INTERACTION OF METAL IONS WITH ELECTRON RICH SPECIES

ABSTRACT OF THE

y/^ THESIS N ^ ^ W

SUBMITTED FOR THE AWARD OF THE DEGREE OF

iQctor of ^I)tIo£(opl)p

SAURABH SHARMA

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2010

•^'1^«,

ABSTRACT

The thesis entitled "Interaction of metal ions with

electron rich species" is comprised of five chapters. The first

chapter describes a general introduction of the subjecfimatter.

The very relevant matters include;

• A brief history of element and its toxicity in environmental

samples.

• Importance of speciation analysis.

• A note on mineralization and sampling techniques.

• A brief description of various analytical techniques and its

applications in the quantitation of metals in soil, natural

water and synthetic samples.

• A note on the interaction of metal ions with electron rich

species.

• A description on process monitoring and validation.

• Importance of statistical analysis on the calibration data in

order to judge the performance and suitability of the

proposed analytical method.

A description of the transition metal ions especially

copper(II), iron(III), uranium(VI) and thorium(IV) based on

the toxicity of the elements on human organs is included. A

brief description of the electron rich species interacting with

copper(II), iron(III), uranium(\/I) and thonum(IV) metal ions

is also included. An abundant and well-connposed list of 146

references is given at the end of this chapter taken from the

world's leading scientific journals in the field.

The second chapter describes a simple and selective

spectrophotometric method has been described for

determination of Fe(III) in synthetic mixture and soil samples.

The method is based on chelation of ferric ion with piroxicam

to produce a coloured metal complex at room temperature

which absorbs maximally at 495 nm. Beer's law is obeyed

(cone, 2.79 - 25.13 ^g mL'^) with apparent molar absorptivity

(5.88 X 10^ L mor^cm"^ ) and Sandell's sensitivity ( 0.019

)ag/cmV 0.001 absorbance unit). Proposed method was

successfully applied in the determination of Fe(III) in synthetic

mixture and soil samples.

In chapter three, a new and novel UV spectrophotometric

method has been discussed for the determination of Cu(II) in

synthetic mixture and water samples. The method is based on

complex formation of Cu(II) with cefixime immediately in 1,4-

dioxan-distilled water medium at room temperature. The

complex showed maximum absorption wavelength at 336 nm.

Beer's law is obeyed in the concentration range of 1.015 -

8.122 ng mL'^ (Linear regression: A = 1.59 x 10"^ + 1.305 x

10"^ C) with apparent molar absorptivity (8.29 x IQ- L mol"

^cm^^ and Sandell's sensitivity (0.008 ^g/cmV 0.001

absorbance unit). The limits of detection and quantitation for

the developed method are 3.19 x 10"^ and 9.65 x lO"^ ^g

mL' \ respectively. Interferences due to Mn(II), Ca(II), Mg(II),

Cd(II), AI(III), Pb(II), Zn(II), Fe(II), Fe(III), Ni(II), Cr(III),

Hg(II) and As(III) was investigated. Nine cations do not

interfere in the determination of Cu(II). Proposed method was

successfully applied to the determination of Cu(II) in synthetic

mixture, sea and well water samples.

The fourth chapter describes a validated

spectrophotometric method for the determination of thorium

(IV) in synthetic mixture and soil samples. The method is

based on the chelation of thorium(IV) with rifampicin at room

temperature which absorbs maximally at 525 nm. Beer's law is

obeyed in the concentration range of 1.16 - 23.2 i g mL"^ with

apparent molar absorptivity and Sandell's sensitivity of 8.23 x

10^ L mol"^ cm"^ and 0.0232 |ig/cmV 0.001 absorbance unit,

respectively. The influence of variables was investigated and

optimized. Interference due to other metal ions was studied

and the tolerance limit was achieved. The proposed method

was applied to the analysis of thorium(IV) in synthetic mixture

containing various other metal ions and soil samples. The

results of analyses of the proposed method were statistically

compared with the reference method showing acceptable

recovery and precision.

The last chapter describes an optimized and validated

spectrophotometric method has been developed for the

determination of uranyl ion in soil samples. The method is

based on the complexation reaction between uranyl ion and

rifampicin in methanol-water medium at room temperature.

The method is followed spectrophotometrically by measuring

the absorbance at 375 nm. Under the optimized experimental

conditions. Beer's law is obeyed in the concentration range of

1.35 - 20.25 |ig mL"^ with apparent molar absorptivity and

Sandell's sensitivity of 8.0 x 10^ L mol'^ cm"^ and 0.042

iag/cmV 0.001 absorbance unit, respectively. The interference

of a large number of anions and cations has been investigated

and the optimized conditions developed have been utilized for

the determination of uranium(VI) in soil samples. The three

Sigma detection limit (n = 9) for uranyl ion was found to be

0.20 fag mL"^ The proposed method was successfully applied

to the determination of uranyl ion in soil samples.

INTERACTION OF METAL IONS WITH ELECTRON RICH SPECIES

THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF

Boctor of ^titlo^optip IN

CHEMISTRY

w

BY

SAURABH SHARMA

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2010

2 r SEP 20W

DR. LUTFULLAH Ph.D. (Alig.)

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (U.P.) INDIA Tel.: +91-571-2703515 (Office) E-mail: lutfullah786®gmail.com

Dftted: 2 3 - ^ -^^ l^

Certificate

This is to certify that the thesis entitled "Interaction of metal Ions

with electron rich species" is the original work of Hr. Saurahh Sharma,

carried out under my supervision and suitable for submission for the award

of the degree of Doctor of Philosophy In Chemistry.

(Dr. tXiifullah)

AaNOWLEDGEMEMTS All praises to Almighty God, The most merciful. The most

beneficent, whose spiritual inspiration and blessing enabled me to

complete this task.

I take this opportunity to extend my heartiest gratitude to my

honourable supervisor. Dr. Lutfullah for his generous consideration,

undaunted help, unceasing support, valuable suggestions and

guidance in the completion of this work. I am also thankful to Dr.

Nafisur Rahman for his constant encouragement and help.

I wish to express my sincere and profound gratitude to

Dr. Syed Najmul Hejaz Azmi for his sympathetic attitude, persistent

interest and consistent encouragement throughout the entire work.

I extend my sincere thanks to The Chairman, Department of

Chemistry, Aligarh Muslim University, Aligarh for providing research

facilities. I am grateful to Aligarh Muslim University for awarding

UGC research fellowship to carry out this work.

I feel short of words to express my heartiest gratitude to my

beloved parents, sisters and brothers who contributed significantly

with their spiritual inspiration and moral support to complete the

work.

(Saurabh Sharma)

List of Publications

List of Tables

List of Figures

CHAPTER 1

CHAPTER 2

CONTENTS

General Introduction References

Spectrophotometric Determination of Fe(lli) via Complexation with Piroxicam in Synthetic Mixture and Soil Samples

i

ii

iv

1 44

53

CHAPTER 3 Interaction of Cu(ll) with Cefixime 75 and Its Application for Determination of Cu(ll) in Water Samples

CHAPTER 4 Spectrophotometric Studies of Thorium(IV)- rifampicin Complex and Its Application in the Determination of Th(IV) in Soil Samples

07

CHAPTER 5 Spectrophotometric Determination of Uranium(VI) via Chelation with Rifampicin in Soil Samples

132

LIST OF PUBLICATIONS

[1] Spectrophotometric Determination of Fe(III) Via

Complexation with Piroxicam in Synthetic Mixture

and Soil Samples.

J. Sci. Indust. Res. 69 (2010) 135.

[2] UV Spectrophotometric Determination of Cu(II) in

Synthetic Mixture and Water Samples.

J. Chin. Chem. Soc. 57 (2010) 622.

[3] Determination of Thorium(I\/) with Rifampicin

in Synthetic Mixture and Soil Samples by

Spectrophotometry.

Acta Chim. Slovenica (2010) (Communicated).

[4] Spectrophotometric Determination of U(VI) Via

Chelation with Rifampicin in Soil Samples.

J. Chin. Chem. Soc. (2010) (Communicated).

LIST OF TABLES

Table 1.1. Determination of metal species in the selected fields of 19 development.

Tabic 1.2. UV-visible spectrophotometric methods for the determination ' I of metal ion in environmental samples.

Table 1.3. Validation characteristics normally evaluated for different ' 2 t>'pes of test procedure and the minimum number of determinations required (if applicable).

Table 1,4. Approaches for determining limits of detection and 36 quantitation.

Table 2.1. Test of accuracy and precision of proposed method. 67

Table 2.2. Optical and regression characteristics of proposed method. 69

Table 2.3. Evaluation of bias: Comparison and applicability of the 71 proposed method with the reference method for the determination of Fe(III) from synthetic mixture sample at 95% confidence level.

Table 2.4. Determination of Fe(III) in soil samples by proposed ""2 spectrophotometric method and reference method.

Table 3,1. Effect of metal ions on the determination of 7.107 [ig niL 9^ Cu(II).

Table 3.2. Precision of the proposed method. 'M

Table 3.3. Test of accuracy in synthetic mixture sample by standard 96 addition method.

Table 3.4. Optical and regression characteristics of the proposed 100 method.

Table 3.5. Point and inter\'al hypothesis tests for the determination of 101 Cu(II) in synthetic mixture sample for the evaluation of bias of the proposed method with that of the reference method at 95% confidence level.

Table 3.6. Determination of Cu(II) in sea and well water samples by the 102 proposed UV spectrophotometric method and the reference method.

Table 4.1. Effect of various metal ions on the determination of 23.2 n<; mL ' thorium(IV).

I l l

Tabic 4.2. Test of precision of the proposed method at three 122 concentration levels of Th(IV).

Table 4.3. Recovery data of thoriuni(IV) in synthetic mixture sam|)le b> 12' standard addition technique.

Table 4.4. Optical and regression characteristics of the proposed and 127 reference methods.

Table 4.5. Evaluation of bias: Applicability of the proposed method in 128 synthetic mixture and soil samples and its comparison with the reference method at 95% confidence level.

Table 5.1. Effect of various metal ions on the determination of 18.9 fig 149 mL"' uranyl ion.

Table 5.2. Optical and regression characteristics of the proposed and 132 reference methods.

Table 5.3. Test of precision of the proposed method.

Table 5.4. Test of accuracy of the proposed method by standard addition method.

154

Table 5.5. Applicability of the proposed method for the determination of 157 uranyl ion in soil samples and the comparison of the results with the reference method at 95% confidence level.

Table 5.6. Comparison of the proposed spectrophotometric method with 158 other methods for the determination of uranyl ion.

LIST OF FIGURES

Fig. 1.1. Importance of analytical instrumentation in speciation of 29 metals.

Fig. 1.2. Linearit'with the values of correlation coefficient. 40

Fig. 2.1. Absorption spectra of (a) 0.2 mL of 5 x 10" M ferric 59 sulphate in distilled water (b) 0.3 mL of 0.016% piroxicani in methanol (c) 0.45 mL of 0.005 M ferric sulphate in water + 2.5 mL of 0.16% piroxicam in methanol. Each solution is diluted up to the mark with ethanol in 10 mL standard volumetric flask.

Fig. 2.2. Limiting logarithmic plot for stoichiometric ratio bet>veen 60 Fe(ni) and piroxicam: (a) log A vs. log [Fe^^], and (b) log A vs. log [piroxicam].

Fig. 2.3. Job's plot of continuous variations for stoichiometric ratio 62

between Fe(III) and piroxicam (1:2).

Fig. 2.4. Reaction sequence of proposed method. 63

Fig. 2.5. Effect of concentration of piroxicam on absorbance of metal 64 complex at 22.34 fa,g mL"' Fe(III).

Fig. 2.6. Effect of solvent on absorbance of coloured complex, 65 [Fe(III)] = 22.34 ^g mL*.

Fig. 2.7. Calibration curve for determination of Fe(Ill). 6H

Fig. 3.1. Absorpt ion spectra of (a) 2 niL of 3.195 x 1 0 ' M copper 85 su lpha te in distilled wa te r (b) 0.05 niL of 0.1 % cefixime in methanol and (c) 0.4 mL of 3.195 x l o " M copper sulphate in distilled wa te r + 1.8 mL of 0 . 1 % cefixime in methanol . Solutions a, b and c a r e diluted up to m a r k with distilled wa te r , methanol and 1,4-dioxan in 10 m L s t anda rd volumet r ic flask, respectively.

Fig. 3.2. Job's plot of continuous variations for stoichiometric ratio 84 between Cu(II) and cefixime (2.205 x 10 ^ M each).

Fig. 3.3a. IR spectrum of free cefixime. 85

Fig. 3.3b. IR spectrum of Cu(II)-cefixime complex. 86

Fig. 3.4. Reaction sequence of the proposed method. 88

Fig. 3.5. Effect of the volume of 0.1 % cefixime. ')<)

Fig. 3.6. Effect of solvent on the absorbance of Cu(Il)-cefixime complex, ')1 |Cu(n) l = 8.122 fig mL"'.

Fig. 3.7. Determination of Cu(Ii) in synthetic mixture sample b\ '5 standard addition method.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig-

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

3.8.

4.1.

4.2.

4.3a.

4.3b.

4.4.

4.5.

4.6.

4.7.

4.8.

5.1.

5.2.

5.3a.

5.3 b.

Calibration curve for the determination of Cu(II).

Absorption spectra: (a) 9.721 x 10"M rifampicin in methanol (b) 2.066 X 10 " M rifampicin in methanol + 7.0 x IQ- M thorium(IV) in distilled water.

Job's plot for thorium-rifampicin complex.

IR spectrum of free rifampicin.

IR spectrum of Th(IV)-rifampicin complex.

Reaction sequence of the proposed spectrophotometric method.

Effect of the concentration of rifampicin on the absorbance of the complex.

Effect of solvent on the absorbance of the coloured complex.

Recovery' of Th(lV) from synthetic mixture by standard addition technique: (a) 6.963 and (b) 13.949 mg mL"'.

Calibration curve for the determination of Th(IV).

Absorption spectra of (a) 1 mL of 1.0 x 10" M uranyl nitrate in distilled water (b) 1 mL of 6.1 x IQ" M rifampicin in methanol and (c) 1.5 mL of 5.0 x 10"'' M uranyl nitrate in distilled water + 2.0 mL of 6.1 X 10"'' M rifampicin in methanol. Solutions a, b and c are diluted up to mark with distilled water, methanol and again methanol in 10 mL standard volumetric llask. respectively.

Job's method of continuous variations of uranyl nitrate-rifampicin complex.

IR spectrum of free rifampicin.

IR spectrum of U02^-rifampicin complex.

M8

112

113

114

115 i

1 r ;

1 18

119

124 I t

126

134 1 i

141

142 1 2

143 j

Fig. 5.4. Reaction sequence of the proposed method. 144

Fig. 5.5. Effect of the volume of 6.1 x 10' M rifampicin with 20.25 ^g 146 mL"' uranyl ion on the absorbance of the complex.

Fig. 5.6. Effect of solvent on the absorbance of the coloured complex. 148

Fig. 5.7. Calibration graph for the determination of uranyl ion. 151

Fig. 5.8. Recovery graph for uranyl ion through standard addition 155 method: (a) 6.75 and (b) 13.5 lg mL"' uranyl ion.

GENERAL INTRODUCTION

Electron rich species are compounds which have generall) nitr^tgen. sulphur,

and oxNgen donor atoms. These atoms are acting as basic centres for interaction wiih

metal ions. These compounds are natural and synthetic. Several of these compounds

ha\e been found biologically active and used as medicine. Many others are useful in

industry. In the hetrocyclic compounds the donor atoms are present in the ring, side

chain and or directly attached to the carbon atom of the ring. Interaction of transition

metal ions with electron rich species results in the formation of complexes. Transition

metal complexes have been used for carcinostatic activity, antitumour activity,

fungicide and insecticide. Many sulphur and nitrogen containing compounds \ iz

pyridine-2-carboxaldehyde thiosemicarbazone (1) and isoquinoline-l-carboxaldehyde

thio.semicarbazone (2) have been found to have a broad range of anticancer activiu

S-. ^CH: :N NH-

/ /

(!) NH,

C H = : N NH C

(2)

/ /

NH,

Metal ion interaction with vitamins, enzymes and nucleic acid leads to the

formation of chelate. These metal chelate on reacting to the normal cell replace the

essential metal ions from the normal enzyme system, thus, metabolic activit\ is

changed. The anticancer drug may deactivate the carcinogenesis by binding the

normal metal ion [2-7]. Many metal chelates of folic acid [8], ribollavine |4]. adciiinc

[10] and thioguanine [11] have also been reported. Bismuthiol-1 is a fi\e membered

hetrocyclic ring system having three suphur and two nitrogen atoms and a derivative

of 1,3,4 thiadiazole (3) in which hydrogen atom attached to carbon atom at 2 and 5

positions are replaced by -S-H group (4).

N- N NH^ NH

J W / \

(3) 14,

Bismuthiol-1 (2,5-dimercapto 1.3.4 thiadiazole) is an important analytical

reagent for bismuth [12]. Several metal complexes of bismuthiol-1 with metal ions

such as Zn(ll), Cu(II), Ni(ll) and Co(Il) have been prepared [13]. It is also used as a

reagent for spectrophotometric determination of palladium [14-16]. Uran\l (UO;' )

and thoriurn(IV) give chelate after interaction with bismuthiol-1 in aqueous methanol

mixture [17]. Other heterocyclic compounds with nitrogen and sulphur coordinating

sites like thiazole (5), and 2 acetamido-5 nitro thiazole (6) are also biologically acti\e

HC N HC N

J W / \

(5) (6)

and form complexes with UO;"^ and Th^ ions. Thiazole. imidazolcthiol on

interaclion witli metal ions form biologically active complexes acting as t'ungicidcs

Man> other complexes are used as anti oxidant [ 13.18,19],

2-Benzimidazolethiol is an industrially important compound. It has nitrogen

and sulphur basic centers for interaction with metal ions. It shows tautomerisms.

C=S - « » - ^C SH

H H

It has good coordinating ability. Certain metallic salt of 2-benzimidazole thiol have

been used as heat stabilizer for polyamide. It is specific reagent for determination of

various metals. Spectrophotometric determination of Pd(II), Ru(lII) have been done

using this compound [20]. Adduct formation with Sn(II), Sn(lV) and Sb(III) have

been reported [21]. Interaction of inner tranisition metal ions such as Y(III). Pr(IIl).

Nd(Il]), Sm(]ll), Gd(Il) and Dy(Ill) with 2-benzimidazole thiol led to the formation of

1:1 complexes in aqueous solution [22].

Schiff bases are an important class of compounds in medicinal and

pharmaceuticals tlelds. They show biological applications including antibacterial [2,1-

25]. antifungal [26.27]. and antitumour activities [28.29]. Many of researchers studied

the synthesis, characterization and structure-activity relationship of SchilT bases

[30.31]. Schiff base complexes with transition metals have also played a prominent

role in the development of coordination chemistry [32].

Transition metal complexes of Schiff bases have been amongst the most

widely studied coordination compounds, since they are found to be useful as

biochemical, analytical and antimicrobial reagents [33-35]. A series of complexes

[36] involving Mn(ll), Co(Il), Ni(Il), Cu(ll), Zn(ll). Cd(ll) and Hg (11) ions and

Schiffs base ligand obtained by condensation of 5-(2"-thiazolylazo) salicyaldeh>de

and p-methox aniline have been reported [36]. The structure of the complexes ma\

be represented as:

OCH.

H3CO

where M= Mn([I), Co(n), Ni(II), Cu(II), Zn(Il), Cd(ll) and Hg(ll).

The tetradentate Schiff bases are interesting complexing reagents 137-41]

because they form highly stable metal chelate compounds with a limited number ol

metal ions such as Cu(Il). Ni(II), Co(Il), Co(ni). Fe(Il), Fe(lll). Pd(ll). Pt(lll).

VO(ll). U02(I1). The metal chelates are extractable in organic solvent. The reagents

bis(salicylaldehyde)tetramethylethylenediimine (H2SA2Ten), bis(salicylaldehyde

ethylenediimine (H2SA2en), and bis(salicylaldehyde) propylenediimine (H2SA2Pn)

have been reported for the determination of uranium, iron, copper and nickel in

mineral ore samples and phosphate rock residues [42-43]. Phenyl group substituted

ligands such as bis(salicylaldehyde)-dl-stilbenediimine (dl-H2SA2S) and

bis(salic>laldehyde)-meso-stilbene-diimine (meso- H2SA2S) have been reported for

HPLC determination of uranium, iron, copper and nickel [44]. The structure of these

complexes, H^SAjTen, H2SA2en, HsSAjPn, dl-H2SA2S and meso- H2SA2S is shown

below:

Where H^SAjen ; R,, R2, R3 and R4 = H

H2SA2Pn ; R, =CH3, R2, R3 and R4 = H

H2SA: fen ; R,, R,, R3 and R4 = CH3

di-112SA2S ; Ri and Rj = C. Hs, R2 and R4 = H

meso-112SA2S ; R, and R3 = CfcHs, R2 and R4 = H

M= Cu(ll), Fe(lll), Ni(I!), UOJCII).

