SMX/SDZ are detailed in the Table V.l.

V.3. Results and discussion

Phenoxazine is an isolog of phenothiazine. It is a part of the chemical

structure of actinomycin D, which is known to exert intensive anticancer activity on

malignant tumors in children [6] and is reported to be more potent and less toxic

chemosensitizer [7]. Phenoxazine derivatives exist in neutral form, as monocations,

as dications and even as trications depending on the environment [8]. Their molecular

structure and luminescent properties have been studied to a great extent [9]. Besides,

they have impressive applications as biological stains [10], as laser dyes [11] and as

redox indicators [12]. Phenoxazine derivatives are nervous system depressants

particularly with sedative, antiepileptic, tranquillizing activity [13], spasmalytic

activity [14], antitubercular activity [15] and anthelmentic activity [16]. In recent

years phenoxazine derivatives are reported to be potential chromophoric compoimds

in host-guest artificial photonic antenna systems [17].

The sulphonamides are analogues of/7-aminobenzoic acid and are known

since 1932. Though, a large number of sulphonamides are synthesized and reported in

the literature, only about two dozens of them have been used in clinical practice [18].

They differ only slightly in their antimicrobial activity but vary greatly in their

pharmacokinetic properties, or rate of excretion. Accordingly, they are classified as

short-, medium-, long- and ultra-long acting drugs [19]. Sulfanilamide (SAA) belongs

to short-acting, sulfamethoxazole (SMX) medium- acting and sulfadoxine (SDX) and

sulfadazine (SDZ) ultra-long acting sulphanomides.

V.3.1. Spectral characteristics

The absorption spectrum of the red colored products with chromium shows a

wavelength of maximum absorption at 540nm for PNZ and 510 nm for CPZ/ TMP.

The reagent blank shows negligible absorption at these wavelengths.

75

Chapter V Phenoxazines chromium(VI)

V.3.2. Optimization of analytical variables

The reagent concentrations and quantity needed as also the reaction conditions

were optimized to arrive at a standard procedure. Each parameter was optimized by

setting other parameters constant.

V.3.3. Order of addition

During the course of the investigation, it was observed that the sequence of

addition of reactants was also important as it influence the intensity and the stability

of the color of the product to a great extent. The sequence (i) Cr(VI) +

SAA/SDX/SMX or SDZ + PNZ/CPZ/TMP +HC1 and (ii) Cr(VI) + PNZ/CPZ/TMP +

HCl + SAA/SDX/SMX or SDZ gave less intense and unstable color. While, (iii)

SAA/SDX/SMX or SDZ + HCl + Cr(VI) + PNZ/CPZ/TMP gave more intense and

stable red color.

V.3.4. Effect of reagents

The effect of PNZ/CPZ/TMP reagent was studied in the range of

0.10 - 5.00 ml of (0.025%, w/v) solution of each to achieve the maximum color

intensity, volume of 0.50 - 3.00 ml of the solution gave good result. Hence, 2 ml of

(0.025 %, w/v) PNZ/CPZ/TMP solutions in 25 ml standard flask was selected for

further studies, under optimized conditions. The maximum intensity of the red color

was achieved in hydrochloric acid medium.

Preliminary investigations showed that hydrochloric acid was better than

sulphuric, phosphoric or acetic acid. Maximum intensity of the red color was

achieved in the range of 1.0-6.0 ml of 2M HCl. Therefore, 1 ml of 2M HCl in 25 ml

standard flask was used for getting the best results. Similarly, the same procedure was

adopted to ascertain the amount of SAA, SDX, SDZ and SMX required for getting

constant and maximum color intensity. It was found that 0.50 - 3.00 ml of the

solution were needed to get good result. Hence, 2 ml of (0.05%, w/v) SAA, SDX,

SMX or SDZ solutions are sufficient to get reproducible resuhs.

Experiments were carried out to optimize temperature and time of the

reaction. It was found that the maximum color developed in 5 min at room

76

Chapter V Phenoxazines chromiunt(VI)

temperature and remained almost stable for about 4 h. Increase in the temperature

decreased the intensity of the red color.

V.3.5. Analytical Parameters

The colored products obeyed Beer's law. The optical characteristics such as

optimum range for the determination of the ions, molar absorptivity, sandell's

sensitivity, slope, intercept, correlation coefficient are presented in Table V.l which

conclusively proves that the reagents are sensitive.

The accuracy and precision of the proposed methods were evaluated by

performing recovery tests by standard addition method. These tests were performed

by adding known amounts of standard solutions at different concentration levels to a

fixed amount of real samples and the mixtures were analyzed by the proposed

procedures. Each test was repeated five times.

V.3.6. Interferences

In order to establish the analytical potential of proposed method, the effect of

some possible interfering ions which often accompany chromium(VI) was examined

in presence of number of other ions by the proposed methods. An ion was considered

to be interfered with the determination if the obtained absorbance values differed by

more than ±3% firom that of chromium(VI) alone. Metals such as iron(III),

vanadium(V) manganese(VII) and cerium(IV), non metals such as bromate, iodate

and periodate were found to interfere severely and caused low recovery of

chromium(VI). However, using appropriate masking agents could eliminate the

interference from these ions. Masking agents such as EDTA, tartrate and citrate did

not interfered in the determination of chromium(VI). The use of a mixture of tartaric

acid (50 mg) and citric acid (5 mg) has been found to have effective masking action

on a large number of foreign metal ions. During the interference studies, if a

precipitate was formed, it was removed by centrifugation. The maximum tolerable

concentrations of different ions (defined as the foreign-ion concentration causing an

error smaller than ±3% for determining the analyte of interest) in the determination of

chromium(VI) are listed in Table V.2.