The chemistry of thiosemicarbazones has received considerable attention in

view of their variable bonding modes, promising biological implications, structural

diversity and ion-sensing ability [45,46]. They have been used as drugs and are

reported lo possess a wide variety of biological activities against bacteria, fungi and

CvTlaii! lype of tumours and they are also useful models for bioinorganic processes

[47J. fhe inhibitory action is attributed due to their chelating properties [48-50]. The

activity of these compounds is strongly dependent on the nature of the hetero atomic

ring and the position of attachment to the ring as well as the form of

ihiosemicarbazone moiety [51]. These are studied extensively due to their flexibility,

their selectivity and sensitivity towards the central metal atoms, and similarities with

natural biological substances due to the presence of imine group (-N=CH-) which

imparls the biological activity [52]. Copper(II) complexes of thiosemicarbazones are

good antimicrobial agents [53]. It has been reported that copperll) chelates of

anthraccne-9-carboxyaldehyde thiosemicarbazones [54] exhibited antitiinioiir

activities whereas copperil) complexes of phenylglyoxal bis(lhiosemicarbazone)

showed cytotoxic activity [55].

Complexes of Co(n), Ni(II) and Cu(II) have been synthesized with 3-amino-2-

phenyl 4(3H) quinazoline thiosemicarbazone (APQT). The complexes have been

formulated as [M(APQT)2]X2where M= Co(II), Ni(II) and Cu(II); X= CI', Br", f,

NO3" and C104' [56]. The ligand APQT acts as a neutral tridentate ligand and

coordination takes place through azomethine N, thione S and primary amino group of

quinazoline. The geometry of these complexes is octahedral in nature and shown as:

HoN

NHo

Where R=^Phenyl

Cephalexin belongs to the first generation cephalosporins. Many drugs possess

modified toxicological and pharmacological properties when they are complexed

8

[57,58]. In this series, metal complexes of Cu(n) and Zn(ll) with Schiff base of

cephalexin (Salicylidene cephalexin) of the type, ML2 , have been prepared [59]. The

conductometric titration revealed that one mole of metal complexed with two moles

of ligand. The physical, analytical and spectral studies of Schiff base and its

complexes confirmed that the coordination of metal to the Schiff base occurred

through phenolic deprolonated oxygen and the imino nitrogen. The structure of the

complexes is shown below.

o

H,C

HO 0

-NH

" ^ 0 / = \

Where M=Cu(lI)/Zn(II).

Makkar and coworkers [60] have reported the synthesis of Fe(III) complexes

of l-(2'-hydroxyphenyl) ethylideneanilines. The structures of these complexes were

proposed on the basis of elemental analysis and IR studies. The metal to ligand

stoichiometry was found to be 1:3 and the possible structure is shown as;

Fe

Where R = H, - CH3, - OCH3, - OC2H5, CI, Br, OH

A simple spectrophotometric method has been reported [6i | for the

determination of Fe(III) based as a thiocyanato mixed complex with N-h\droxy N(-p-

chloro)-phenyI. N"-p- tolyl-m-toluamidine. The red-orange complex is quantitali\cl\

extracted into benzene. The benzene extract showed an intense peak at 465 nm with

molar absorptivity of 1.34 x 10"* L mor'cm"'. Beer's law was obeyed in the

concentration range of 0.2 to 4.2 ppm.

It was reported in the literature [62] that lansoprazole, an antiulcer drug, was

found to react with Cu(Il), Co(ll), Ni(ll), Hg (II), Zn(II) and Cd(ll) resulting in the

formation of complexes. The structure of these complexes is shown below.

CH,

O CF^

Where M = Cu(Il). Ni(ir). Co(ll). Hg (II). Zn(II) and Cd(ll)

10

Melal-ligand stability constants fall in the order of Cu(II) > Co(ll) * Ni(li); Hg(ll) •

Znn)>Cd(ll).

There has been a growing interest in the synthesis of uranium(lV) complexes

|63-()(i]. As a further contribution to this area. Schiff base coniplcxes of 2-

aminiobenzoyi hdrazones with uranium(lV) acetate have been synthesized |67|

These complexes were characterized on the basis of elemental anah sis. conductivit).

magnetic and infrared spectral data and hence the following structure was proposed.

R

Ihe enxironment around uranium(lV) consists of six oxygen and two nitrogen

atoms. Of the six oxygens, four belong to two acetate moieties. The uranium atom is

likely to be above the plane of the ligand-coordinating atoms. The interaction of

uranium(VI) with hydrazones led to the formation of coloured complexes. These

reactions have been studied spectrophotometrically [68,69]. Biradar and Angadi [70J

have reported the synthesis and spectral studies of uranium(VI) complexes with

aro\ Ihsdrazones. The resulting reddish brown complexes are soluble in N.N-dimeth\ 1

formamide and dimethyl sulphoxide. These complexes have 1:2 stoichiometr> and

can be represented as:

11

The coordination properties [71] of five dyes of alizarin green series. Viz..

compounds 1-V, with uranyl ions were studied in the presence of cationoid surfactants

(cetylpyridinium bromide, carbethoxypentadecyltrimethyl ammonium bromide).

SOgNa

OH

12

il, r = S03Na, R' = R^=R^ = H

HI, R^ = R = S03Na, R' = R^ = H

IV, R^= R-* = SOsNa, R' = R = H

V, R ' = R^ = SOaNa, R = R = H

Ternary complexes of the composition UO2L2S2, UO2L2S4, UO2L2S6 and ,

IJO2L iSg where L is dye and S is surfactant are formed in weekly acid solutions. 1 he

i'ormaiion constants of the complexes were established at pH 4.3-4.8, 4.4-4.9 and 5.2-

5.9 for dyes 11, 111 and IV, respectively. The stability of the complexes is sufficient to

enable a direct photometric determination of uranium in excess dye and surfactant.

Th(IV) chemistry presents an excellent area of research because of its

possibility of formation of compounds with high coordination number. Th(IV) with

ionic radius of 0.99A and a charge of 4+ fulfills the optimum conditions required for a

high coordination. The coordination chemistry of thorium(IV) has been reviewed [72-

74]. Agarwal and Prasad [75] have reported the synthesis of some mixed ligand

complexes of thorium (IV) derived from 4[N-(2'-hydroxy-r-naphthalidene) amino]

aiilip\rene semicarbazone (HNAAPS) or 4[N-Cinnamalidene) amino] anlipvrine

scmicarbazone (GAAPS) as primary ligand and diphenyl sulfoxide (DPSO) as

secondary ligand with the general composition ThX4.n(L).DPS0 (n=l, X = CI, Br,

NCS or N03~; n = 2, X = 1 or CIO4, L = HNAAPS or GAAPS). The infrared studies

revealed that semicarbazone behaves as neutral tridentate (N, N, O) while DPSO

coordinates through its oxygen atom. The nitrates are bicovalently bonded, while

thiocyanate are N-coordinated in these compounds. The structures of thorium(IV)

complexes are given below:

13

-Ttv

X X o

H,C, 51^6 -6^5

[ThX4(L)DPSO]; X = CI, Br or NCS; L = HNAAPS or GAAPS

H5C6 CgHs

[Th(N03)4 (L)DPSO]; L = HNAAPS or GAAPS

H^c;

H.c:

(CIO,)^

[Th(L)2DPSO](C104)4; L = HNAAPS or GAAPS

14

The complexation of Th(IV) with desmethyldesferrithiocin (H2DMDFT). a

derivative of siderophore desferrithiocin (H2DFT) was studied [76] by potentiometn.

spectrophotometry and NMR. Three protonation constants of the Hgand were

determined by potentiometric titrations and ' H N M R and assigned to the phenolalc

group, the nitrogen at the hydroxypyridine ring and the carbox\iate group. Ihe

spectrophotometric studies suggested a 1:2 Th/ desmethylferrithiocin complex. The

formation constant of 1:2 complex. Th (DMDFT)2, was determined (log p; = 26.7).

II

^ ^ ^ / / . - . .OH

" N " ' ^ - ^ N COOH ^N-^^^^^-^^ 1 Y \ \ COOH ^"•^-^ CH3 S ^

H2DFT H2DMDFT

The interaction of Th(IV) with N-hydroxy ethylethylenediaminetriacetic acid

(HEDTA) and with the related ligands nitrilotriacetic acid (Nl'.A). \'-

h_\droxethyliminodiacetic acid (HIMDA). N-meth_\ liminodiacelic acid (MIMD.X).

o\_\bis (ethylenenitrile) tetraacetic acid (OETA) and eth\ienebis (ox>eth>lencniirilc)

letraacetic acid (EOTA) has been studied potentiometrically [77]. Evidence is

presented for hydrolysis reactions of Th(IV) chelates of all these ligands as the pH

increased. The equations for the hydrolysis reactions are presented as:

Til L (H2O)" ^—^— Th (OH) L + H^

2 Th L (UjOf ~—^— Th L(0H)2 Th L + 2H*

2 Th (OH) L - ^ - Til (0H)2 Th E

The formation constant of 1:1 Th(lV) - HEDTA chelate was determined to be

10'^ \ It was found that rh(iV) reacts with l-phenyl-3-methyl-4-benzoyi-5-

15

pyrazolone [78,79] to yield a stable complex. This reagent is used mainl> for

separation of Th(lV) from other metals to determine its content in rock and soil.

This thesis deals with:

(i) interaction of Fe(lll) with piroxicam. The resulting citiourcd complex ha--

been utilized for spectrophotometric determination of Fe(IlI) in soil samples,

(ii) Interaction of Cu(II) with cefixime. The Cu(II)-cefixime complex has been

studied spectrophotometrically to determine the Cu(Il) content in water

samples,

(iii) Interaction of Th(IV) with rifampicin resulted in the formation of coloured

complex. A spectrophotometric method was developed to determine the

rh(IV) in soil samples,

(iv) Interaction of U(V1) with rifampicin. The lJ(Vl)-rifampicin complex was

exploited to develop a spectrophotometric method for determination of

U(V1) in soil samples.

The use of metals in human history has yielded great benefits as well as

unexpected harmful consequences. The generic term metal refers to roughh 70

electropositive elements in the periodic table. As a group, they share some common

physical, chemical, and electrical properties. Further, while sharing common

properties, metals exhibit wide ranges with respect to one another, in both chemical

behaviour and the measured values of those common properties. Historically, it has

been the exploitation of these properties of metals which has led to successi\c waves

of progress in the development of modern technological society and its dependence

on, and increasing appetite for metals. The chemical and radioactive behaviour of

some metals affects the biological s\ stems and poses the serious health problems. The

elements of greatest concern include Pb (Group 14), transition elements: Pd, Ni, Cr.

16

Cii. Fe. Cd and Hg. and As (Group 15). Other different types of metals, like Cs (an

alkali metal). Be, Sr, and Ba (alkaline earth metals), and U and Th (rare earth meials)

also pose health problems [80].

Man's perturbation of nature's slowly occurring life cycle of metals includes

(i) the extraction, smelting, and processing of metal bearing ores into products, (ii) the

distribution and use of these products by industry and consumers, and (iii) the return

of these metals in a concentrated form to the natural environment through disposal of

processing wastes and the discard of spent products, 'fhe metal or metals thus become

contaminants in the receiving environments. Part of the reason the\- become

contaminants is seen within the description of the man-made life cycles above. The\

include (i) the rapidity of the man-made cycles relative to natural ones, (ii) the

transfer of the metals from mines to random environmental locations where higher

potentials of direct exposure occur, (iii) the relatively high concentrations of the

metals in discarded products compared to those in the receiving environment, and (iv)

the chemical form, or species, in which a metal is found in the receixing

en\ ironmcntal s\'stem.

fjivironmental concerns drove an interest in metal species determination after

the accidental mercury poisoning in Minamata. Japan in the I95()s. Other

environmental sectors such as waste water treatment and waste management will be

reliant on metal species determinations. Environmental issues will be driven with the

introduction of environmental regulations. These regulations may be set when

commercial instrumentation is available and further communication between

ecotoxicologists, environmental and analytical chemists is improved. Therefore, the

concept of trace metal or elemental speciation has aroused increasing interest among

the inorganic analytical chemistr> community during the last 30 >ears [8 I J. Together

17

with the progress in analytical instrumentation and analytical schemes, the need for

species idenlification will bring new insights regarding the use and applicabilit\ oi

trace metal species determination. This knowledge is the result of the continiuuis

e\olution of approaches developed by environmental chemists, gcochcniistx aiiJ

biologically oriented chemists relying on solid chemical reasoning.

Environmental research has now clearly established that metals and metalloids

occur in the environment as a large array of chemical species. They occur at low

concentrations as free metal ions and are most commonly found in combined forms.

For instance, these may be found as inorganic salts, oxyhydroxides, and charged or

neutral organometallic entities. They can also be found integrated into larger

biological molecular structures such as amino acids, proteins and metalloenzymes. ,\ll

of these various chemical species undergo continuous chemical changes with kinetic

and thermodxnamic forces to constantly change the eco.sstem. It has now been well

established that the chemical species of a metal closely regulates its fundamental

physico-chemical properties (liquid to solid and liquid to gas partitioning) and hence

its mode and the occurrence of its bioaccumulation in the food chain. The type oi'

species present also directly determines the toxicity of the metal in biota [82-88].

The toxicity of trace metals in environmental sciences is an area of increasing

interest. In the environment, many diverse species of an element can be present, and

different species of the same element can possess ver\' different degrees of toxicit).

Monitoring elemental species requires an analytical method that is sensitive and

specific enough to resolve and quantify the individual species may constitute onl\ a

fraction of an element's total concentration in a sample.

The primary requirements for metal and metalloid species determination are

related to the toxicity aspects regarding biological systems. This attitude towards

18

meial speciation is. however, changing and the demand for speciation now comes

from areas other than environmental analysis. Demands are now increasing in

industrial process control, the food industry, and the biomedical and the

pharmaceutical fields. However, metal species determinations should be understood

as a way to increase and improve the qualit' of industrial processes at lower cost.

Increasing information exchange between academic research groups and their

industrial partners will facilitate enhanced metal speciation analyses. The nuclear

industry should also benefit from the development of chemical species identification.

In this field, the concept of determining chemical species has been developed.

However, most of the analytical results are instrument-limited and rel\ on

radiochemical procedures using gamma spectroscopic detection. The rapid

development of uhrasensitive techniques using coupled systems with ICP-MS

detection should bring new insight into the field of chemical species determination of

actinides. Table 1.1 lists different types of applications where chemical species

determination is playing a fundamental role in the evolution of a particular subfield.

Hundreds of mineral species contain iron as a constituent, and igneous rocks

average about 5 percent iron content. The major iron ores are the minerals licmatitc

(Fe^Oj). magnetite (Fe304), limonite (FeO(OH)-/7H20). and siderite (FeCC),).

Iron is the most useful of all metals. It is also the cheapest available metal.

Most is used to manufacture steel. Ordinary carbon steel is an alloy of iron with

carbon (about 1.5%), with small amounts of other elements. Alloy steels are carbon

steels with other additives such as nickel and chromium. Wrought iron is iron

containing a very small amount of carbon, and is tough, malleable and less fusible

than pure iron. Pig iron is an alloy containing about 3% carbon with varying amounts

of sulfur, silicon, manganese and phosphorus. It is hard, brittle, fairly fusible and is

19

Table 1.1. Determination of metal species in the selected fields of development.

Element Al

Sb

As

Cd Cr

Cu

Fe

Pb

uL_ ..._ Hg

Pt

Se

Sn

Th

U

Selected field Industrial Biomedical Food Environmental Food

Environmental Biomedical Food Industrial hygiene

Environmental Environmental Food Environmental Food Environmental Food Biomedical Environmental industrial hygiene Industrial hygiene Environmental Industrial hygiene Food Environmental Pharmaceutical Biomedical Environmental Food Environmental Industrial Industrial Environmental Industrial En\ ironmental

Applications Polymerisation products Chemical forms in serum and biological tliiiJ^ Chemical forms in foodstuffs Redox and organometallic species in sea and soil Redox and organometals in foodstuffs Redox and organometallic species As in proteins, haemoglobin and biological fluids As in foodstuffs Workplace atmosphere Cd complexes and metalloproteins Redox species in the environment Food stuffs Redox species in soil and water samples Species in foodstuffs Species in soil and water samples Species in foodstuffs Fe in blood, serum and proteins Species in soil and water samples Species in the atmosphere Phosphine in workplace Species in soil and water samples Species in workplace Species in foodstuffs Species in soil and water samples Cis-Pt formulations Cis-Pt in cancer therapy Redox and organometallic species Species in foodstuffs Species in soil and water samples Formulations in catalysts Species in effluents and waste waters i Species in soil, water and nuclear sites ' Species in effluents and waste waters Species in soil, water and nuclear sites

20

used lo produce other alloys including steel. Therefore, Iron is one the most iniportaiu

constituent of metallurgicii! industry. Hence determination of iron is \ery important,

Zaijun et al. and Sarma et al. have analyzed Fe(IlI) by forming complex of Fe(ill)

with dimethyldithiocarbamate [89] and pyridoxal-4-phenyl-3-thioscmicarbazone

[90]. respectively.

in sufficient amounts, copper salts can be poisonous to higher organisms as

well. Copper is a naturally occurring element that is present in drinking water.

Stagnation of water in pipes and plumbing fixtures containing copper and copper

alloNS in distribution systems and household plumbing allows leaching and increases

water copper levels. Characteristics of the water, including increased acidit\.

increased temperature, and reduced hardness, can increase the leaching of copper into

the water. Acute ingestion of excess copper in drinking water is associated with

adverse health effects, including acute gastrointestinal disturbances, and liver toxicit\.

A model for liver toxicity might be derived from patients with Wilson disease, which

causes abnormal copper regulatory mechanisms that result in accumulation of excess

copper. The current EPA MCLG of 1.3 milligrams per liter (mg/L) for copper in

drinking water is based on the need to protect against ad\erse gastrointestinal effects,

fhus. the determination of Cu(II) is becoming important due to the ill efTects o\'

excess copper in drinking water and soil. Therefore, there is a need to de\elop no\el

methods for the determination of Cu(II) that might be important to know the exact

concentration of Cu(ll) in soil and water system. So far. researchers have developed

methods for estimating Cu(l!) by forming Cu(II) complexes with electron rich species

such as 2-ethanolimino-2-pentylidino-4-one [91]. l-(2-quinolylazo)-2,4.5-

trihydro.xybenzene [92], l,l,l-trifluoro-3-(2-theno\l)acetone [93]. diacetyl

21

monooxime [94]. These metal-ligand complexes were monitored

spectrophometricallx and utilized to know the concentration of Cu(ll).

Uranium is a silvery-white metallic chemical element in the actinidc series of

the periodic table. The actinides refer to the fourteen elements with atomic numbers

90 through 103 i.e. from thorium to lawrencium through uranium (.Atomic number

92). Uranium is radioactive, long-lived, and highly toxic element. Uranium-238. the

most prevalent isotope in uranium ore, has a half-life of about 4.5 billion years.

Uranium-238 decays by alpha emission into thorium-234, which itself decays b beta

emission to protactinium-234, which decays by beta emission to uranium-234. and so

on. The various decay products, (sometimes referred to as "progeny" or "daughters")

form a series starting at uranium-238. After several more alpha and beta decays, the

series ends with the stable isotope lead-206. As long as it remains outside the body,

uranium poses little health hazard (mainly from the gamma-rays). If inhaled or

ingested, however, its radioactivity poses increased risks of lung cancer and bone

cancer. Uranium is also chemicall_\ toxic at high concentrations and can cause damage

to internal organs, notably the kidneys. Uranium content of land waters in excess oi' i

ppb (Ijag L"') is regarded as anomaly.

Uranium occurs in +III, +IV, +V and +VI oxidation states. However

compounds of U(VI) is of considerable importance to analytical-inorganic chemists.

Uranium species of interest is the uranyl ion, U02^^ which is stable, highly soluble

and mobile in aqueous phase. Uranyl ion can be found in soils and in low pH-water

runoff in and around nuclear waste sites and processing facilities. The uranyl unit

consists of a uranium centre with a formal charge of+6 coordinated to two double

bonded oxygen atoms for a linear dioxo cation. This unit is highly stable and binds to

22

other ligands \ ia the formation of U-0 bonds in a plane perpendicular to the axis o\

the uranvl ion.

The spectrophotomelric methods are virtuaiK the commonest methods in

chemical anal) sis. Colour reactions of practical value have now been found lor aiinosi

all inorganic ions. An increasing number of analytical methods based on metal-ligand

interactions are based on the corresponding colour reactions. Electron rich species

such as o-hydroxypropiophenone isonicotinoyl hydrazone [95], 2-

hydroxybenzohydroxamic acid [96], 4-(2-pyridylazo)resorcinol [97], 2-(2-

thiazolylazo)-p-cresol [98] were reacted with U(VI) to form coloured complexes and

thus exploited for the determination of U(V1).

Thorium is a naturally occurring, slightly radioactive metal, it is estimated to be

about three to four times more abundant than uranium in the earth's crust. It has been

considered a waste product in mining rare earths, so its abundance is high and cost

low. Monazite is the primary source of thorium. Thorium is present in very small

quantities in virtually all rock, soil, water, plants and animals. Where high

concentrations occur in rock, thorium may be mined and refined, producing waste

products such as mill tailings. If not properly controlled, wind and water can

introduce the tailings into the wide environment [99.100]. If inhaled as a dust, some

thorium may remain in the lungs for long periods of time, depending on its chemical

form. If ingested, thorium typically leaves the body through face and urine within

several days. The small amount of thorium left in the body will enter the blood stream

and be deposited on the bones where it may remain for many years. Studies have

shown thai inhaling thorium dust causes an increased risk of developing lung cancer,

and cancer of the pancreas. Bones cancer risk is also increased because thorium may

be stored in bone [101-103].

23

Thorium occurs in +111 and +IV oxidation slates. Th(IV) oxidation stale is the

only state observed in aqueous solution [104]. This is untypical of the actinides where

HI! tends to be dominant state. The precipitation with oxalate from dilute acid

solution is a principal means of separation from most elements including zirconium

and titanium.

Spectrophotometry enjoys a significant role in the determination of traces of

metals due to simplicity and accuracy. The problem of chemical analysis generally

follows two steps: (i) Separation of the desired constituent and (ii) determination of

the desired analyle. Much research has been carried out for the determination i iiIV)

by employing specific electron rich species such as 2-hydroxy-l-napthaldehyde

isonicotinoylhydrazone [105], Semi-xylenol orange [106], 2-carboxy maleianilic acid

[107], N-(2-hydroxyethyl)ethylenediaminetriacetic acid [108] and four azo

compounds based on l-phenyl-2, 3-dimethylpyrazoline-5-one nucleus namely 4-

phenylazo- (2-hydroxy, 5-x) l-phenyl-2, 3-dimethyl-pyrazoline-5-one, where x= H

(1), OH (II), COOH (III) and NH2 (IV) [109]. These Th(IV)-ligand complexes have

given characterstic colour reactions. This fact of complex formation was exploited

and thereby utilized for the determination of Th(lV) b> spectrophotometric method.

Sampling

There is no general agreement on the solutions preferred for the various

components in sediment or soils to be extracted, due mostly to the matrix effect

involved in the heterogeneous chemical processes [110]. All factors have to be

critically considered when an extractant for a specific investigation is chosen.

Important factors are the aim of the study, the type of solid materials, and the

clemenls of interest. Partial dissolution techniques should include reagents that are

sensitive to only one of the various components signitkant in trace metal binding.

24

Whatever extraction procedure is selected, the validity of selective extraction results

primarily depends on the sample collection and preservation prior to anal> sis.

A sampling plan has to be established prior to sampling. The purpose and

e.xpecUilion ol a sampling program must be realistic and can nc\cr surpass ihc

measurement and sample limitations. Moreover, costs and benefits must be

considered in the design of ever)' measurement program. The total variance of an

analysis (,v tmai) is expressed as:

2 = 2 , 2 ^ total ^ measurement '" ^ sampling

where .v measurement and .s' sampiing are the variances due to the measurement and

sampling, respectively [111]. The measurement, sampling plans and operations must

be designed and accomplished so that the individual components may be evaluated.

Sampling uncertainty may contain systematic and random components arising troin

the sampling procedure. In environmental sampling, the act of sample removal trom

its natural environment can disturb stable or meta-stable equilibria, if the test portion

is not representative of the original material, it will not be possible to relate the

anahtical result to the original material, no matter how good the analytical method is

nor how carefully the analysis is performed. Further, sampling errors cannot be

controlled by the use of standards or reference materials.

Sampling of sediments and soils

Because of the heterogeneity and complex nature of sediments, care should be

taken during sampling and analysis to minimize changes in speciation due to changes

in the environmental conditions of the system. Sampling for pollution mapping has to

consider the heterogeneity of the deposit by methods such as particle size analysis and

geochemical normalization. Sediment sampling must avoid alteration of natural

biogeochemical processes, which would affect results by the unrepresentativeness of

25

the original equilibrium. Consequently, sampling variance and artifacts introduced

during processing of samples can be more than an order of magnitude greater than

analytical measurement variances in trace element speciation [112]. Schoer [113j has

studied the effect of particle size of sediments on the adsorption capaciix. Variaiion.s

in the behavior of different elements with particle size is attributed largeK to

differences in their relative potential for sorption on clay minerals, hydrous oxides,

and organic matter surfaces, all of which tend to be concentrated in smaller grain

sizes. The maximum concentration of organic carbon in the sediment samples was

found in a size range of 2-6.3 |tm, whereas smaller fractions showed onl\ traces o\'

organic carbon. On the other hand, easily reducible manganese reached its highest

concentration in the fraction of <2 |im. Appropriate comparability among oxide

sediment samples collected at different times and places from a given aquatic s\ stem

and between different systems can be obtained most easily by analyzing the fme-

grained fraction of sediment. Some investigations have also pointed to a relation

between specific surface, grain size fraction, and the speciation of trace elements in

sediments. .Amorphous Fe-oxide precipitates appear to be most signit^icant in affecting

both surface area and sediment trace metal levels. It was found that externdl surlacc

area, determined by Brunauer-Emmett-Teller (BET) method, is a function of both

grain size and of composition of geochemical phase [114]. Suspended particulate

matter sampling is mainly carried out by filtration. Such samples are of limited utility

for studies of the speciation of elements in solids. In recent years, suspended sediment

recovery by continuous-flow centrifugation has commonly been used to obtain

sufficient sample for speciation, up to a few grams to carry out all the anahsis:

panicle size distribution, mineralog_\. total and sequential extractions conlcnl.

ITchehcr ct al. |I15] provided a comparative slud\ of suspended particle mailer

26

separation by filtration, continuous-flow centrifugation. and shallow water sedimeiil

traps. Although particles were separated by density, rather than size, the continuous-

tlow centrifugation technique was preferred due to its speed and high rcco\cr_\ rale.

The conlinuous-flow separation technique is simpler \o use especial!) on the open ^ca.

where suspended sediment concentrations are low. Trace elements in Nuspciidcd

particulate matter from open North Sea have been measured for particle size

distribution, specific surface, bulk concentration, and partitioning between fi\c

sequential extraction fractions [116].

The trace element concentration in soils and sediments are normally much

higher than those for water samples, many precautionary steps taken relating to

sample container preparations and sampling of waters are equalh applicable to soil

and sediments. Soil composition may vary greatly over a small area. Samples have lo

be taken from a number of locations to obtain a suitable average composition studies,

the source of contamination and its mobility within the soil should be taken into

account. Often pollutants deposited from the atmosphere are immobile and will

remain within the surface layer. If the soil is disturbed, sample should be taken from

the whole of the disturbed area. For landfill sites, samples should be taken over the

complete depth of the land fill. The samples collected should be representative of the

sampled land. In many cases it is important to take sub samples within a defined area

in order to provide a representative composite sample.

Sediment sampling is normall' carried out using a tube corer or a grab

sampler. Core samplers are used for shallow areas. The tube is immersed with \al\c

SNSiem open. The valve is then closed to permit the sample to be withdrawn, .lust

before breaking the surface of the water, the tube is sealed to preserve the sediment

structure so that sections corresponding to different depths in the sediments can be

27

analyzed. Grab samples are used where the sediment is loose so that there is iin

vertical structure.

Sample preparation

Soil and sediment samples should be stored in sealed polythene containers at

40 C until arrival at the laboratorj'. The samples are seperated into particle sizes b\

wet or dry sieving using 63 jxm or 20 |j.m nylon sieves. Moist sediments are often used

for metal specification analysis. Such samples should be sealed under the nitrogen in

polyethylene containers and frozen. Before chemical analysis, the samples should be

0

homogenised and dried (30 - 60 C) to a constant weight, Homogenisation is achiexed

by a grinding mill or agate pestle and mortar. The powdered sample is poured into a

cone shaped heap, divided into four equal parts. Two opposite quarters are combined

and reconed. The process is repeated until the amount of the sample is reduced to that

required for analysis.

Analytical methodology

Total metals determination requires the extraction of the element from a

complex matrix. The detection step using atomic absorption spectroscopic techniques

is usual! performed without matrix removal, which ma\ lead to se\ere inierfercncc

problems. Most of the analytical instrumental development has been associated with

the spectroscopic reduction of these interferences in order to yield improved qualit)

data. Despite the important instrumental progress, interlaboratory comparison

exercises show major discrepancies in the results obtained from the laboratories

involved. Metal species determination requires careful extraction and preservation of

the analytes. These steps considerably minimise the final amount of material

introduced at the detection stage and hence considerably reduce spectroscopic

28

interferences compared with total metals determination. European Economic

Community interlaboratory exercises have frequently shown that a better agreement is

obtained b) participants performing species determination compared with those

measuring the total metals content for the certification of reference materials. Pan ol

the future development in this area will be closeK related to the introduction vM

commercial instrumentation for speciation. Man>' chemical approaches can be suUed

with simple low-cost instrumentation. There is, however, a concern with man\

methods being developed that use expensive mass spectrometric detection systems

only available in certain laboratories. Fig. 1.1 illustrates the importance of analytical

instrumentation in environmental decision making processes. Biological systems can

be responded to chemical species and the species integrity can be maintained using

appropriate instrumentation. Regulation and control are correct tools so that decision

making ma_\ be rationalised. Applications of metal and meialloid species

determinations are numerous and speciation analyses should soon be substituted for

current total metal determinations. Many areas, however, cannot \\aii for the

implementation of regulations. The food, pharmaceutical, biomedical and chemical

industries will rapidly require high-quality information obtained by metal species

determination to improve and consolidate their developments.

Man> organic compounds (natural and synthetic) possess electron rich centres

due to the presence of N, O and S are capable of forming metal complexes. Metal

complexes have spectacular colors. These colors arise from the absorption of light

which excites electrons within the metal's orbilals or. in the case of charge-transfer,

e.xcitcs electrons from metal-based orbitals to ligand-based orbitals (or the rc\ersc).

fhe interaction of metal ions with electron rich species leading to the formation (»f

metal complexes is exploited to develop analytical procedures for the estimation of

29

Fig. LI. Importance of analytical instrumentation in speciation of metals.

30

metal ions in soil, sea, sewage and natural waters. This aspect of stud\' is being

helpful in monitoring the pollution level. Instrumental analysis is opened many doors

to scientific progress in support of both traditional core areas of chemi.stry, and ne\\

ones like biotechnology, material chemistry, environmental chcmislr, chemical

toxicology and small domain chemistry.

UV-visible spectrophotometric methods continue to be popular for carr\ing

out single component assays on a variety of metal ions via complex formation with

suitable ligands. Represantative examples of UV-visible spectrophotometric methods

of metal analysis [117-137] that have been published are given in Table 1.2.

VALIDATION

The method to be fit for the determination of metal ions in soil and natural

vater samples, the method must meet the following validation characteristics. Typical

alidation characteristics (Table 1.3), which should be considered are as follows:

Seiectivity/ Specificity

Linearity

Range

.Accuracy

Precision

Limits of detection and quantitation

Robustness / ruggedness

Selectivitj'/ specificit>'. Selectivty of a method refers to the extent to which it can

determine particular analyte(s) in a complex mixture without interference from other

components in the mixture. The terms selectivit_\ and specificit ha\e been used

interchangeabl). The term specific generally refers to a method that produces a

3 1

Table 1.2. UV-v

ion in cnvironin

isible spectrophotometric methods for the determination of metal

ental samples.

Metal ions

Cu(ll)

I'(VI)

U(V1) " "

Fe(lII)

Cu(ll)

Ni(ll)

Bi(III)

Pb(II) Tl(III) As(lll)

Ni(ll) Hg(ll)

iF<I') V(V) ^ Pdn)

Cr(Vl) and V(V) U(VI) La(IlI) Au(I!) Pb(II) Cr(VI)

Reagents used

Chlor(phen\ 1) glyoxime

Meloxicam

Piroxicam

Piroxicam

Cefixime

2-Hydroxy-3-methoxybenzaldehyde

thiosemicarbazone

l-amino-4,4,6-trimethyl(lH,4H) pyrimidine 2-thiol Benzoic acid azo phenylcalix[4]arene 4-(4'-A''..'V-dimethylaminophen\ i)ura/ole 2-(5-bromo-2-pyridylazo)-5-di-ethylaminophenol Dibromo-/?-methyl-carboxyazo Diphenylthiocarbazone 2,2"-pyridine Variamine blue 2-(2-quinolylazo)-5-diethylaminobenzoic acid 3,4-Dihydroxyben2aldehyde isonicotinoyi hydrazone ArsenazoIII 1 -(-2-pyridylazo)-2-naphthol 2-carboxyl-1 -naphthalthiorhodanine Chromazurol S FeiToin

>.,„a,(nm)

290.5

398

390

495

336

410

470

440 514 560

625 488 522 570 628

400 & 360 651 530 540 520 510

References

|118|

[119]

[120]

[121]

[122]

[123]

[124] [125] [126]

J\21\ ' [128] [129] [130] [131]

[132]

[133] [134] [135] [136] [137]

32

Table 1.3. Validation characteristics nonnaily evaluated for different types of

test procedure and the minimum number of determinations required (if

applicable).

Validation characteristics

Specificity Linearity Range Accuracy

Precision Repeatability

Intermediate Precision/ rep­roducibility Detection limit Quantitation limit

Minimum number

-5 concentrations -9 determinations over 3 concentration levels (e.g. 3 x 3 )

6 determinations at 100% or 9 determinations over 3concentration levels (e.g. 3 X 3) 2-series

--

Test procedure

Identity

Yes No

Tlo J No

No

No

No No

Impurities

Quantitative

Yes Yes Yes Yes

Yes

Yes

No Yes

Limit

Yes No No No

No

No

Yes No

Assay

Yes Yes Yes Yes

Yes

Yes

No No

33

response for a single analylc only, while the term seleelive refers to a method, \siiich

provides responses for a number oi' chemical entities that ma\ or ma> not be

distinguished from each other. If the response is distinguished from all other

responses the method is said to be selective. Since there are very few methods that

respond to only one analyte, the term selectivity is usually more appropriate than

specificity. The International Union of Pure and Applied Chemistry (lUPAC) has

expressed the view that "Specificity is the ultimate of selectivity". The lUPAC

discourages the use of the term specificity and encourages the use of the term

selectivity.

Linearity. The linearity is the ability of the method to produce test results which are

proportional to the concentration (amount) of analyte in samples within a given

concentration range, either directly or by means of a well-defined mathematical

transformation. Linearity should be determined by using a minimum of five standards

whose concentration span 80-120% of the expected concentration range. The linearity

of a method should be established by the inspection of the plot of the instrumental

response versus the initial concentration of analyte. If there is a linear relationship,

test results should be evaluated by appropriate statistical methods, for example, by

calculation of the regression line using lea.st square method.

Range. The specified range is derived from the linearity studies. The range of the

proposed procedure is the interval between the upper and lower concentration

(amount) of analyte in the sample for which it has been demonstrated that the

analytical method has suitable levels of precision, accuracy and linearity.

Accuracy. The accuracy of a method is defined as the degree to which the determined

value of analyte in a sample corresponds to the true value. Accuracy may be measured

34

in different ways and the method should be appropriate to the matrix. The accurac of

an analtical method may be determined by any of the following ways:

• Analyzing a sample of known concentration and comparing the measured value to

the "true" \alue. However, a well characterized sample (e.g. reference standard)

must be used.

• Standard addition method. In the standard addition method, a sample is assayed,

a known amount of pure active constituent is added, and the sample is again

assayed. The difference between the results of the two assays is compared with the

expected answer.

Precision. According to International Conference on Harmonisation (ICH). the

precision is the closeness of agreement (degree of scatter) between a series of

measurements obtained from multiple sampling of the same homogeneous sample

under the prescribed conditions and may be considered at three levels:

• repeatability

• intermediate precision

• reprt)ducibilit>

• Repeatability'. It is the precision obtained b>' independent test results with the

same method on identical test material in the same laborator\ by the same

operator using the same equipment within short interval of time. It is also termed

as intra-assay precision [138]. Sometimes it is also termed as within run or within

da\ precision.

• Intermediate precision. It expresses within-laboratories variations: different

da\s. different analysts, different equipment etc. The International Organization

for Standardization (ISO) definitions used the term •'M-factor diffciem

intermediate precision" where the M-factor expresses the number of tactors

35

(reference standard, operator, equipment, laboratory or time) that differ between

successive determinations [139]. Intermediate precision is sometimes also called

between-run, between-day or inter-assay precision.

• Reproducibility. It is the precision obtained within the ^amc method on

identical test material in different laboratories with different operators using

different equipments [140].

Limits of detection and quantitation. Limit of detection (LOD) determines the

lowest amount of analyte that can be detected, as it (the analyte) yields instrumental

response greater than a blank, but cannot be quantified. It is a parameter of "limit test""

and expected to produce a response, which is significantly different from that of a

blank. On the other hand limit of quantitation (LOQ) is a parameter of "deicrminaiion

test"" and can be defined as the lowest concentration of the anahte that can be

measured and quantified with acceptable precision and accuracy.

The most common definition of LOD and LOQ is the anaUte concentration

for which the signal exceeds that for a realistic analytical blank by three and ten times

of the standard deviation, respectively. Several approaches have been gi\en in the

ICH guidelines to determine the detection and quantitation limits (Table L 4).

Robustness/ruggedness. The "robustness / ruggedness" of an analytical procedure is

defined [141] as a measure of its capacity to remain unaffected b> small, but

deliberate variation in method parameters and provides an indication of its reliahilit\

during normal usage. Ruggedness is a measure for the susceptibilit of a method to

small changes that might occur during routine analysis like small changes of pH

values, mobile phase composition, temperature etc. Full validation must not

neces.sarily include ruggedness testing; it can, however, be very helpful during the

36

Table 1.4. Approaches for determining limits of detection and quantitation.

Approach Detection limit Quantitation limit

Visual evolution

Signal-to-noise

Standard deviation of

the response (So)' and ^ ^ x (S )/b the slope(b)

Minimum level detection Minimum level quanliflabic

3:lor2:l 10:1

10.0 X (S,)/b

^ Standard deviation of the blank, residual standard deviation of the calibration line, or standard deviation of the intercept.

37

melhod development / prevalidation phase, as problems that may occur during

validation are often detected in advance. Ruggedness should be tested, if a method is

supposed to be transferred to another laboratory.

STATISTICAL ANALYSIS

Recent trend in the determination of metal emphasizes the use of staiisiical

analysis for evaluation of the method performance which includes the following

parameters [142,143]:

Mean

Standard deviation, variance & standard analytical error

Relative standard deviation

Coefficient of correlation

Regression line

Variance

Errors in the slope and the intercept

Conildence limit for the slope and the intercept

Error in the concentration

Equivalence testing

Interval hpothesis

Mean. It is the sum of all the measurements divided by the number of measurements.

It is calculated by the following expression:

X = y 'x . /n

Standard deviation, variance & standard analytical error. The most useful

measure of spread is the standard deviation, S.D. This is defined by the formula:

38

S.D.= ] y (.V. — yi ) - / i. iJ — i )

The square of S.D. is a very important quantity known as the variance which is uselui

in propagation of error.

The standard analytical error (SAE) of the mean is calculated by SAE = S.D. / Vn

Relative standard deviation. It is calculated by

RSD = ^^ly_ X 180

The RSD (also called coefficient of variation), the units of which are percent is an

example of relative error.

Coefficient of correlation. When using instrumental methods, it is necessar\ to carr

out a calibration process by using a series of samples (standard) each having a known

concentration of analyte. Two statistical procedures should be applied to the

calibration curve:

• Test whether the graph is linear or in the form of a curve

• Find the best straight line (or curve) through the data points

Linearity is Judged by correlation coefficient, "r". which can be calculated for a

calibration curve to ascertain the degree of correlation between the measured

instrumental variable and the sample concentration.

V[«Xx-:-(Zx,)^][«Ejf-(i:>^,)^]

Where n = number of data points

The maximum value of r is T When this occurs there is exact correlation

between the two variables (x and y). When the value of r is zero (x = 0). there is

complete independence of the variables. The minimum value of r is -I. indicates thai

39

the assumed dependence is opposite to what exists (Fig. 1.2). As a general rule, 0.90 <

r < 0.95 indicates a fair curve, 0.95 < r < 0.99 as a good curve, and r > 0.99 includes

excellent linearity.

Regression line. The best straight line through a series of experimental points is thai

line for which the sum of the squares of the deviation of the points from the line is

minimum. Besides, determining a straight line, uncertainties in the use of calibration

graph for analysis of unknown samples can be specified by this method of least

squares. The equation of the straight line is

A = a + b C

Where A = instrumental response (i.e. absorbance), b = slope, a = intercept. C =

concentration of the standards.

To obtain the regression line A on C, the slope 'b ' of the line and the intercept

'a" on llic y-axis are given by the following equations:

Zx'-iilxf/n]

a = y - bx

Where x = mean of all the values of x, and y = mean of all the values of j , .