77

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Chapter V Phenoxazines chromium(VI)

Table V. 2. Effect of diverse species in the determination chromium(VI)

Tolerance limit Foreign ions (^g ml' )

Bi , Ba , Ti'* , EDTA, Br", citrate, tartarate, oxalate

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V.3.7. Applications

V.3.7.1, Collection, preparation and determination of soil samples

Soil samples were collected about 0.5 km. away from waste treatment plant of

a factory, which used chromium extensively in plating baths. The treated industrial

effluents (water) from the factory were released into the neighboring fields, which

were ultimately used for irrigation purposes. Static sampling procedure was adopted

and six soil samples were collected at random from depth of 0-20 cm with a distance

of about 50 m between each sampling site.

Known amoimt of soil sample (5.0 g) was taken in a platinum crucible and

then heated it for 3 h in a muffle furnace at 55''C. After cooling, transferred the

sample into a platinum basin and added 2.0 ml of double distilled water, 1.0 ml of

concentrated sulphuric acid and 10.0 ml of concentrated hydrofluoric acid and heated

on a sand-bath until vapours of SO3 appear. The residue was dissolved in 5.0 ml of

double distilled water acidified with hydrochloric acid to pH 3 [20]. The pH of the

solution was raised to -10 by the addition of concentrated ammonia solution to

precipitate the iron and aluminum. Filtered off the precipitate with a suitable filter

paper and washed it with 3-ml portions of double distilled water. Evaporated the

filtrate to ~ 20 ml, cooled, acidified the solution to pH 3 with hydrochloric acid,

transferred it into a 25-ml standard flask and diluted up to the mark with distilled

79

Chapter V Phenoxazines chromium(VI)

water. Iron, which is commonly found in most of the soil samples, interferes and its

elimination is necessary. Methods reported in the literature for the elimination of

iron(III) interference, include: either precipitation as hydroxide or use of masking

agents. The former method is extensively used [21]. We have reported a simple spot

test to know the presence of iron(III) in filtrate. This involves the use of KSCN

solution which gives red color with iron(III) [22] (1 drop of filtrate + 1 drop of 2N

H2SO4 + 1 drop of thiocyanate, HCl is avoided as it gives dense fimies of NH4CI).

Absence of red color is an indication that iron (III) precipitation process is almost

complete.

About 2.5 ml or other suitable aliquot of the above prepared solution was

taken and oxidized fi-om chromium(III) to chromium(VI) [9] by adding 3 ml of

bromine water and boiled for 3 min to ensure that all chromium(III) was oxidized to

chromium(VI). The solution was cooled and diluted to volume in a 100-ml standard

flask. An aliquot of this solution was taken for analysis by the proposed method. The

resuhs are presented in TableV.3.

V.3.7.2. Waste waters from industries (plating baths)

The solution were obtained fi*om three different waste chromium baths

and was determined by taking 2.5 ml of the sample and diluting to 25 ml with

distilled water. Bromine water (3 ml) was added and boiled for 3 min to ensure that

all chromium (III) was oxidized to chromium(VI). The solution was cooled and made

up to 100 ml in a standard flask. To enhance the reliability of the method standard

addition test was conducted.

The above solutions were transferred to ten 25-ml standard flasks and

were analyzed by proposed methods. The results are presented in Table V.3.

V.3.7.3.Biological samples.

As mentioned earlier, chromium is an essential trace nutrient for humans.

Mertz [23] has proposed urinary chromium excretion as a means of accessing the

chromium nutritional status of individuals. Determination of chromium in biological

materials is a serious problem and the reported values for the same are different

matrices differed by over an order of magnitude.

80

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Chapter V Phenoxavnes chromium(VI)

Most of the values lie in the 2-20 ng ml' range, this presented a serious

dilemma from the nutritional standpoint. The amount of chromium absorbed had been

determined to be of the order of 0.5-1% by radiotracer experiments [24], so daily

urinary excretions of the order of 10 ng/day meant that dietary chromium intake had

to be more than 1 mg/day. So far no reasonable diets in the U.S. were found which

could supply more than 100 ng/day. Hence, chromium is supplemented through

B-complex formulations.

V.3.7.4. Sampling

Urine samples were collected from healthy individuals and individuals who

were on supplementary chromium intake in the form of B-complex formulation.

These samples varied considerably in color, salt content and chromiimi concentration

and are believed to be of representative samples of various types of subjects that are

likely to be encountered in metabolic studies.

. Five ml of each of urine sample were transferred into five 25-ml standard

flasks. To four standard flasks 1 ml each of the solutions containing 1.2, 1.3, 1.4 and

1.5 ng Cr ml'' was added. Aliquots of this solution were analyzed by proposed

method. The results are presented in Table V.4.

V.6. Conclusion

The proposed methods are simple, inexpensive, sensitive and precise also has

the advantage of determination without the need for extraction or heating. The

method does not involve complicated reaction conditions. This is the first time that

spectrophotometric methods are being reported using phenoxazines as chromogenic

agent and SAA, SDX, SMX and SDZ as electrophilic coupling agent for

determination cliromiimi(VI) in environmental and biological samples. Statistical

analysis of the results revealed that the proposed method yield accurate and

reproducible values in the determination of chromium(VI) in various soils and

industrial effluent matrices. Applications of the proposed method in the determination

of chromium(VI) in a variety of real samples have demonstrated their practical utility.

82

Chapter V Phenoxa^nes chromium(VI)

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J. Mater. Chem. 12(2002)1.

19. E.H. Northey, The Sulfonamides and Allied Compounds, ACS Monogr. Ser. American

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