Errors in the slope and the intercept. The determination of errors in the slope (b)

and intercept (a) of the regression line may be calculated by first calculating S,,

(standard deviation of the calibration line) from the following equation:

S„=^I.y,-yY/r,~2)

where i' values are obtained from calculated regression line for gi\en values oi" \;

once the value So has been obtained, both the standard deviations of the slope Si, and

40

Fig. 1.2. Linearitj' with the values of correlation coefficient.

4 1

the intercept S;, can be obtained from the following equations

s, = 5J^x,-.x)-

Confidence limit for the slope and the intercept. It determines whether the slope

and'or intercept of a line differ significantly from a particular or predicted vakie. It

can be calculated [144] in the following manner:

• b ± / Sb (for slope)

• a ± / S;, (for intercept)

where / = tabulated 't" value at desired confidence level for (n-2) degrees of freedom.

Error in the concentration. The determination of the error in the x-value can be

calculated b\ the following formula:

' b 1 ^ . (>•->')-n b'Zx-xy-

where x and v are the average concentration and absorbance values. respectivcK.

for 'n' standard solutions.

Equivalence testing. An important propert>' of an analytical method is that it should

be free from the systematic error (bias). Determining bias involves anahzing one or

more standard reference materials whose analyte concentration is known. Howe\er,

random errors make it unlikely that, the measured amount will equal to the known

amoum e\en when no systematic errors are present. In order to decide wiielhcr the

ditlerence between the observed and standard values can be accounted for b\ random

42

variation, a statistical test i.e. a significance test is used for the interpretation ot

analytical data.

• Student's t-test. Here comparison is made between \\\o sets of rcplicalc

measiiremcnls made by two different methods; one is the lest method \shilc oihci

is accepted (reference method).

where

A'l = mean from the test method

v, = mean from the accepted (reference) method

ni and n- = number of measurements

Sp = pooled standard deviation of the individual measurements of two sets is given b\

^ ^ n,-i)Sl+u,~\)S:

.A statistical /-value is calculated and compared with a tabulated value for the

given number of tests at the desired confidence level. If i^^i > ab then there is

significant difference between the results obtained b> the two methods at the gi\en

confidence level, but if /cai < Aab then there is no significant difference between the

methods. It is an accuracy-indicating test.

43

F-Tcst. This test indicates whether there is a significant difference between the

t ^o methods (i.e. the new method and the accepted relerence method). It can be

represented as:

S

where S,' > S;

If i-'cai Fuib at the selected confidence level, then there is a signiilcant

difference between the variances of the two methods.

Interval hypothesis. For a method, a bias of ± 2.0 % is acceptable [145] and can be

calculated statistically [146] using the following quadratic equation:

where x, and x. are the means of methods 1 and 2. based on n and nj

measurcnients, respectively. Sp is the pooled standard deviation and /i,! is ihc

tabulated one sided /-value, with ni + n2- 2 degrees of freedom at the specitled kncl

of signillcance.

44

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136] E. E. Kostenko, J. Anal. Chem. 65 (2010) 366.

52

[137] S. P. Arya. A. Bansal. Fres. Z. Anal. Cliem. 348 (1994) 772.

[138] M. Thompson, S. Ellison, R. Word, Pure Appl. Chem.74 (2002) 835.

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Precision) of Measurement Methods and Results. ISO/DIS (1994) 5725.

[140] R. Causon, J. Chromatogr. B. 689 (1997) 175.

[141] H. T. Karnes, G. Shiu, V. P. Shahi, Pharm. Res. 8 (1991) 421.

[142] G. B. Limentani, M. C. Ringo, F. Ye, M. L. Bergquist, E. O. McSorley. Anal.

Chem. 77(2005)221A.

[143] D. R. Bristol, Commun. Statist. Theory Methods 22 (1993) 1953.

[144] R. Cassidy, M. Janoski, LC-GC 10 (1992) 692.

[145] Canada Health Protection Branch, Drug Directorate Guidelines: Acceptable

methods. Ministry of National Health and Welfare, Draft, Ottawa, Canada

(1992).

[146] C. Hartmann. J. Smeyers-Verbeke, W. Pinninckx, Y. V. Heyden. P.

Vankcerberghen. D. L. Massart. Anal. Chem. 67 (1995) 4491.

53

Spectrophotometric determination of Fe(III) via

complexation with piroxicam in synthetic

mixture and soil samples

54

INTRODUCTION

iron is one of the most important constituent of biological systems as well as of

metallurgical industry. It reacts with di-antipyrene methane to form a red coloured

complex, which absorbs maximally at 520 nm [I]. Trace amount of iron(lll) was

detemiined by complexation with potassium thiocyanate and methyl violet in

presence of polyvinyl alcohol [2]. Colour reaction between Fe(lII) and p-diethyl-

amine phenyl fluorine at pH 9.0 was utilized for the determination of Fe(lil) [3] in the

concentration range of 0.056 - 6.7 \ig I 25 ml. A number of kinetic

spectrophotometric methods have been utilized for Fe(lll) determination using melh\l

thymol blue at pH 3.5 [4], azocarmine B and potassium periodate [5J.

chlorophosphonazo and potassium bromate [6], methyl orange and potassium bromate

[7]. [4.4'-bis(dimethyl-amino) diphenyl-methane], potassium bromate and potassium

iodide [8], orange G and potassium periodate [9], methylene blue and hydrogen

peroxide [10]. Various reagents such as 4-aminoantipyrene [11], 2-hydro.\yl-5-meth\l

benzophenone oxime [12], 2-hydroxy-4-n-butox\- propiophenone oximc [13]. 3-

h_\drox> l-3-phenyi-l-m-chlorophenyl iriazene [14]. thioglycolic acid [I5|. 1.2-

dih>droxy-3.4-dikeloc\clo butane [16]. diformyl hxdrazine [17]. and 1.3-diphcnl -4-

carboethoxN pyrazole-5-one [18] have been used for determination of Fe(lll). Other

spectrophotometric methods for determination of Fe(IIl) have been reviewed [19].

Derivative spectrophotometric procedures have also been reported for determination

of iron [20,21]. This chapter presents a more simple, rapid and selective

spectrophotometric method for determination of Fe(III) in synthetic mixture and soil

samples. Proposed method is based on chelation of Fe(in) with piroxicam in ethanol-

water medium which absorbs maximalK' at 495 nm.

Apparatus % - - - - y - . ^ h . /

EXPERIMENTAL » ^ ^

Ml absorbance measurements were made on a Speclronic 201)

speeli\>pholoiiicter (Milton Ro\ Compan\. USA) with I cm matched glass cclK,

Absorption spectra were recorded on a Shimadzu UV-visible speclrophtitomeler (UV-

160 A. Shimadzu Corporation. Kyoto. Japan) with matched quartz cells.

Reagents and standards

All reagents used were of analytical reagent grade. 0.005 M Ferric sulphate

(CAS: 10028-22-5, M.W.: 399.88, Fluka Chemie AG, Switzerland) solution was

freshly prepared in distilled water. 6.038 x 10" M (0.20%) Piroxicam (CAS: 36322-

90-4: M.W.: 331.35, Sigma Chemical Company, St. Louis. USA) solution was

prepared in methanol. The solution was stable up to 3 days. Soil samples were

collected from Department of Chemistry, Aligarh Muslim Universitx (A.M.U.).

Aligarh. India.

Determination of Fe(III)

Into a series of standard volumetric flasks (10 mL). aliquots (0.05 - 0.45 mL) of

5.0 > 10"' M standard ferric sulphate solution were pipetted. Then to each volumetric

flask, 6.038 x 10' M piro.xicam (2.6 mL) was added and diluted up to the mark with

ethanol. Contents of each flask were mixed well at room temperature (25 ± TC) and

absorbance was measured at 495 nm against reagent blank prepared simi!arl_\ except

Fe(lll) within stability period (24 h). Concentration of Fe(Ill) was calculated either

from a calibration curve or regression equation.

Determination of Fe(ni) in synthetic mixture

S\nthetic mixture of iron(lll) was prepared by taking 200 mg of ferric sulphate

with 13.9 mg FeS04.7H20, 192.3 mg 3CdS04.8H20, 90.0 mg Pb(N03)2, 23.01 mg

56

ZnS0,.7ll:0, 222.18 mg MgSOa.VH.O and 84.5 mg MnSOa.H.O in 100 niL slandarJ

volumetric llask and diluted up to the mark with distilled water. Fc(iilj was

determined by proposed procedure.

Determination of Fe(III) in soil sample

Air-dried finely powdered soil sample (2 g) of AMU locality was shaken with

0.5 M HCl (15 mL) for 24 h to extract iron [22]. After filtration, volume of filtrate

was made up to 25 mL with distilled water. Of this solution, 15 mL was percolated

through column packed with Amberlite IR 400 (CI" form) because Fe(IH) was found

to be completel)' adsorbed on resin in 4M HCl. Column was washed with 2.5 M HCl

to remove unadsorbed species and non targeted metal ions. Fe(lll) v\as eluted with

0.05 M HCl (flow rate, 2 mL /min). Effluent was evaporated and residue was

dissolved in 5 ml of distilled water. pH of solution was adjusted to 2.6 b\ addition of

ammonia solution and final volume of solution was maintained to 15 mL. Fe(lll) was

estimated by both proposed and reference methods [11].

Procedure for reference method

Appropriate volumes of standard solution of Fe(IIl) corresponding to 0.2 - 4.8

pg mL"' were transferred to a series of 25 mL volumetric flasks. To each flask. LO

mL of IN HCl. 1.0 inL of 0.1% 4-aminoantipyrene and 2.0 ml of 0.1 »o

iminodibenzyl were added. The contents were mixed thoroughly and diluted to

volume with ethanol. Absorbance of the resulting coloured complex was measured at

620 nm against corresponding reagent blank.

VALIDATION

Proposed method has been validated for specificity and selectivity, accuracy

and precision, robustness, linearity and evaluation of bias. Specificity and selecli\it\

of proposed method was evaluated by determining concentration of l-eHil) (22.34 fig

57

mL"') in the presence of various metal ions added in the form of FeS04.7Ii20.

NiCl26H20. Al(NOj)3.9H20, 3CdS04.8H20, Pb(N03)2, ZnS04.7H20, MgS04.7H20.

CaCb, MnS04.H20 and CuC^. Accuracy and precision of proposed method was

c\aluaied b_\ replicate analysis (n = 5) of calibration standards at three conccnlraiinn

le\els ( 5.59. 13.96 and 25.13 ).ig mL" ) Five sample solutions of each conccnira!iuii

Nsere anal\7.ed within one day (intra day precision) and in five consecutive da_\s (iiucr

day precision).

Robustness of proposed method relative to each operational parameter was

judged by analyzing content of Fe(Ill) in synthetic sample by observing influence of

small variations of concentration of piroxicam. A synthetic mixture sample containing

25.13 fig mL'' of Fe(lII) was assayed five times using proposed method. Mean

percentage recovery and relative standard deviation were calculated b> standard

methods.

Linearity of the proposed method was evaluated at nine concentration le\els

(2.79. 5.59. 8,38. 11.17, 13.96. 16.76. 19.55. 22.34. and 25.13 |ig mL"'). Lacli

concentration level was analyzed repeatedly for five times. Absorbance obtained at

each concentration was plotted against initial concentration of Fe(IlI) and linear

regression equation was evaluated by statistical treatment of calibration data. Other

regression characteristics were calculated using Origin Software. Limits of detection

(LOD) and limit of quantitation (LOQ) were calculated as:

S / , ( D - 3 . 3 x - i

h

b

58

where S,, is standard deviation of calibration line and b is the slope.

Bias has been exaluated b\ means of point and interval liNpothesis tests |2.^|.

Where test method is compared with reference method and considered to be

acceptable if mean recovery is within ± 2.0 % of that of reference method. 0, and

6,. are lower and upper acceptance limits, respectively and can be calculated as

where x, and x, are mean values based on ni and n2 measurements respective!). S,,

is pooled standard de\iation and t,ai is tabulated one-sided t-value. with n. + n; - 2

degrees of freedom at 95% confidence level.

RESULTS AND DISCUSSION

Piroxicam was found to react with Fe(lll) at 25±1°C resulting in the formation

of coloured complex. The absorption spectra of Fe(Ill), piroxicam. and Fedll)-

piroxicam complex are illustrated in Fig. 2.1. The absorption spectrum of Fe(lll)-

piroxicam complex showed two absorption bands peaking at 430 nm and 495 nm.

Absorbance measurement at 495 nm as a function of initial concentration of Fe(Ill)

was utilized to de\'elop a rapid, simple and selective spectrophotometric method for

determination of iron(lll).

Stoichionietry

Stoichiometry of reaction between Fe(ill) and piroxicam was evaluated b>

limiting logarithmic method [24], where in two sets of experiments were performed.

In first set. concentration of Fe(ni) was varied keeping a constant concentration of

piroxicam. while in second set. concentration of Fe(III) was kept constant and

piroxicam concentration was varied. Log absorbance versus log [ferric sulphate] or

[piroxicam] (Fig. 2.2) was plotted to evaluate slope of respective line. Slope was

59

200 240 280 320 360 400 440 480 520 560 600 540 660

Wavelength (nm)

Fig. 2.1. Absorption spectra of (a) 0.2 mL of 5 xlO'^ M ferric sulphate in

distilled water (b) 0.3 mL of 0.016% piroxicam in methanol (c) 0.45 mL of 0.005

M ferric sulphate in water + 2.5 mL of 0.16% piroxicam in methanol. Each

solution is diluted up to the mark with etianol in 10 mL standard volumetric

flask.

60

-0,3 -

-0.4

0) o c m () i _

() i/i

< O) o

-0.5

-0.6

-0.7

-0.9 -48 -4.6 -4.4 -4.2 -4.0 -3.8 -3.6 -3.4

log [Ferric sulphate] or [Piroxicam]

-3.2 -3.0

Fig. 2.2. Limiting logarithmic plot for stoichiometric ratio between Fe(III) and

3+ piroxicam: (a) log A vs. log [Fe J, and (b) log A vs. log [piroxicam].

6 1

continuous variation also indicated that one mole of Fe(lll) reacted witli 2 moles of

piroxicam (Fig. 2.3). Formation constant of complex was calculated and found to be

6.12 X !()'". Chelating reaction of piroxicam with metal ions can be expected through

three coordination sites (-0H, -CONH and-^N functional groups). Therefore,

reaction sequence of proposed method is given as in Fig. 2.4.

Optimization of Variables

Optimization of variables was assessed by testing reaction time, concentration

of piroxicam and solvents.

Effect of reaction time on absorbance of metal complex and its stabilit\ was

investigated. Complex got stabilized immediately at 25 ± 1 °C after mixing analyte

and reagent. Complex remained stable for 24 h.

Concentration of piroxicam for method development was optimized. For this

purpose, absorbance of metal complex was investigated at 22.34 i g mL' Fe(IIl) with

piroxicam (cone. 3.018 xlO"' - 1.691 xio"' M). Highest absorbance \\a^ obtained

with 1.45 ^10"' M piroxicam and remained constant up to 1.70 xJO"' M piroxicam

(Fig 2.5). I'herefore. 1.57 ^lO'' M piroxicam was used for the determination process.

Elfect of solvents (ethanol. acetone, methanoL 1.4-dioxan and

dimethylsulphoxide) was investigated on absorbance of colored complex, which

showed maximum absorbance in ethanol (Fig 2.6). Therefore, ethanol was best

solvent for dilution of reaction mixture in determination process of Fe(III).

Validation

Varying concentrations of each ion with fixed concentration of Fe(lll) were

taken and absorbance was recorded to know concentration of Fe(III). Tolerated

62

025

Mole fraction of Fe(lll)

Fig. 2.3. Job's plot of continuous variations for stoichiometric ratio bet\vecn

Fe(III) and piroxicam (1:2).

63

Steiii

2Fe''^ + 3804"

Step 2

Piroxicam

Sl B..

Fe'^

Ethanol-H20 medium

0

2H + 2

0 ^ / 0

HsC^ ,S O

O / • ^ o ^^'

Pink coloured metal chelate

Fig. 2.4. Reaction sequence of proposed method.

64

(D O C 03

J3

<

1.3

1,2

1.1 -

1 0 -

0.9 -

0.8

0 7 -\

0.6

0 5

0 4

0.3 H

0.2

0.1

0.0 I I 1 i 1

2 3 4 5 6 7 -n 1 1 1 1 1 1 1 1 1

9 10 11 12 13 14 15 16 17 15

[Piroxicam] x 10'', M

Fig. 2.5. Effect of concentration of piroxicam on absorbance of metal complex at

22.34 ^g mL ' Fe(III).

1.4

1.2

1 :

S 0.8 TO

I 0,6 <

0.4

0.2

^ ^ ^ _<?•''

#

65

.^ ^

.^ <5>

Solvent

Fig. 2.6. Effect of solvent on absorbance of coloured complex, [Fe(III)] = 22.34 |j,g

niL''.

66

amounts of each ion, which is concentration value tested that caused less than ± 2%

absorbance alteration, has been found as follows: Fe'^, 0.50; Cd , 84.31; Pb^\ 62.16;

Zn^^ 52.31; Mg''^ 21.80 and Mn^^ 27.47 tg mL"'. However, metal ions K\-\ Al'".

Ca^', and Cu~*) interfere in determination of Fe^ .

Accuracy and precision were evaluated within the same da\ and on tl\c

consecutive days. Recovery and RSD (%) were found between 100.04 -lOO.O.S "o:

0.22 - 0,74 % for intra day and 99.93-100.11%; 0.28-0.81% for inter day precision.

respectively (Table 2.1).

Robustness of proposed method was established by deliberately changing

concentration of piroxicam as:

• volume of 6.038 x lO'^M piroxicam, 2.6 ml (± 0.2 mL)

Under optimized experimental conditions, Fe(III) solution containing 25.13 |.ig

mL ' Fe(lll) was analyzed by proposed method. Mean recovery (%) and RSD (%j

were found to be 100.04 % and 0.22 %, respectixely. indicating robustness oi

proposed method.

Calibration cur\e was constructed by plotting absorbance against initial

concentration of Fe(III) (Fig. 2.7). Beer's law was obeyed (cone, 2.79 ~ 25.13 [ig

mL"') with apparent molar absorptivity (5.88 x 10 L mol'cm"') and Sandell's

sensitivitx (0.019 (j.g/cm' /O.OOl absorbance unit).Calibration data was fitted to the

equation. A = a + b C, where A is absorbance at 495 nm. C is concentration in ^g mL

b is slope and a is intercept of calibration. Calibration data (n = 9) were treated

statisticailx. High value of correlation coefficient (0.9999) indicated excellent

linearitv (Table 2.2). In order to verify that proposed method is free from procedural

error, experimental intercept of calibration line was tested for significance of

deviation from theoretical intercept (zero). For this justification, values oft-calculated

67

Table 2.1. Test of accuracy and precision of proposed method.

Parameters Intra day assay Inter day assay

Concentration taken, ^g mL"' 5.59 13.96 25.13 5.59 13,96 25.13

Conceiilralion found . |.ig mL"' 5.59 13.97 25.14 5.586 13.974 25.13

Standard deviation', |ug mL"' 0.041 0.043 0.055 0.045 0.049 0.070

Recovery. % 100.07 100.08 100.04 99.93 100.11 100.UO

Relative standard deviation, % 0.74 0.30 0.22 0.81 0.35 0.28

"Mean for five independent determinations.

''Confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).

68

1.2 -

1 , 0 •

J3

O </> < 0.6 H

0.4

0.0 •

10 15 20 25

[Fe(lll)], ng ml'

30

Fig. 2.7. Calibration curve for determination of Fe(ni).

69

Table 2.2. Optical and regression characteristics of proposed method.

Parameters

Wavelength . nm

Beer's law limit, p.g mL''

Molar absorptivity, L mor'cm"'

SandelPs sensitivity

LJnear regression equation

±tSa

±tSb

Correlation coefficient (r)

Variance (So") of calibration line

Detection limit, jag mL"

Quantitation limit, |ag mL'

Proposed method

495

2.79-25.13

5.88 X 10

0.019 ^g/cm^/0.001 absorbance

unit

A = 5.55 X 10""*+ 1.52 C

2.814 X 10-'

1.791 X IQ-

0.9999

2.690 X 10''

0.10

0.31

±t Sa and ±1 Sb are confidence limits for intercept and slope, respectively.

70

from relation, t = a / S;, [25] and found to be 1.27, which did not exceed theoretical t-

value (2.365) at 95% confidence level. This indicated that intercept for proposed

method is not significantly different from zero.

Applicability of proposed method for determination of FeIIl) in sxnthetic

mixture and soil samples has been tested. Results of the proposed method were

statistically compared with those of reference method [11] using point and inicrxal

hypothesis tests. The paired t- and F-values at 95 % confidence level were calculated

and found to be less than tabulated t- (2.036 at u = 8) and F-values (6.39 at u == 4.4) at

95 % confidence level [26], thus confirming no significant difference between

performance of proposed method and reference method (Table 2.3). Thus, bias

evaluated by interval hypothesis test by means of lower limit (0L) and upper limit (6i)

were in the range of 0.98 - 1.02.

Performance of proposed procedure is also judged by analysis of soils taken

from different locations. Results were found in good agreement with reference

method (Tabic 2.4). Thus, proposed method is suitable for routine analysis of Fcdll)

in real samples of soil. The speed of analysis of proposed method is as good as that of

reference method.

CONCLUSIONS

Proposed spectrophotometric method is found simple, selective and accurate for

determination of Fe(in). The method has advantage of using a commonly available

solvent (ethanol with the use of one reagent i.e. piroxicam). Proposed method, which

has avoided use of acid, buffer solution and heating of reaction mixture, can be used

as an alternate method for routine quality control analysis of Fe(III) in soil samples.

71

Table 2.3. Evaluation of bias: Comparison and applicabilitA' of the proposed

method with the reference method for the determination of Fe(III) from

synthetic mixture sample at 95% confidence level.

Samples Proposed method Reference method t- 1" t),'

value'" \akie"

Recovery'' RSD Recovery'' RSD

(%) (%) (%) (%)

Synthetic 100.01 0.91 99.87 0.83 0.253 1.200 0.999 l.OO: mixture

^Mean for 5 independent analyses.

''Theoretical / (v= 8) and F-values (v= 4, 4) at 95 % confidence level are 2.306 and

6.39, respectively.

'A bias, based on recover' experiments, of ± 2% is acceptable.

72

Table 2.4. Determination of Fe(III) in soil samples by proposed spectrophotometric method and reference method.

Sample

Soil 1

Soil 2

Concentration ofFe^'digmL"')

Proposed method Reference method

8.379 8.382

8.382 8.375

73

REFERENCES

[I] X. Qi, J. Huang. W. Zhao, Guangdong weiliang Yansue kexue 14 (2007) 49.

|2 | W. Sun. M. Lu. yejin fen.xi 26 (2006) 67.

[3] Xuc-Hua sun. 11. Ma. R, Tian. 11. Chai. Y. Liu. Hua.xuc shiji 29 (2U07) 29:-,.

[4J M. Hasani, A. Razace. H. Ahdollahi, Spectrochim. Acta 68A (2007) 414.

[5] G. Yin, X. Ma, S. Kang, Lihua Jiyanyan Huasue Fence 41 (2005) 515.

[6] C. Han. S. Feng, Yejin Fenxi 25 (2005) 63.

[7] Z. Li, Y. Li, J. Wang, Fenxi Shiyanshi 25 (2006) 59.

[8] Y. Wei, K. Zhao, Q.Yu, J.Li, Guangpu, Shiyanshi 22 (2005) 1007.

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[lOJ G. Sharen. Nei Menggu shida xuefao. Ziran keaul 34 (2005)194.

11 IJ S. .\4anjula, S. Khan. A. A.Syed. Science Asia 33 (2007) 455.

[12] S. Shingadia. K.K. Desai, Acta Ciencia Indica Chemistry 33 (2007) 347.

[13] K. Purohit, K.K. Desai, J. Saudi Chem. Soc. 10 (2006) 129.

[14] N. Singh, R.S. Chauhan, P.K. Gupta, Asian J. Chem. 17(2007) 265.

[15] M. G. Gioia. A. M. Di Peter, R. Gatti, J. Pharm. Biomed. Anal. 29 (2002) 1159.

[16] C. D. Stalikas, A. C. Pappas, M. I. Karayannis , P. G. Veitsistas. Microchim.

Acta 142(2003)43.

[17] B. M. Nagabhushana, G. T. Chandrappa. B. Nagappa, N. H. Nagaraj. .Anal.

Bioanal Chem. 373 (2002) 299.

[18] R.K. Patil, D. G. Dhuley. Indian J. Chem. 39A (2000) 1105.

[19] S. Khan. R. Dashora. A.K. Goswami, D. N. Purohit, Rev. Anal. Chem. 23 (2004)

[20] G. A. Shar. G. A. Soomro, J. Chem. Soc. Pakistan 28 (2006) 39.

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[211 V. K. Reddy. S. M. Reddy. R. P. Reddy, T. S. Reddy. Chem. Anal. (Warsaw ). 46

(2001)687.

[221 C. D. Staiikas. G. A. Pilidis and S. M. Tzouwara-Karayanni, Sci. Total Fnviron.

236(1999)7.

[23] C. Hartmann. J. Smeyers-Verbeke, W. Pinninckx, Y, V. Heyden. P.

Vankeerberghen. D. L. Massart, Anal. Chem. 67 (1995) 4491.

[24j J. Rose. Advanced Physico-chemical Experiments; Pitman, London. UK (1964)

p. 67.

[25] V. V. Nalimov. The Application of Mathematical Statistics to Chemical

Analysis, Pergmon Press, Oxford (1963) p. 167.

[26] J. Mendham, R. C. Denney, J. D. Barnes, M. Thomas, Statistics: Introduclion to

Chemometrics. In Vogel's Textbook of Quantitative Chemical Analysis. 6th ed..

Pearson Education, Singapore (2002) p. 137.

75

Interaction of Cu(II) with cefixime and its

application for determination of Cu(II) in water

samples

76

INTRODUCTION

Copper has received considerable attention owing to its technological and

biological significance, h is an essential constituent of abotit thirt) enzymes and

gl>coproteins. It is required for the synthesis of hemoglobin and for some biological

processes [1,2]. The concentration of copper plays a very important role which

decides its vitality and toxicity for many biological systems [3,4]. It has been reported

that the toxic copper species are Cu(OH)" , Cu2(OH)2^^ and CUCO3. Therefore, copper

ions present in various aqueous systems are considered to be the most toxic of

dissoUed copper species [1,2]. The excess concentration of copper in water s\ stems is

harmful to human beings, affects the self-purification of bulk water [5] and disturbs

the microbiological treatment of waste water [6]. Thus, the determination of trace

amounts of Cu(II) is becoming increasingly important because of the increased

interest in environmental pollution [7]. In chemical analysis, metal chelation followed

by solvent extraction and spectrophotometric determination is considered as the most

preferred mode of analysis for a number of metal ions in different matrices |8].

Several spectrophotometric methods have been reported in which the sol\cm

extraction step is replaced by the use of surfactant [9-13]. Micellar media are mainl\

used to simplity the sxstem and replacing the extraction step. Man

spectrophotometric methods have been developed for the determination of Cu(H)

based on reaction with reagents such as l-nitroso-2-naphthol [14], 1-phenyl-1,2-

propanedione-2-oxime thiosemicarbazone [15], 2-hydroxy-l-naphthaldehyde

benzoylhydrazone [16], N-ethyl-2-naphthylamine [17], ethyl violet [18]. mixture of

1,10-phenanthroline and neocuproine [19], 1,5-diphenyl carbazone [20], 2-ketobutyric

acid thiosemicarbazone [21]. pyruvic acid thiosemicarbazone [22]. 2-acet\ I

thiophene-4-phen_\ l-3-thiosemicarbazone [23]. 4-\anillidene amino-3-nielh\ 1-5-

77

mercaplo-i.2,4-triazole [24], dimethyl glyoxime [25], variamine blue [26]. Most of

the reported spectrophotometric methods are time consuming, emploxing man\

reagents to develop the colour and extraction of copper complex into organic solvent.

Spcctropholometr\ is the good tool for determining metal ion concentration in lcaf\

vegetables, pharmaceuticals, natural water and soil samples due to itN low co^i.

simplicit and adaptability. Analytical methods based on spectrophotomctr 127-2^)

were published in reputed journals. Therefore, it is decided to exploit this technique to

develop an optimized and validated UV spectrophotometric method for the

determination of Cu(ll) in synthetic mixture and water samples. This chapter presents

UV spectrophotometric method based on the complex formation of Cu(II) with

cefixime in 1,4-dioxan-distilled water medium at room temperature (25 ± TC). The

formed complex showed maxirnum absorbance at 336 nin. The reaction condition.s arc

optimized and validated as per International Conference on Harmonisation ISA)

[30].

EXPERIMENTAL

Apparatus

.Ml spectral and absorbance measurements were made on a Shimadzu UV-

visibie 160 A spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with 1 cm

matched quartz cells.

IR spectra were recorded on a Perkin-Elmer FTIR 1650 spectrophotometer in

wave number region 4000-400 cm'' using KBr pellet technique.

An Elico LI 120 pH meter (Hyderabad, India) was used for pH measurement.

Reagents and standard.s

All reagents used were of analvtical reagent arade.

78

• 3.195 < 10""' M copper sulphate (M.W.: 159.61. Suprachem prodiicls Ltd.

r-nglaiid) solution (203.06 ppm Cu) was prepared b dissoKing 0.05 1 g in lOii

niL distilled water.

• 2.205 ^ 10""' M (0.1%) cefixime (CAS: 79350-37-1, M.W.: 453.45. Sigma

Chemical Company, St. Louis, USA) solution was freshly prepared in melhanoL

The solulion was stable up to 12 h.

• Buffer solutions ranging from 3.72-5.57 were prepared by mixing varying

volumes of 0.2 M acetic acid (9 -1 mL) and 0.2 M sodium acetate (1-9 mL) in 10

ml standard volumetric tlask [31].

Sea and well water samples were collected from Muscat. Oman.

Procedure for the determination of Cu(n)

Into a series of 10 mL standard volumetric flask, aliquots (0.05-0.4 mL) of

3.195 X 10"' M standard copper sulphate aqueous solution corresponding to 1.015-

8.122 f g mL"' were pipetted. To each flask, 1.8 mL of cefixime (0.1%) solution was

added and diluted up to the mark with L4-dioxan. The contents of the flask were

mixed well and the absorbance was measured at 336 nm against reagent blank

prepared similarl> except Cu(II) within stability period (6 h). Lhe amount of C udli

was obtained either from the calibration graph or the regression equation.

Deterniiniition of Cu(II) in synthetic mixture sample

Synthetic mi.xture of Cu(II) sulphate sample solution was prepared b> taking 5 I

mg of copper sulphate with 200 mg MgS04, 7.28 mg HgCb, 200 mg Cr^O?, 290 mg

Al2(S04)3.16H20, 51 mg ZnS04.7H20, 51 mg MnS04.H20, 51 mg CaClj. 51 mg

Pb(N03)2 and 72.8 mg Fe2(S04)3 in 100 mL standard volumetric flask and diluted up

to the mark with distilled water. The amount of Cu(ll) was determined b\ the

proposed procedure.

79

Determination of Cu(II) in water samples

100 niL of sea and well water samples were collected and transferred into

cleaned pohethylene bottles. The sea and the well water samples were filtered

through a Millipore 0.45 yim pore size membrane and analyzed within 6 h ot

collection. .Xliquot (0.5 mL) of sea and well water samples was pipetted into a 10 ml

standard volumetric Hask with 1.8 mL of cefixime (0.1%) solution. The conlenis oi

each tlask were mi.xed well at room temperature and diluted up to the mark with 1.4-

dioxan. Ihe absorbance of each solution was recorded at 336 nm against the reagent

blank prepared similarly except Cu(ll). The amount of Cu(Il) in sea and well water

samples was obtained either from the calibration graph or the regression equation.

The amount of Cu(Il) was also estimated by the reference method [32].

Procedure for reference method

.Miquots (0.025 - 0.25 mL) of standard copper sulphate (1.598 - 10 M(

solution corresponding to 0.5 - 5 f.tg mL' were pipetted into a series o\ ^i) ml

separating funnels. To each funnel. 3.0 mL of universal buffer mixture (100 ml oi' j

solution of mixed acids, being 0.04 M H3PO4, 0.04 M acetic acid and 0.04 M bon^

acid: neutralized 56.76 mL of 0.2 M NaOH and made up to 200 mL) of pH 7.5. 3.0

mL of 1.98 ^ 10"' M l,5-diphenyl-l,4-pentadien-3-oxime and 1.0 mL of magnesium

sulphate were added and mixed well. The contents of the separating funnel were

shaken vigorously with 5 mL of chloroform for 30 s and then allowed to separate the

two layers, fhe absorbance of the organic layer was recorded at 503 nm. The amouni

of Cu(ll) was obtained either from the calibration graph or the regression equation.

Detcrminatiini of stoicliiometry

T he stoichiometry of the reaction was studied b\ .lob's method of continuous

variations [33]. For this purpose, different volumes (0, 0.2, 0.4. 0.6, 0.8. 1.0. 1.2. 1.35.

80

1.5. 1.8. 2.0 ml.) of 2.205 x 10'" M copper sulphate was added with different volumes

(2.0. 1.8. 1.6. 1.4. 1.0, 0.8. 0.65. 0.5. 0.2, 0 mL) of 2.205 x lO"' .VI celiximc and

diluted with l.4-dio.\aii in 10 mi standard volumetric flask, f he absorbancc \\a^

recorded ai .336 nm and plotted against the mole fraction of copper sulphate.

V'alidation

The present method has been validated for specificity, precision. acciirac>.

linearity, robustness and evaluation of bias.

The specificity of the proposed method was evaluated by determining 7.107 ^g

mL"' Cu(ll) in the presence of metal ions such as Mn(II), Ca(II), Mg(I[). Cd(ll).

Al(lll). Pbdl). Zn(ll). Fe(Il), Fe(III), Ni(II), Cr(III). Hg(ll) and As(III) added in the

form of .MnSO4.H:0. CaCl.. MgSOa. 3CdS04.8ITO. Al2(S04):,.16H2(). r NNO.) ;.

ZnS04.7H20. FeS04.7H20, Fe2(S04)j. Ni(N03)2.6H20. Cr203. HgCl;. and AS2O;.

The precision of the proposed method was evaluated by intra-da\ and inter-da

precisions. Standard Cu(Il) solution at three concentration levels (2.031. 4.061 and

8.122 |ag ml.'') was assessed with five replicates (n=5) for each of three working

sample concentrations in a single day (intra-day precision) and over five days (inter-

da) precision).

The accuracN of the proposed method was determined b\ standard addition

method. For this purpose. 0.15 mL synthetic mixture sample solution corresponding

to 3.046 ug mL'' Cu(ll) was spiked with 0. 0.05, 0.1. 0.15 and 0.2 mL standard Cu(ll)

solution corresponding to 0, 1.015, 2.031, 3.046 and 4.061 |ag mL'' Cu(ll).

The robustness of proposed method was assessed by analyzing 5 \xg mL'' Cu(ll)

in s\nthetic mi.xture sample by varying the volume of ceflxime (1.8 ± 0.2 mL) at

room temperature (25 ± PC).

8 1

Lincaritx of ihe proposed method was assessed al seven concentration levels

(1,015. 2.031. 3.046. 4.061, 6.092, 7.107 and 8.122 ).ig mL"'). Each concentration

level was independently analyzed repeatedly for five times. The instrumental response

i.e. absorbance obtained at each concentration was plotted against initial concentration

of CLI(II). The linear regression equation was evaluated by least square treatment ol

the calibration data. The other statistical parameters for the proposed method were

calculated using Origin Software. Limits of detection and quantitation were calculaied

[34J.

Point and interval hypothesis tests have been utilized to evaluate the bias of the

proposed method and the reference method [35]. The proposed method is compared

w ith the reference method and considered to be acceptable if mean recovery of the

proposed method is within ± 2.0 % of that of the reference method. The lower (0 )

and the upper (6'(,) acceptance limits can be calculated by the following quadratic

equation:

e'x; -SpJ •, )-f^(-2^^) + (.v; -.V;;,„; OiJ=.0

where .Y, and .v, are mean values at ni and n2 measurements respectiveh. S,, is tiic

pooled standard deviation and t,,,i is the tabulated one-sided t-value at 95% conlldence

level.

RESULTS AND DISCUSSION

The aqueous solution of copper sulphate absorbed maximally at 206 nm. The

methanolic solution of cefixime was peaking at 210 and 290 nm. When the two

solutions were mi.xed together, a red shift in the wa\elength is observed due to the

complexaiioii reaction of Cu(ll) and cefixime. Thus, a complex was obtained

immediaieh' in 1.4-dioxan-distilled water medium with /.„,av = 336 niii. Ihe I \

82

spectra for copper sulphate, cefixime and Cu(n)-cefixime complex are shown in

Fig. 3.1. U is clear from the graph that the maximum absorbancc \\a\elcnglh ot'liic

Cu(ll)-ccfiximc complex is different to that of cefixime. 'fhus. ihc absorbaiiLC

measLiremcnl at 336 nm as a function of initial concentration of C"u(ll) is cxploiicd to

dexelop a new and novel UV spectrophotometric method for the determination o\

Cu(ll) in synthetic mixture and water samples. The reaction was carried out at room

temperature and the complex was stable up to 6 h.

Stoichiometry

The stoichiometric ratio between Cu(II) and cefixime was evaluated by .lob's

method of continuous variations. Varied volumes of equimolar (2.205 < 10 ' M)

solutions of Cufll) and cefixime were taken, keeping the total volume of Cu(Il) and

cefixime con.stant at 2 mL in 10 ml. standard volumetric flask and diluted up U' ihc

mark witii 1.4-dioxan. The absorbancc of each set is recorded and plotted against the

mole fraction of Cu(Il) (Fig. 3.2). The plot of absorbancc versus mole fraction of

Cu(II) has confirmed that 1 mol of Cu(II) reacted with 1 mol of cefixime. The

resulting Cu(]l)-cefixime complex remained stable for about 6 h. Thus, the

stoichiometrx of the complex is established and found to be 1:1.The LR. spectra of

free cefixime and Cu(II)-cefixime complex are shown in Fig. 3.3 a and b.

respectivcl_\. Cefixime has -NH2, -COOH. -CONH and C=0 lactam groups wliich arc

the potential sites for coordination with metal ions. Comparison of IR spectrum of the

complex with those of free cefixime indicates that the lactam (C=0) band appears at

1766 cm' in the free cefixime while the complex shows this band again at 1 766 cnf'

suggesting that no coordination occurs with copper ion. The amide carbonyl band.

\'(C=())-N11 in the free cefixime appears at 1674 cm"' with a weak shoulder at 1635

cm ' while tiie Cu(ll)-cefixime complex shows this band at 1676 cm"' with a

83

(D O C

re

o CO

<

210 225 240 255 270 285 300 315 330 345 360 375 390 405

Wavelength, nm

Fig. 3.1. Absorption spectra of (a) 2 niL of 3.195 x 10 M copper sulphate in distilled water (b) 0.05 niL of 0.1 % cefixinie in methanol and (c) 0.4 niL of 3.195 X 10^ M copper sulphate in distilled water + 1.8 niL of 0.1% cefixinie in methanol. Solutions a, b and c are diluted up to mark with distilled waJcr. methanol and l,4-dio\an in 10 mL standard volumetric flask, res()ecti\ely.

8 4

0.0 • -

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 O.i

Mole fraction of copper sulphate

0.9 1 0 11

Fig. 3.2. Job's plot of continuous variations for stoichiometric ratio between Cu(II) and cefixime (2.205 x 10 ^ M each).

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prominent peak at 1631 cm' indicating the coordination of cefixime ni(h C'u(ll)

through nitrogen. The asymmetrical and symmetrical stretching bands of carhoxylatc

groups change from 1533 cm"' to 1543 cm' and 1373 to 1379 cm''. respectiveK due

to the coordination. The Cu-N stretching vibration occurs at 428 cm ' [36]. A tentatixe

mechanism for the complexation between Cull) and cefixime is given in Fig. 3.4.

The apparent formation constant (Kf) for the complex foirnation between Cu(ll)

and cefixime is calculated using the following expression [37]:

Kf- Mth^At)'^ where Aobs and Aextp are observed and extrapolated absorbance values for the

complex, respectively. CM and CL are the initial concentration of Cu(II) and cefixime

in mol L ' , respectively. € is the limiting concentration. Thus, Kf for the complex is

found to be 1.723 x 10 . The apparent Gibbs free energy ( A G ) is calculated using

AG = - 2.303 RT log Kf and found to be - 35.59 kJ mof' confirming the feasibilit

of the reaction.

Optimization of Variables

The optimization of variables was investigated by testing reaction time,

concentration of cefixime, solvents and sodium acetate-acetic acid buffer solutions of

different pH.

The effect of reaction time on the absorbance of Cu(II)-cefixime complex and

its stability was investigated. The Cu(II)-cefixime complex got stabilized immediateh

at 25 ± 1 °C after mixing cefixime and 1,4-dioxan as solvent. Complex remained

stable for 6 h.

Step

88

Cu(S04) -*- Cu-" + S04'-

Step 2

H,N^ ,S w \ /) NOCH2COH

\ C -N-

H il 0 0

0 HzN-^S \\ .

V N -s^

Cefixime COOH

C=CH2 H ^

H II 0 0 -N

COOH C=CH2 H

Step 3

H,N^ ,S 0

Cu^

H20-1.4-dioxan medium

,\ /) NOCH2CO

0 0-

COOH C=CH2 H

H,N^ ,S 1 ) NOCH2CO

if " 0 0 '

-N

COOH

Cu(Il)-cefixime complex

C=CH2 H ^

Fig. 3.4. Reaction sequence of the proposed method.

89

The volume of 0.1% cefixime was optimized for metiiod deveiopmenl. The

absorbance of the metal complex was investigated at 8,122 ^g mi./ Cu(li) uilh

volume of cefixime in the range 0.4-2.0 mL. It is clear from Fig. 3.5 thai the

maximum absorbance was obtained with 1.6 mL cefixime. Above this volume up to

2.0 mL of 0.1% cefixime, the absorbance remained unchanged. Therefore. 1.8 mL of

0.1% cefixime was used in further measurement of Cu(II) in synthetic mixture and

water samples.

The effect of solvents such as methanol, acetone, dimethylsulphoxide (DMSO).

acetonitrile. ethanol, 1,4-dioxan and distilled water was investigated at 8.122 ^g mL"'

Cu(ll) on the absorbance of Cu(ll)-cefixime complex. The reaction mixture uas

turbid in ethanol, acetone and distilled water. The absorbance for Cu(ll)-cellxime

complex in other solvents is shown in Fig. 3.6. It is clear from the figure thai the

highest absorbance was obtained in 1,4-dioxan. Therefore, 1,4-dioxan was the best

solvent for dilution of the reaction mixture of Cu(II)-cefixime complex in

determination process of Cu(II) in synthetic mixture and water samples.

The pH of the complex was measured and found to be 4.68. The effect of pH on

the absorbance of the complex was investigated using sodium acetate-HCI (range

3.72-5.57 buffer solutions. A constant absorbance was obtained in the pH range of

3.72-5.57. but the absorbance value in this pH range was found to be less than thai

obtained without buffer solution when dilution is made with 1,4-dioxan. Therefore, all

absorbance measurements of the complex were made in 1,4-dioxan solvent without

the invohement of buffer solution.

90

1.1 -

1.0

0.9

0.8

o

TO 0.7 -Q i _ O i/>

JD < 0.6

0 5 -

0.4

0.3 -

0.2 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.

Volume of 0.1% cefixime, mL

2.0 2.2

Fig. 3.5. Effect of the volume of 0.1% cefixime.

9 1

DMSO Acetonitrile Methanol 1,4-dioxan

Solvent

Fig. 3.6. Effect of solvent on the absorbance of Cu(II)-cefixime complex, Cu(II)] = 8.122 Jig inL ' .

9 2

Validation

Varying concentrations of metal ions such as Mn(II), Ca(ll), Mg(II), Cd(ir).

Al(lll), Pb(ll). Zn(ll), Fe(H), Fe(III). Ni(]l), Cr(in), HgCII) and As!li) with 7.107 ^L;

ml."' CLI(II) were tai<en and the absorbance was recorded to know the coacenlraiion ut'

CLi(li). The tolerated amount of each metal ion in |.ig ml.'' wa.s determined (Tabic

3.1), when the absorbance value did not exceed ±2% on addition of cations. Howe\er.

metal ions such as Ni(II), Cd(II), Fe(II) and As(III) interfere in determination of

Cu(ll).

The intra-day and inter day precisions were evaluated by determining the

concentration of Cu(II) at lower, middle and upper concentration levels for five

repeated times within the same day and on five consecutive days, respectively (Table

3.2). It can be seen from the table that percentage recover) and RSD (intra day and

inter da\ precisions) were in the ranges of 99.93-100.08 % and 0.14-0.67 %.

respectiveK. It is evident from the table that percentage recovery and RSD values

were precise and can be used to determine Cu(Il) in synthetic mi.xture and water

samples.

Standard addition method is one of the methods to show the accuracy of the

proposed method just by the addition of the number of standardized aliquots to a real

sample in order to raise its concentration by a known amount. Thus, the accuracy of

the proposed method was investigated by performing recovery experiments through

standard addition method. The absorbance for each solution is recorded at 336 nm and

plotted as shown in Fig. 3.7. The results of analyses are summarized in Table 3.i. it

is clear from the table and the graph that the linearity of the regression line for

93

Table 3.1. Effect of metal ions on the determination of 7.107 fxg niL '

Cu(II).

Metal ions Added as Tolerance limit (|ag mir ' )

Ca-^

Fe- ^

MgS04

HgCb

Cr203

Al2(S04)3T6H20

ZnS04.7H20

MnS04.H20

CaCb

Pb(N03)2

Fe2(S04)3

15.44

1.88

26.25

4.36

3.48

4.97

5.50

12.76

3.56

9 4

Table 3.2. Precision of the proposed method.

Parameters Intra d

2.031

2.023

0.012

lay assa_\

4.061

4.062

0.011

8.122

8.125

0.011

Inter da

2,031

2.033

0.014

. assa\

4.061

4.059

0.012

8.122

8.120

0.014

Concentration taken, |.ig mL'

Concentration found' , jug mL"'

Standard deviation, |xg mL"'

Recovery', % 99.93 100.02 100.04 100.08 99.95 99.98

Relative standard deviation, % 0.57 0.28 0.14 0.67 0.31 0.17

" Mean for five independent analysis.

9 5

1 0

2 3 4

S t a n d a r d A d d e d

C o n c e n t r a t i o n , ng mL'^

Fig. 3.7. Determination of Cu(II) in synthetic mixture sample by standard addition method.

96

Table 3.3. Test of accuracy in synthetic mixture sample by standard addition method.

Concentration (fig mL"') Linear regression parameters Recovery

(%)

Sample Standard Nominal Error Intercept slope f

Added (S^ ,)

3.046 0.1.015.2.031. 3.049 0.020 0.3982 0.1306 0.9999 100.10

3.046.4.061

^Coefficient of correlation.

97

synthetic mixture sample was good. As can be seen from the tlgure that the

concentration of Cu(ll) in synthetic mixture sample is given by intercept slope. Vhc

ratio of the intercept and the slope of the regression line is subjected to error (S^, i.

thus S ^ is calculated from the following expression

+ " b'T (X, - X)'

and found to be 0.02 |j.g mL''. The confidence limit for the concentration of Cu(II) in

synthetic mixture sample is calculated by x^ ± /S ^ at n - 2 degrees of freedom and

found to be 3.049 ± 0.062. The most attractive feature of the proposed method usiny

standard addition method is its relative freedom from various non targeted cations.

The robustness of the proposed method was established by deliberate!)

changing the volume of 2.205 x lO" M cefixime, 1.8 mL (± 0.2 mL) for the

determination of Cu(II). The synthetic mixture sample solution containing 5.0 jig mL

' Cu(ll) was analyzed five times repeatedly by the proposed method. Percentage

recovery and RSD were found to be 99.84 % and 0.15 %, respectively, indicating

robustness ol'the proposed method.

Under the optimized experimental conditions, the calibration graph b\

considering 7 independent concentration levels of Cu(ll) was constructed by plotting

the absorbance against initial concentration of Cu(II) (Fig. 3.8). Beer's law is obeyed

in the concentration ranges of 1.015- 8.122 )ag mL'' with apparent molar absorptivit)

of 8.29 X 10 L mor'cm'' and Sandell's sensitivity (0.008 fig/cm^/0.001 absorbance

98

0) o c TO

o 0)

<

1 1 -

1.0 -

0.9 -

0.8 -

0.7 -

0.6 -

0.5 -

0.4 -

0.3 -

0.2 -

0 1 -

/

/

0 0

[Cu(ll)], ng mL'

Fig. 3.8. Calibration curve for the determination of Cu(II).

99

unit). Linear regression equation is obtained by statistical treatment of the calibration

data (n = 7) vvhicli is fitted with the straight line equation in the form o\' A = a - b( .

where A is absorbance at 336 nm, C is concentration in f.ig niL' . b is slope and a is

intercept of calibration. High xalue of correlation coefficient (0.9999) indicated

excellent linearity (Tabic 3.4). The experimental intercept of the calibration line was

tested for significance of deviation from the theoretical intercept, i.e. zero. For this

justification, t-value calculated from relation, t = a / Sa[38] found to be 1.61, did not

exceed the tabulated t-value (2.571, v=5) at 95% confidence level. This indicated that

the intercept in the calibration equation of the proposed method is not significantl\

different from zero. Thus, the proposed method is free from procedural error.

The applicability of the proposed method for the determination of Cu(ll) in

synthetic mixture sample has been tested. Results of the proposed method were

statistically compared with those of reference method [32] using point and interval

hypothesis tests. The paired t- and the F-values at 95 % confidence level were

calculated and found to be less than the tabulated t- (2.036 at D = 8) and F- (6.39 at u

= 4.4) values at 95 % confidence level [39], thus confirming no significant difference

between the performance of the proposed method and the reference method (Table

3.5). Thus, the bias calculated by interval hypothesis test in the form of lower limit

(0() and upper limit (0u) were in the range of 0.98 - 1.02.

The performance of proposed method is also tested in sea and weii water

samples collected from Muscat, Oman. Results of analysis were found in good

agreement with reference method (Table 3.6). Thus, the proposed method is suitable

for routine analysis of Cu(n) in real samples of water. The speed of analysis and less

100

Table 3.4. Optical and regression characteristics of the proposed method.

Parameiers Analytical data

Kax (nm)

Beer"s law limit (|jg mL"')

Molar absorptivity (L mol'dn"')

SandelTs sensitivity

Linear regression equation'*

Sa

±tSa

Sb

±tSh

Correlation coefficient (r)

Variance (S„")

LOD(|ag ml/ ')

LOQlugmL;')

336 nm

1.015-8.122

8.293 X 10'

0.008 |ag/cm^/ 0.001 absorbance unit

A=1 .59x 10" +1.305 X 10"' C

9.850 X 10-

2.533 X 10"

1.919 X 10'

4.933 X 10"'

0.9999

1.588 X ](y''

0.032

0.095

'With respect to A= a + bC, where C is the concentration in ).ig mL" and A is

absorbance.

±tSa and ±tSi, are the confidence limits for intercept and slope, respectively.

101

Table 3.5. Point and interval hypothesis tests for the determination of Cu(II) in

synthetic mixture sample for the evaluation of bias of the proposed method with

that of the reference method at 95% confidence level.

Proposed method Reference method Paired t- F-value 9L 6 d

value

Recovery' RSD Recovery' RSD

(%) (%) (%) (%)

99.84 0.15 99.91 0.15 0.721 1.01 0.998 1.003

'Mean for 5 independent analyses.

''Theoretical; (v= 8) and F-values (v= 4, 4) at 95 % confidence level are 2.306 and

6.39. respectively.

'A bias, based on recovery experiments, of ± 2% is acceptable.

102

Table 3.6. Determination of Cu(II) in sea and well water samples by the proposed UV spectrophotometric method and the reference method.

Sample Concentration of Cu(II) in i g mL

Proposed method Reference metiiod

Sea water 2.164 2.16:

Well water 1.022 1.021

103

number of reagents utilized in the proposed method are the main advantages of the

proposed method as compared to reference method.

CONCLUSIONS

riic proposed method is a direct UV-spectropholomelrie method as coinparcd i.

relerence method (extractive spectrophotometric method). The proposed mcliiv J is a

simple and accurate for the determination of Cu(Il) in synthetic mixture and water

samples. The method has advantage of using a commonly available solvent i.e. 1.4-

dioxan with the use of one reagent, i.e. cefixime. The proposed method has avoided

the use of acid, buffer solution and heating of reaction mixture, can be used as an

alternate method for routine quality control analysis of Cu(II) in soil, vegetable and

pharmaceutical samples.

104

REFERENCES

[ l ]M. M. Freemantle. Chemistr\ in Action. Macmilian Educalion Ltd.. London

(1989).

|2] E. B. M. Sorensen, Metal Poisoning in Fisli, CRC Press, Boston. MA. USA

(1991),

[3] \. H. Scheinberg, A. G. Morell, Ceruloplasmin, In "Inorganic Biochemistry", Vol

1, G. L. Eichhom (Ed.), Elsevier, New York (1973) p. 306.

[4] N. N. Greenwood, A. Earnshow, Chemistry of the Elements. Buttcrworth-

Heinemann. Oxford, 2005: p. 1197.

[5] T. Yubin, C. Fangyan, Z. Honglin, Adsorp. Sci. Tech. 16 (1998) 595.

[6] S. E. Ghazy, S. E. Samra, S. M. El-Morsy, Adsorp. Sci. Tech. 19 (2001) 175.

[7] Y. Yamini. A. Tamaddon, Talanta 49 (1999) 119.

[8] N. Y. Sreedhar, Y. Jyothi, J. Indian Chem. Soc. 82 (2005)1038.

[9] G. A. Shar, G.A. Soomro, The nucleus 41 (2004) 77.

[10] S. E. Ghazy, R. M. El-Shazly. M. S. El-Shahawi. G, A. A. Al-Hazmi, A. A, El-

Asmy, .L Iranian Chem, Soc. 3 (2006) 140.

[11] .l.Yun, H.Choi,Talanta 52 (2000) 893.

[12] G. A. Shar, M. 1. Bhanger. J. Chem. Soc. Pak. 24 (2002) 176.

[13] M. P. San Andres, M. L. Marina, S.Vera, Talanata41 (1994) 179.

[14] H, Abdollahi, M. S. Panahi, M. R, Khoshayand, Iranian J. Pharm. Res. (2003)

207.

[15] N. B. L. Prasad, K. H. Reddy, J. Indian Chem. Soc. 84 (2007) 474.

[16] V. K. Reddy, S. M. Reddy, P. R. Reddy, T. S. Reddy, J. Anal. Chem. 55 (2000)

435.

105

[17] i. Mori, T. Fujimoto, Y. Fujita, T. Matsuo. Talanta 42 (1995) 77.

[18] K. Yamamoto, T. Kumamaru, Anal. Sci. 11 (1995) 307.

[19] A. Safavi, M. R. H. Nezhad, Can. J. Anal. Sci. Spectrosc. 49 (2004) 210.

[20] M. R. Shishehbore, N. Nasirizadeh. A. M. H. Shabani. M. Tabalabaec, Can. ,1

Anal. Sci. Spectrosc. 50 (2005) 130.

[21] L. E. Attah, Indian J. Chem. Tech. 16 (2009) 351.

[22] L. E. Attah, J. Chem. Soc. Nigeria 29 (2004) 71.

[23] N. B. L. Prasad, K. H. Reddy, T. S. Reddy, Ind. J. Chem. A 42 (2003) 112.

[24] R. A. Nazarath, B. Narayana, N. V. Sreekumar, Indian J. Chem. A 40 (2001)

1016.

[25] U. Muralikrishna, A. Shivaramakrishna, Asian J. Chem. A 13 (2001) 289.

[26] B. Narayana, M. Mathew, N. V. Sreekumar, K. Vipin, J. Indian. Chem. Soc. 80

(2003) 937.

[27] H. Z. Mousavi. N. Pourreza. J. Chin. Chem. Soc. 55 (2008) 750.

[28] K, Zarei. .M. .-Xtabati. M. Safaei, J. Chin. Chem. Soc. 54 (2007)1395.

[29] A. Shokrollahi. M. Ghaedi, H. Ghaedi, J. Chin. Chem. Soc. 54 (2007) 933.

[30] International Conference on Harmonisation, Food and Drug Administration.

Rockville. MD, USA. ICH Harmonised Tripartite Guideline - Text on

Validation of Analytical Procedures, Fed. Regist. 60 (1995)11260.

[31] H. T. S. Britton, Solutions of Known Hydrogen Ion Concentration, In Hydrogen

Ions. Volume I. Chapman and Hail Ltd, London (1942) p. 305.

[32] N. Y. Sreedhar. Y. Jyothi, J. Indian Chem. Soc. 82 (2005) 1038.

[33] W. Likussar. D. F. Boltz. Anal. Chem. 43 (1971) 1265.

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21(2005)167.

106

[35] C. Harlmann, J. Smeyers-Verbeke, W. Pinninckx, Y. V. He>dcn, P.

Vankeerbcrghen. D. L. Massail. Anal. Chem. 67 (1995) 4491.

[36J K. Nakamoto, Infrared Spectra of inorganic and Coordinalion Compounds. John

Wiley & Sons. New York (1963) p. 149.

[37] B. K Seal, H. Sil, M. Banerjee, D. C. Mukherjee, Bull. Chem. Soc. Jpn. 55

(1982)1620.

[38] V. V. Nalimov, The Application of Mathematical Statistics to Chemical

Analysis, Pergamon Press, Oxford (1963) p. 167.

[39] J. Mendham, R. C. Denney, J. D. Barnes, M. Thomas. Statistics: Introduclion to

Chernometrics, In Vogefs Textbook of Quantitative Chemical Analysis. 6!ii cd.

Pearson Education, Singapore (2002) p. 137.

107

Spectrophotometric studies of Thorium(rV)-

rifampicin complex and its application in the

determination of Tli(IV) in soil samples

108

INTRODUCTION

Thorium is a naturally occurring actinide element found in environment or

associated with other metal ions in different complex matrices, nuclear fission

products, monazite sands and geological materials. Thorium is known to cause acute

toxicological effects for human and progressive and irreversible renal injury [1]. The

determination of thorium(IV) in presence of various metal ions found in soil and

rivers is of special interest. Various analytical techniques such as thin la\cr

chromatography [2], gravimetry [3], titrimelry [4]. reversed phase liquid

chromatography [5], fluorimetry [6], potentiometry [7,8], X-ray fluorescence [9], and

inductively coupled plasma mass spectrometry [10] have been reported for Th(lV)

determination. These reported methods such as liquid chromatography, X-ra\

fluorescence, and inductively coupled plasma mass spectrometry are sensitive but

expensive due to high cost and laborious cleanup procedure [5] required prior to

analysis of thorium(IV). Thorium(IV) has been determined spectrophotometricalK

based on reaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol in presence

of sodium acetate-acetic acid buffer solution of pH 4.86 at 580 nm [II]. sodium 4.8-

diamino-i.5-dihvdrox>anthraquinone-2.6-disulphonate in acidic pH at 685 nni (12].

sodium 5-(4-dieth>l-amino-2- hydroxyl phenyl azo) 1.2,4-triazole-3-carboxlate in

acidic pH at 535 nm [13], and bromocresol orange at 560 nm [14], Two extractive

spectrophotometric methods have been utilized for the estimation of thorium(IV)

based on isoam\i alcohol extractable complex of thorium(lV) with 2-hydroxy-l-

napthaldehyde isonicotinoyl hydrazone at pH 3 ( X,niax420 nm) [15] and chloroform

extractable complex with 8-quinolinol in presence of acidic buffer at 390 nm [16].

The present chapter describes a simple and sensitive spectrophotometric method for

determination of Th(IV). The method is based on the reclion of Tii(iV) with

109

rifampicin resulting in the formation of a coloured complex which absorbs maximal!)

at 525 nm.

EXPERIMENTAL

Materials and reagents

All absorbance measurements were made on a speclronic 201)

spectrophotometer (Milton Roy Company, USA) with 1-cm matched glass cells. An

Elico model LI-10 pH meter (Hyderabad, India) was used to measure pH of the

solutions.

All chemicals and solvents used were of analytical reagent grade. Glass distilled

deionized water was used throughout the experiment.

• 1.0 X 10" M thorium nitrate pentahydrate (CAS: 16065-92-2, M.W. = 570.13.

Fluka Chemie AG, Darmstadt. Germany) was prepared in distilled water.

• 1.125 X 10" M (0.1%) rifampicin (CAS: 13292-46-1. M.W. = 822.94. Merck.

USA) was prepared in methanol.

Procedure for the determination of thorium(IV)

Into a series of 10 mL volumetric flasks, different aliquots (0.05 - 1.0 mL) of

1.0 X 10" M standard thorium nitrate solution were pipetted. To each flask 1.7 mL of

1.125 X 10 ' M rifampicin was added and diluted up to the mark with distilled water

(pH 3.5). The contents of each flask were mixed well at room temperature (25 ± TC)

and the absorbance was measured at 525 nm against the reagent blank prepared

similarly except thorium(IV) within the stability time period of 24 h. The

concentration of thorium(IV) was calculated either from a calibration curve or

regression equation.

110

Determination of thorium(IV) in synthetic mixture sample

Sxiilhetic sample of thorium was prepared by taking 570.13 mg of tiioriiini

nitrate pentahydrate with 596 mg Pb(N03)2, 580 mg ZrOCl.. 8H:0. 523 mg

Ba(N03)2. 480 mg CrCl3.6H20, 222 mg CaCi, 2H2O, 119 mg NiCl2.6H20. 65 mg

FeCb and 341 mg CuCU 2H2O ,in 100 mL standard volumetric flask and diluted up to

the mark with distilled water. The amount of thorium(IV) was determined b the

proposed procedure.

Determination of thorium(IV) in synthetic soil sample

Soil samples were taken from Aligarh district of Uttar Pradesh. The digested

soil samples was analyzed for thorium(IV) but tested negative. Therefore, air-dried

finely powdered soil sample (500 mg) with 570.13 mg of thorium nitrate pentahydrate

was digested with 2 mL of concentrated H2SO4 in a closed platinum crucible

following the method reported by Hughes and Carswell [17]. After digestion, the

content of the crucible was cooled and transferred to ice-cold water. The mixture was

stirred until all the soluble matters had dissolved and then filtered through Whatmann

No. 42 filter paper (Whatmann International Limited, Kent, UK) in 100 mL standard

volumetric fiask. The filtrate was diluted up to the mark w ith distilled water. A 20 mL

portion of this solution was percolated through the column packed with .A.mberlite IR

400. The column was washed with 150 mL of 0.1 M H2SO4 to remove unadsorbed

species. The thorium(lV) was eluted with 0.5M H2SO4 at a flow rate of 2 mL per

minute. The effluent was evaporated to dryness. After evaporation, 10 mL of distilled

water was added and the pH of the solution was adjusted to 3.5 by the addition of

ammonia. The final volume of the solution was maintained to 20 mL. The

concentration of thorium(IV) was determined by the proposed procedure.

I l l

RESULTS AND DISCUSSION

A pink coloured complex was obtained with Xmax at 525 nm due to the

interaction of tiiorium(IV) with rifampicin while the rifampicin in methanol-water

medium showed absorbance peak at 470 nm (Fig. 4.1). The reaction was carried out

at room temperature The absorbance measurement at 525 nm as a function of initial

concentration of thorium(lV) was utilized to develop a rapid and selccti\c

spectrophotometric method for the determination of thorium(lV).

The stoichiometry of the reaction between thorium(IV) and rifampicin was

studied by Job's method of continuous variations [18] using equimolar concentrations

(3.037 X 10"' M each). As can be seen from Job's plot (Fig. 4.2) that one mole of

thorium(IV) reacted with one mole of rifampicin. Thus, the combining molar ratio

between thorium(IV) and rifampicin is 1:1.

The I.R. spectra of free rifampicin and Th(lV)-rifampicin complex are shown in

Fig. 4.3a and b. respectively. Rifampicin has -OH and ~C=0 potential sites for

coordination with metal ions. Comparison of IR spectrum of the complex with those

of free rifampicin indicates that phenolic v(C-OH) band appears at 2364 cm ' in the

free rifampicin while the complex does not show this band suggesting that there is

coordination occurs with Th(IV) at this potential site. The carbonyl band, v(C=0) in

the free rifampicin appears at 1746 cm"' with a weak shoulder at 1663 cm"', while the

Th(IV)-rifampicin complex shows this band at 1648 cm' indicating that the

complexation occurs through the coordination of oxygen atom. The phenolic 0-H at

carbon 9 in rifampicin is most deshielded [19]. So, it is deprotonated followed b\

chelate formation through o.xygen atoms of keto and phenolic C-O gropus. The fh-O

stretching vibration occurs at 416 cm"' [20]. A tentative mechanism for the

112

<

375 390 405 420 435 450 465 480 495 510 525 540 555 570 585 600 615

Wavelength, nm

Fig. 4.1. Absorption spectra: (a) 9.721 x 10'^M rifainpicin in methanol (b) 2.066

10 ^M rifampicin in methanol + 7.0 x 10 ^M thorium(IV) in distilled water.

113

0.4 0.6 O.i

Mole fraction of Th(IV)

Fig. 4.2. Job's plot for thorium-rifampicin complex.

r 1

J c i S 3 | . » - - o r o i 4

" i i^^jft—-

o O

5 C t K * • iS S t n

3 -—

» "U V- *- » « ^. i .^ ^ o

o CM

'f.^

\

; t e w s * * • f iK i 'SSJ i ) •

=. 8&08 •.»y • i > f '-r: t

; < *i£S«*. 9«- ' S» .'»• 'i >

• < l „

> t »i!:2!«^»' WOS/ t ' t

S U -*-u

a.

Cf5 o o o C3

o

do

lA

tcfooc- • «5 0Si i

o

I 8 cs 0 4

'A

%

1.1

f I ?t.; pt esse <

o o

i E

s I £

is' H o E

: i-VPlii- ' t'l ZViZ

o £ 0

C-4 o

C3

o

116

complexation between Th(IV) and rifampicin is given in Fig. 4.4.

Optimization of Variables

Tlic optimization of variables was assessed b\' testing several parameters such

as reaction time, concentration of rifampicin, and solvents.

The effect of the reaction time on the absorbance of the pink coloured

complex and its stability was investigated. The complex got stabilized immediatel\ at

25 ± 1 °C after mixing the analyte and reagent. The complex remained stable for about

24 hour.

The effect of the concentration of rifampicin on the absorbance of the colored

complex was investigated in the range of 6.08x10'^ - 2.43x10''' M rifampicin. The

results (Fig. 4.5) showed that the highest absorbance was obtained with 1.70 xlO"* M

rifampicin and remained constant up to 2.43x10' M. Therefore. 2.07x10" M

rifampicin was taken as the optimum concentration for the determination of

thorium(l V).

The effect of solvents such as acetone, acetonitrile. ethanol.

dimethyisulphoxide. 1,4-dioxan, methanol and water were investigated on the

absorbance of the coloured complex. The resuhs are shown in the bar graph (Fig. 4.6).

As can be seen from the figure that the complex showed maximum absorbance in

demineralized water and the pH of the complex in this medium was found to be 3.5.

Therefore, al' measurements were done in demineralized water (DMW) at pH 3.5.

Effect of foreign ion.s

A slud) of the effect of competing metal ions showed that the determination o1'

Th(IV) was not subsequently affected by a range of metal ions, i.e. Pb"" (25-fold

excess). Zr*' (25-fold excess), Ba""" (22-fold excess), Cr ^ (20-fold excess), CTr' (14-

117

Step 1

riHN03)45H20 Th 4+

4NO3 511.0

OH

Step 2

CH3.

CH3. . O 1 ^ "OH - O

CH3 / CH3

H.ca

OH

H3C p l ^ H O NH

•K >--4 OH

O ' O CH3 ^

Rifampicin

H'

CH=N—N N - C H .

Step

Th 4+

Methanoi-H20 medium At25"C

CH3.

CH3. O f "OH " O- , II J ^ H3C OH HO NH

CH< /^Ch,\^^r^^

H3CO' CH=N—N N-CH-,

CH3, -" ^^ - V - ^ ^ C H a

0H3~ / ^ CH3 OH 0=\

CH3' H3C0'

// - r ^ H3C 9 H H O NH r. CH=N—N N-CH3

O

°CH3°^^

Pink complex species

Fig. 4,4. Reaction sequence of the proposed spectrophotometric method.

118

0 2 4 6 10 12 14 16 18 20 22 24 26

[Rifampicin] X 10 M

Fig. 4.5. Effect of the concentration of rifampicin on the absorbance of the

complex.

119

Acetone Acetonitrile Ethanol Methanol DMSO 1,4-dioxan DMW

Solvent

Fig. 4.6. Kffect of solvent on the absorbance of the coloured complex.

120

fold excess), Ca " (9-fold excess), Ni " (5-fold excess) and Fe^^ (2.8-fold excess). The

results are summarized in Table 4.1. However, metal ions such as Mn ' \ 2x\~\ Hg' .

Cd^\ Mg"' and Sr"" interfere in the determination of Th(IV). The interference caused

by metal ions can be removed through a column packed with Amberlite IR 400 resin.

.Most of the metal ions except Th''^ and UO^"^ are washed out on passing 0.1 M

H.SO4. w hereas Th(lV) was eluted with 0.5 M H2SO4.

Validation

The intra- and inter day precisions were evaluated within the same day and on

five consecutive days. The results are summarized in Table 4.2. It can be seen from

the table that the value of RSD (%) were in the range of 0.34-3.03 %.

The accuracy of the proposed method was evaluated by performing recovery

experiments through standard addition technique. The recovery of thorium(IV) in

synthetic sample at two concentration levels was investigated and the results are

summarized in Table 4.3. It is evident from the table and the graph (Fig. 4.7) that the

linearity of ihe regression line of the standard addition method uas good. This

indicated thai the proposed method is precise one and accurate.

The ruggedness of the proposed method was established by deliberateh

changing the concentration of the reagent as:

• Volume of 1.215 x 10" M rifampicin, 1.7 mL (± 0.3 mL)

Under the optimal conditions, the thorium(IV) solution containing 23.2 |j.g mL"'

thorium(IV) (synthetic sample) was analyzed by the proposed method. The results

showed the mean percent recovery and RSD of 100.13 and 0.34%, respectively. The

results of :hc anal\sis indicated the ruggedness of the proposed method.

121

Table 4.1. Effect of various metal ions on the determination of 23.2 ng niL '

tlioriumiV).

Metal

Pb"

Zr^-

Ba-

Cr'"

Cu'^

Ca ^

Ni^^

Fe^^

ions Added as

Pb(N03)2

ZrOCb.SH.O

Ba(N03)2

CrCl3.6H20

CuCb 2H2O

CaCb 2H2O

NiCl2.6H20

FeCb

Tolerance limit (mg ml. )

0.596

0.580

0.523

0.480

0.341

0.222

0.119

0.065

122

Table 4.2. Test of precision of the proposed method at three concentration le\els

ofTh(IV).

Parameters Intra day assay Inter day assa\

Concentration taken (^g mL"') 2.3200 11.600 23.200 2.3200 11.600 23.200"

Concentration found'(^gmL"') 2.3204 11.596 23.229 2.3250 11.596 23.234

Standard deviation g mL"') 0.065 0.051 0.079 0.070 0.056 0.100

Relative standard deviation (%) 2.79 0.438 0.341 3.028 0.482 0.429

Standard analytical error (%) 0.029 0.023 0.035 0.029 0.025 0.045

Confidence limit'' 0.080 0.063 0.098 0.080 0.069 0.124

•"Mean for five independent determinations.

''Confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).

123

Table 4.3. Recovery data of thorium(IV) in synthetic mixture sample by standard addition technique.

Concentration (|.ig niL ) Coefficients of linear regression Rcco\cr\

equation of standard addition ("o)

Synthetic Standard Nominal Intercept slope

6.96 0,1.16,2.32, 6.962 0.30031 0.04313 0.99996 100.04

4.64, 6.96

13.92 0,1.16,2.32, 13.94 0.60022 0.04303 0.99998 100.20

4.64, 6.96

Coefficient of correlation.

Mean for five independent analyses.

124

-4-1-

Nominal Concentrat ion, ^g mL Standard added

Fig. 4.7. Kccoxerv of rh(IV) from synthetic mixture b\ standard addition

technique; (a; 6.963 and (b) 13.949 ng mL 1

125

The calibration curve was constructed by plotting absorbance against the

concentration of thoriuiTi(IV) in fig niL"' (Fig. 4.8). Beer's law was obeyed over the

concentralioii range of 1.16 - 23.2 |.ig niL' with molar absorptivit_\ and SandcH's

sensilixii of 8.23 >' 10' L moT cm' and 0.023 |.ig/cm"/ 0.001 abiorlxnice unit.

respective is. The calibration data (n = 9) were treated statistical l_\ and the results are

summarized in Table 4.4.

The applicabilit' of the proposed method for the determination of thorium(lVj

in synthetic mixture and soil samples has been tested. The results of the proposed

method were statistically compared with those of the reference spectrophotometric

method [16] using point and interval hypothesis tests. The t- (paired) and the F-

values at 95 % confidence level were calculated and found to be less than the

tabulated t- (2.036 at u = 8) and the F-values (6.39 at u = 4,4) at 95 °.o confidence

level [21 [ thus confirming no significant difference between the performance of the

proposed method and the reference method (Table 4.5). It is also clear from the table

that the bias evaluated by interval hypothesis test [22] by means of lov\er limit (OL)

and upper limit (9u) were in the range of 0.98-1.02. Thus, the proposed method is

suitable for routine analysis of thorium(lV) in nuclear process laboratories where time

and economy are essential.

CONCLUSION

The proposed method is simple and accurate for the determination of Th(IV). The

proposed method has the advantages of having low limit of detection (0.099 |.tg ml, ')

and low cost of analysis. In addition, the proposed method is based on complexation

of Th(IV) w iih rifampicin at room temperature showing the involvement of one

126

- I — 1 — I — 1 — I — I — I — I — I — I — I — I — \ — I — I — 1 — t -

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

[Th(IV)], Jig m f '

Fig. 4.8. Calibration curve for the determination of Th(IV).

127

Table 4.4. Optical and regression characteristics of the proposed and reference

methods.

Parameters

Wavelength (iiin)

Beer's lav limil (|.ig inL" )

Molar absorptivity (L mol'cm"')

SandelTs sensitivity

Linear regression equation

±tSa

±tSb

Correlation coefficient (r)

Variance (So) of calibration line

Deleclioji iin;il (|j.g ml"' )

Quantitation limit ().ig ml"')

Proposed m

525

1.16-23.2

8.23 X 10

ethod

0.0232 ^g/cm^/0.001

absorbance unit

A = 5.917 X

xlO"^C

1.88 X 10"

1.32 X 10"

0.99999

I.69X 10"'

0.099

0.301

10- + 4.31

Reference methcKl

390

1.0-20

9.70 X 10'

0.0239 iig/cm^/ 0.00

absorbance unit

A = 4.685 X 10' +4.

xlO"^C

1.722 X 10"'

1.440 X 10"

0.99999

1.369 X 10""

0.092

0.280

±t Sa and :tt Sb are confidence limits for intercept and slope, respectively.

128

Table 4.5. Evaluation of bias: Applicability of the proposed method in synthetic

mixture and soil samples and its comparison with the reference method at 95"/)

confidence level.

Samples Proposed method Reference method Paired

t - & F

values

G, 0,>'

Recovery" RSD

(%) (%)

Recovery' RSD

(%) (%)

*Synthetic

mixture

Soil samolc:

99.97 0.438 100.18 0.492

100.05 0.498 100.18 0.492

t = 0.699

F= 1.123

t = 0.403

F= 1.024

0.989 .006

0.990 1.007

*Synthetic mixture of thorium(IV) contains: 1 mg thorium nitrate pentahydrate + 596

mg ?b-\ 580 mg Zr^\ 523 mg Ba'^ 480 mg Cr , 222 mg Ca^^ 119 mg Ni" . 65 mg

Fe and 341 mg Cu" in 100 mL distilled water.

''Mean loi 5 independent anaKses.

'Fheorctical / ( i/= 8) and F-values (1^= 4, 4) at 95 % confidence level are 2.306 and

6.39, respectively.

A bias, based on recovery experiments, of ± 2% is acceptable.

129

reagent only and there is no use of buffer solution. Hence, the proposed method can

be used for routine analysis of Th(IV) in real samples as the interferences caused b>

some metal ions can be easily removed by anion exchange resin.

130

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[16] K. Goto. D. S. Russell, S. S. Berman, Anal. Chem. 38 (1966) 493.

[17] K. C. Hughes, D. J. Carswell, Analyst 95 (1970) 302.

[18] W. Likussar. D. F. Boltz, Anal. Chem. 43 (1971) 1265.

[19] S. Sadeghi, E. Karimi, Chem. Pharm. Bull. 54 (2006) 1107.

131

[20] K. Nakainoto, Infrared Spectra of Inorganic and Coordination Compounds, John

Wiley & Sons, New York (1963) p. 149.

[21] J. Mendham, R. C. Denney, J. D. Barnes, M. Thomas, Statistics: Introduction to

Chemometrics. In VogeTs Textbook of Quantitative Chemical Analxsis. 6lh ed..

Pearson Education, Singapore (2002) p. 137.

[22] C. Harlmann. J. Smeyers-Verbeke, W. Pinninck.x, Y. V. Heyden. P.

Vankeerbcrghen. D. L. Massart. Anal. Chem. 67 (1995) 4491.

132

Spectrophotometric determination of U(VI) via

chelation with rifampicin in soil samples

133

INTRODUCTION

Uranium dioxide powder is a starting material for the manufacture of fuel

pellets which are used in nuclear power reactors. The uranyl ion, U02"^ can be found

in soils and in low pH-water run off in and around nuclear waste sites and processing

facilities. It is reported that U(V1) is leached out of rocks into water streams. In view

of this extraction and determination of U(Vr) from surface and ground uater has

become a matter of great interest [1,2].

The literature citation revealed that there are various analytical techniques for

the estimation of uranium which include thin layer chromatography [3], gravimetr\

[4], titrimetry [5], fluorimetry [6,7], potentiometry [8], polarography [9], X-ra\

fluorescence [10], inductively coupled plasma mass spectrometry [11]. In addition,

spectrophotometric methods have also been employed to determine U(VI) in presence

of thorium(IV) [12], ore leachates [13], natural waters [14-16], process streams of a

uranium extraction plant [17] and soil [18]. Moreover, the gravimetric method

generally requires the prior separation of interfering elements whereas multiple steps

are in\oi\cd in the titrimetric method. Generally, the electroanaKlical methods are

employed to determine U(V1) on a routine basis. Spectrophotometry is the technique

of choice in process control due to its simplicity, adaptability and reasonable

sensitivit with significant economical advantages. Therefore, the methods based on

speclrophotoinetry [19-21] were published in reputed journals. Therefore, there is a

need for a simple and rapid spectrophotometric method for the determination of

uranium(VI) in soil samples in the presence of some other metal ions. The proposed

meihoa is based on the complexation of uranyl ion with rifampicin in methanol-waier

medium al room temperature (25 ± \°C). The resulting \eilo\\ coloured complex

134

absorbs maximally at 375 nm. The reaction conditions of tiie proposed metiiod are

opiimized and \alidated [22].

EXPERIMENTAL

Apparatus

Ail absorbance measurements were made on Spectronic 20 D

spectropiiotometer (Milton Roy Company, USA). An Elico model Li-10 pH meter

was used to measure pH of the solutions.

IR spectra were recorded on a Perkin-Elmer FTIR 1650 spectrophotometer in

wave number region 4000-400 cm"' using KBr pellet technique.

Soil samples were collected from Chemistry Department, Aligarh Muslim

Universily, Aligarh, India.

Reagents and standards

All chemicals and solvents used were of analytical reagent grade.

• 5 X 10"' M uranyl nitrate hexahydrate (CAS: 13520-83-7, Fluka Chemie AG.

Darmstadt. Germany) was prepared in distilled water. The apparent purit\ of

uran) 1 nitrate solution was checked by spectrophotometric titration using

potassium ferrocyanide [23].

• 6.1 X 10" M rifampicin (CAS: 13292-46-1, M.W.: 822.94. Merck. USA) was

prepared in methanol.

• Buffer solutions ranging from 3.72-5.57 were prepared by mixing var_\ing

volumes of 0.2 M acetic acid (9 -1 mL) and 0.2 M sodium acetate (1-9 mL) in 10

mL standard volumetric flask [24].

135

Procedure for the determination of uranyl ion

DilTcreni aliquots (0.1 - 1.5 mL) of 5 x 10" M uranyl nitrate solution were

pipetted into a series of 10 mL standard volumetric flasks. To each tlask. 2.0 ml. o1

6.1 X 10' M rifampicin solution was added and diluted to volume with methanol. The

contents of each flask were mixed well at room temperature (25 ± TC) and the

absorbance was measured at 375 nm against the reagent blank prepared similaiK

within the stability time period of Id. The concentration of uranyl ion was calculated

from the corresponding regression equation.

Determiaation of uranyl ion in soil sample

The accuracy of the proposed method for the analysis of uranyl ion in soil was

tested with synthetic mixture of known composition of uranyl ion plus soil from our

locality. Therefore, air-dried soil sample (500 mg) of our locality was treated with 25

mg of uranyl nitrate and decomposed, by heating with 2.0 mL concentrated H2SO4 in

a closed platinum crucible [25]. After cooling the content of the crucible (reaction

mixture) was transferred to 50 mL of ice-cold water and stirred until all the soluble

matters Lad dissolved. Silica and other insoluble materials were filtered off through,

Whatmann No. 42 filter paper (Whatmann International Limited. Kent. LK) in 100

mL standard volumetric flask. The filter paper was washed with distilled water and

the filtrate was diluted up to the mark with distilled water. 20 mL of this solution was

percolated through the column packed with Amberlite IR 400 because uranium was

found to be completely adsorbed on the resin at acidities below 10" M sulphuric acid

(pH > 1.5) [26]. The column was washed with OTM H2SO4 to remove unadsorbed

species, fhe uranyl ion was eluted with 2M H2SO4 at a flow rate of 2 mL per min.

The et'llucnl was evaporated and the residiie was dissolved in 10 iiiL of disiillcd

water. Ilic pll of the solution was adjusted to 3.9 by the addition of ammonia soluticMi

136

and the final volume of the solution was maintained to 20 mL. The amount of uran\l

ion was also estimated by the reference method [12].

Procedur-. for reference method [12]

Aliquois (0.125-1.5 mL) of 1.992 x lO'' M uran>i nitrate were pipetted uMo a

series ol'25 mL standard volumetric flasks. To each flask, 10 mL of 0.2 M IIC'I. 2.0

mL of 10% SnCl2.2H20 in 1.16 M HCl and 7 mL of 6.57 M NH4SCN were added and

diluted up to the mark with distilled water. The absorbance was measured at 365 nm

against the reagent blank prepared similarly and the amount of the uranyl ion in a

given sample was obtained either from the calibration graph or the regression

equation.

Determiiialion of stoichiometry

The reaction stoichiometry of the proposed method was studied by Job's method

of continiious sariations [27]. For this purpose, different volumes (0. 0.2. 0.4. 0.6. 0.8.

1.0. 1.2. 1.4. 1.6, 1.8, 2.0 mL) of 1.1 x 10" M uranyl nitrate was added with different

volumes (2.0. 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0 mL) of 1.1 x 10 ' M

rifampicin and diluted with methanol in 10 mL standard volumetric flask. The

absorbance was recorded at 375 nm and plotted against the mole fraction of uran\ I

ion.

Validation

The proposed method has been validated for specificity, precision, accuracy,

linearity, lobusiness and evaluation of bias.

The selectivit was ascertained by determining 18.9 ^g mL"' uranyl ion in the

presence of metal ions such as Pb^^ Ni^^ Cr^\ Zr''^ Ca^^ and Ba"^ which are

commoni\ found in soils.

137

The liiiearilx was assessed by analyzing the uranyl ion content at nine

concentration levels: 1.35,2.70.5.40,6.75, 10.8, 13.5. 17.55, 18.9 and 20.25 ng ml/ ' .

Each concentration level was independently analyzed for five times. The absorbance

was plotted against initial concentration of uranyl ion. The regression characteristics

were calculated using OriginPro 6.1 Software. Limits of detection and quantitation

were calculated [28].

The intra-da) and inter-day (n= 5) precisions were evaluated at three

concentrations levels (5.4, 10.8 and 18.9 |ig mL"'). Five sample solutions of each

concentration were analyzed within one da\ (intra-day precision) and in fixe

consecutive days (inter day precession).

The recovery of uranyl ion from soil samples was estimated by the standard

addition method. For this purpose, 5 mL (or 10 mL) of sample solution obtained b\

separation on a column loaded with Amberlite IR 400 was spiked with 0, 1, 2, 3 and 4

mL of reference standard solution of uranyl nitrate (5x10"^ M) in a 100 mL standard

volumetric flask and the solution was diluted up to the mark with methanol. The

absorbance for all these solutions was recorded at 375 nm and the corresponding

regression line was evaluated in order to get slope and intercept using Originl'ro b.l

Software. Each level was repealed 5 times. The concentration of urain I ion m tlic

sample solution was calculated by the ratio of the intercept and the slope of the

regression line. The error in the concentration was evaluated using the following

expression [29]:

S, -^^"^ ^^~ b

i Z-

where S , is the standard deviation of the extrapolated \-\alue (xi

138

The robustness was evaluated by analyzing the content of uranyl ion in soil

sample. A soil sample solution containing 16.20 |ag mL" of uranyl ion was anal\zed

five times using the proposed method. Mean percentage recovery ± relative standard

de\ ialion (RSD) was calculated.

The bias [30] has been evaluated to compare recover results of the proposed

method with those of the reference method at 95% confidence level using the

following quadratic equation.

where Sp is the pooled standard deviation and /tab is the tabulated one sided /-value

with ni + n2 - 2 degrees of freedom at the specified level of significance.

RESULTS AND DISCUSSION

The uranyl ion is known for its ability to form complexes with a variel\ oi

ligands such as H2O, CI", NO3', acetic acid, lactic acid and oxalic acid |3l-33j.

Rifampicin contains more than one electron centers and assumed to be an cxccllcni

ligand for comple.xation reaction. Uranyl ion in distilled water absorbs maximally at

195 nm while rifampicin in methanol gives ?imax at 240 nm. In the present stud,

uranyl ion reacts with rifampicin in methanol-water medium immediately at 25 ± 1"C

resulting in the formation of yellow colored complex which absorbed maximally at

375 nm. The measurement of absorbance at 375 nm lead to the development of a

rapid and selective spectrophotometric method for determination of U(V1). The

absorption spectra for urani ion in distilled water, rifampicin in methanol and uranl

ion-rifampicin complex in methanol-water medium are shown in Fig. 5.1. It is clear

from the graph that the maximum absorbance wavelength of the U(Vl)-rifampicin

complex is dilTerent to that of rifampicin. The reaction complex was stable up to id.

139

-1 1 1 r

200 220 240 260 280 300 320 340 360 380 400 420 440

Wavelength, nm

1-4 Fig. 5.1. Absorption spectra of (a) 1 mL of 1.0 x 10' M uranyl nitrate in distilled

water (b) 1 inL of 6.1 x 10^ M rifampicin in methanol and (c) 1.5 mL of 5.0 x 10 ^

M uranyl nitrate in distilled water + 2.0 mL of 6.1 x 10"* M rifampicin in

methanol. Solutions a, b and c are diluted up to mark with distilled water,

methanol and again methanol in 10 mL standard volumetric flask, respectively.

140

Stoichiometrj'

Tlie composition of the yellow complex between uranyl ion and rifampicin was

determined by Job's method of continuous variations using identical molar

concentrations of uranyl ion and rifampicin (1.10 x 10'"' M each). The absorbancc was

recorded at 375 nm and plotted against the mole fraction of uran\ I nitrate (Ki*4. 5.2). ii

is apparent from the figure that the combining molar ratio between uranyl ion and

rifampicin is 1:1.

The I.R. spectra of free rifampicin and UO2 ^-rifampicin complex are shown in

Fig. 5.3a and b, respectively. Rifampicin has-OH and -C=0 potential sites for

coordination with metal ions. Comparison of IR spectrum of the complex with those

of free rifampicin indicates that phenolic v(C-OH) band appears at 2364 cm"' in the

free rifampicin while the complex does not show this band suggesting that there is

coordination of uranyl ion at this potential site. The carbonyl band. v(C=0) in the free

rifampicin appears at 1746 cm"' while this band in the U(Vi)-rifampicin complex is

shifted to 1719 cm"' indicating that the uranyl ion is chelated through coordinaliv)ii of

oxvgen atoms of the keto group (dihydrofuranone) and the neighboring phenolic

group of rifampicin molecule. The phenolic 0-H at carbon 9 in rifampicin is most

deshielded [34], thus deprotonated and followed by chelate formation through oxygen

atoms of keto and phenolic C-0 groups. The characteristic v(U=0) frequenc> the

complex appears at 901 cm" whereas this frequency is not present in free rifampicin.

The U(VI)-0 stretching vibration occurs at 404 cm"' [35]. Therefore, on the basis of

these experimental findings, a reaction sequence of the proposed method is shown in

Fig. 5.4.

1 4 1

0.25

0.20 -

0) o c CD

XI

o (/5

<

0.15 -

0.10

0.05

0.00

0,0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Mole fraction of uranyl nitrate

0.8 0.9 1.0

Fig. 5.2. Job's method of continuous variations of uranyl nitrate-rifampicin

complex.

-

/

< >

\

!

1 ! 1 ) 1 ;

t t > I t

,ir-:=-^-. I ': ( EISS t-t" • 52>SEJ i

V ' : : ; ; i ^ ^ ; ; ; ; j i

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A , ' ' ' ' ' ;

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1 1' 1 (..atZeSV,' 08 A l f C ;

I *-^— 1 ! •

,.--r ! 1 , , s"' ' ' ; ' '

C5 0 0 0 T - 0 3 CO

o O

o o o

o o o CO

o o o

o CO

S

I 3

u

a

( f 99e>Z ' 69 09t ' ) -

y

( 2530 W ' I-f l £ S ) -

(t'csacz '00/z6 ) — ( sioesz ' Sf090i ) ~

(82t'S-ez 'ortsei. ) —

( f69S>Z ' WULV ) —

o o

o o o

\

X \ ^

s (26t8zz 'zi'-ieez )-(eeeezz 'zisiez)- o

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w

E o w

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o o o

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eJD

1 4 4

Step 1

CH3.

Step 2

CH3 T OH 0=

H3C OH HO NH '

CH=N—N N-CH3

Rifampicin

UO-,

CH3 OH 0=

H3C 9 ^ HO NH

Methanol-H20 medium At25'^C

CH=N—N N-CH3

CH=N—M N-CH 3

CH=N—N N-CH3

Yellow complex species

Fi«4. 5.4. Reaction sequence of the proposed method.

145

The apparent formation constant (Kf) for the complex between uranyl ion and

rifampicin was calculated using the following equation [36]:

iif = r^„.. [c.-^cnc-^

where Aobs and Aextp arc observed and extrapolated absorbance values for the complex.

respectively. CM and CL are the initial concentration of U(VI) and rifampicin in mol 1.' .

respectively. KfOf the complex is found to be 1.51 x 10 . The apparent Gibbs free energy

(AG) was calculated using AG° = - 2.303 RT log Kf and found to be - 23.84 kJ mol"'

confirming the feasibility of the reaction.

Optimization of V^ariabies

The optimization of variables was investigated by testing reaction lime.

concentration of rifampicin and solvents.

The effect of reaction time on the absorbance of U(VI)-cefixime complex and its

stability was investigated. The U(VI)- rifampicin complex got stabilized immedialeh at

25 ± 1 "C after mixing rifampicin and methanol as solvent. Complex remained stable for

1 d.

The concentration of rifampicin was optimized via a series of experiments for the

maximum absorbance of the uranyl-rifampicin complex. The effect of the volume of 6.1

X 10" M rifampicin was studied in the range of 0.2 - 2.0 mL. The results are shown in

Fig. 5.5. It is clear from the figure that the maximum absorbance was obtained with 1.6

mL of 6.1 X IO'^'M rifampicin; above this volume upto 2.0 mL, the absorbance remained

constant. Therefore, 1.8 mL of 6.1 x 10" M rifampicin was used in all measurements.

146

0.50

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

,-4 Volume of 6.1 X 10" M rifampicin, mL

Fig. 5.5. Effect of the volume of 6.1 x 10 ' ' M rifampicin with 20.25 ng mL ' uranyl

ion on the absorbance of the complex.

147

The effect of solvents such as methanol, acetone, acetonitrile, dimethyl sulphoxide

(DMSO) and 1,4-dioxan was tested at 20.25 jig mL'' U(VI) on the absorhance of uranyl-

rifampicin complex. It was observed that the maximum absorbance was obtained in

methanol Vig. 5.6). Therefore, methanol is preferred to be the best sohenl for diUnion ot~

the reaction mixture of the complex. The pH of the complex was measured and found lo

be 3.9, The elTect of pH on the absorbance of the complex was investigated usinu sodium

acetate-acetic acid buffer solution in the pH range of 3.72-5.57. A constant absorbance

was obtained in the pH range of 3.72-5.57, but the absorbance of the complex in this pH

range was found to be less than that obtained without buffer solution using methanol as

the diluting solvent in the reaction mixture. Therefore, all absorbance measurements were

made in methanol solvent without the use of buffer solution.

Validation

The effeci of potential interfering ions on the determination of V\'\ was

investigated by adding known concentrations of each ion in a solution containing anahte

and then determining the latter. The results are summarized in Table 5.1. The tolerated

amounts of each ion were the concentration values tested that caused less than rr 2% the

absorbance alteration. These results showed that the proposed method is selective for the

determination of uranyl ion in the presence of Pb " , Ni " , Cr' ' , Zr''^, Ca ^ and Ba'^.

However, the method was not selective in the presence of Cu^ , Mn'^. be' . 7A\^' Cd" .

N4g' and Sr" . The interference of these ions can be eliminated by passing the sample

solution through a column packed w ith Amberlite IR 400 resin.

148

0 o c CO

o X! <

Methanol Acetone Acetonitrile 1,4-dioxan DMSO

Solvent

Fig. 5.6. Effect of solvent on the absorbance of the coloured complex.

149

Table 5.1. Effect of various metal ions on the determination of 18.9 ng mL uranyl

ion.

Metal ions

Pb^

Zr-"

Cr'

Ca ^

Ni- "

Ba-

Added as

Pb(N03)2

ZrOCb.SH.O

CrCl3.6H20

Ca(N03)2.4H20

NiCl2.6H20

Ba(N03)2

Toler ance limit (mg mL )

0.662

0.580

0.534

0.155

0.095

0.052

150

Ihider the optimized experimental conditions, the absorbance against the initial

concentration of uran\l ion was plotted (Fig. 5.7) and found to be linear o\cv the

concentration range 1.350 - 20.25 |jg mL' with apparent molar absorpli\it\ and

Sandell's sensitivity of 8.0 x 10' L mof'cni"' and 0.042 (.ig/enr/ 0.001 absorbance unit.

respecti\'ely. The calibration data (n = 9) were treated statistically to c\aluate intercept

(a), slope (b). coefficient of correlation (r), confidence limits of intercept (± tSa) and slope

(± tSb) at 95 % confidence level and n-2 degrees of freedom, variance and limits of

detection and quantitation. The results are summarized in Table 5.2. The high value of

correlation coefficient (0.9999) for the proposed method indicated excellent linearit. fhe

low value of variance (2.1 >'- 10' |ig mL" ) of calibration line pointed towards the higher

reproducibility of the proposed method.

The intra-day and inter day precisions of the proposed method v\ere e\a!uated at

three concentration levels: 5.4, 10.8 and 16.2 |ig mL' within the same da)' and on i'lxe

consecuti\'e days, respectively. The results of the analysis are summarized in Table 5.3.

It is evident from the table that the percent relative error and relative standard deviation

were in the range of 0.06 - 0.74 % and 0,44 - 2.57 %. respectively.

The accuracy of the proposed method was investigated by performing reco\cr\

experiments through standard addition method. The results of the reco\ery experiments

are summarized in Table 5.4 and Fig. 5.8. It is evident from the table that the recover) is

satisfactory' (100.31 -10.43 %) and the most attractive feature of the method is its relati\e

freedom from various non targeted cations. The recovery experiments have confirmed

that the proposed method is accurate.

1 5 1

0) o c CD

o (/)

<

1 1 ! 1 1 1 1 i ! ! 1 1 1 1 1 1 1 1 r

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Fig. 5.7. Calibration graph for the determination of uranyl ion.

152

Table 5.2. Optical and regression characteristics of the proposed and reicrence

methods.

Parameters Proposed method Reference meihoci

Wavelength (nm)

Beer's law limit (|.ig mL' )

Molar absorptivity (L moP'cm') 8.0 x 10

Sandell's sensitivity

Linear regression equation

i t s ,

±tSh

Correlation coefficient (r)

Variance (So") of calibration line

Detection limit (|ig mL' )

Quantitation limit (fig niL" )

±t Sa and ±t Sh are confidence limits for intercept and slope, respectively.

375

1.35-20.25

8.0 X 10

0.042 ng/cm^/0.001

absorbance unit

A=1 .39x 10- + 2.37

xlO'^C

2.16 X 10"

1.70 X 10-

0.9999

2.10 X 10''

0.20

0.61

365

5.0-60

6.25 X 10'

-

A = 7.I53 :

10" C

4.60 X 10'-'

1.28 X 10"'

0.9998

6.20 - 10""

1.10

3.33

153

Table 5.3. Test of precision of the proposed method.

Parameters Intra day assay Inter day assa\

Concentration taken (ng mL" ) 5.40 10.80 16.20 5.40 10.80 16.20

Concentration found^ (|ig mL" ) 5.41 10.79 16.23 5.36 10.87 16.19

Standard deviation (ng mL" ) 0.14 0.12 0.07 0.14 0.17 0.14

Relative error (%) 0.19 0.09 0.19 0.74 0.65 0.06

Relative standard deviation (%) 2.57 1.12 0.44 2.67 1.58 0.87

Standard analytical error (%) 0.06 0.05 0.03 0.06 0.08 0.06

Confidence limit'' 0.17 0.15 0.09 0.17 0.21 0.18

^Mean for five independent determinations.

" Confidence limit at 95% confidence level and four degrees of freedom (t - l.llb).

T5T

Table 5.4. Test of accuracy of the proposed method by standard addition

method.

Concentration (fig mL"') Coefficients of regression line Recover-^

Soil 1 Standard Nominal Error Intercept slope r"

"6?75 0. 1.35,2.7,4.05, 6 J7 o!05 0T6 2.36x 10" 0.99997 100.37

5.40

13.5 0,1.35,2.7,4.05, 13.55 0.91 0.32 2.36x10"- 0.99987 100.43

5.40

^ Coefficient of correlation.

'Mean lor fi\ e independent analyses.

155

^T^e-

I—'—1 1 1 1 1——1 r"—1 1 1 1 1 0.00—I 1 1 1 1 r

14-13-12-11-10 -9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 0 1 2 3 4 5 6 7

1 Nominal Concentration, ^g mL" Spiked

Fig. 5.8. Recovery graph for uranyl ion through standard addition nieihod:

(a) 6.75 and (b) 13.5 |xg mL'' uranyl ion.

T55^

The robustness of the proposed method was critically examined b\ determining

uranyl ion contents (20.25 fig mL"') in synthetic environmental soil sample under

deliberate small changes in experimental conditions. The results showed thai the mean

% recovery ± RSD was found to be 100.18 ± 0.44 that suggested the robusuicss ofihc

proposed melhod.

The proposed method has been successfully applied to the deierminaiJoii o\

uranyl ion in synthetic soil samples containing LJ(Vl). The results obtained b\ the

proposed method were compared with those obtained by the reference method'" using

point and interval hypothesis tests in terms of Student's paired t-test, the variance

ratio F-test, lower (9L) and upper bias (6u) limits at 95 % confidence level. The results

are summarized in Table 5.5. The calculated paired t- and F-values are less than the

tabulated t- and F- values at 95 % confidence level [37] suggesting thai there is no

signitlcant difference between the performance of the proposed method and the

reference method. This confirmed that the proposed method is accurate, precise and

reproducible. The interval hypothesis test has confirmed that the true bias (G| and Q )

for the analysis of U(VI) in soil samples is smaller than ±2.0 %; indicating that there

is no significant difference between the performances of the methods compared.

The performance of the proposed spectrophotometric method was compared

with other methods (Table 5.6). It can be seen from the table that TLC method is time

consuming and tedious. The precision is somewhat poor. ICP-MS melhod is sensitixe

but expensive enough and requires solid phase extraction of U(VI) before anal\sis.

The precision of the proposed spectrophotometric method (RSD 0.44-2.67"o) as

compared to other spectrophotometric methods (RSD 0.91-8.21%) is tjuile

satisfactory. The proposed method is simple, rapid, precise and accurate, thus can be

used as alternate method for the determination of U(VI) in soil samples.

157

Table 5.5. Applicability of the proposed method for (he determination of uranyi

ion in soil samples and the comparison of the results with the reference metiiod

at 95% confidence level.

Sample of

iiranyl ion

Proposed method Reference method Paired

t- & F values'

Recovery' RSD

(%) (%)

Recovery' RSD

(%) (%)

Ou

Soil 1 100.18 0.44 100.09 0.83 1=1.339 0.981 1.016

F= 3.503

Soil 2 99.94 0.43 100.09 0.83 t = 2.248 0.989 1.014

F= 3.664

''Mean for 5 independent anaKses.

''Theoretical i v= 8) and F-values (i/= 4. 4) at 95 % confidence level are 2.306 and

6.39. respectively.

A bias, based on recovery experiments, of ± 2% is acceptable.

1 5 8

Table 5.6. Comparison of the proposed spectrophotoinetric method witis oih

methods for the determination of uranyl ion.

Reaoents X,„ax Beer's law RSD (%) Analysis time Referent

(nm) limit (^igiTiL'') (min)

Spectrophotometrv

p-Carboxychlorophosphonazo

SnClj.H.O and NH4SCN

Chromazurol S and

Cetylpyridnium bromide

Rifampicin

TLC

714

365

625

375

4 - 12

5-60

0- 100

1.35-20.25

0.91-8.21

-

<3

0.44-2.67

immediateh

immediately

15

immediately

at 25 ^ r C

[14]

[12r

[16]

This work

Mobile phase: 0-xylene:

methyl ethyl ketone: N.N'-

dimethylformamide: Iso-

propyldithiophosphotic acid

ICP-MS

Duolite XAD76!, 9-phenyl-3-

fluorone

254 2.5-30 60

;2.3 30 I I

"Reference method.

159

CONCLUSIONS

The proposed method has the advantage of being simple, rapid and selccli\c lor

routine anal\sis of uranyl ion in soil samples, in addition, the proposed nicliiud i

utilizing one reagent with less expensive solvent (methanol) for the deterniinalion

proeess. The proposed method has avoided the use of acid, buffer solution and heating

of reaction mixture. In addition the method is useful due to high tolerance limits from

cations and anions. Hence, the proposed method is an effective method for the

quantitative analysis of uranyl ion in soil samples.

160

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Journal of Scienlific & Industrial Research Vol. 69, February 2010, pp 135-141

Spectrophotometric determination of Fe(III) via complexation with piroxicam in synthetic mixture and soil samples

Lutfullah'*,Saurabh Sharma',Nafisur Rahman', SyedNajmul Hejaz AzIni^ Hanan JumaSaid Al Hidaif 1' and Masha' El Mansoor Abdulrahman AlQasmi^

'Department of Chemistry, Ahgarh Muslim University (AMU), Aligarh 202 002, India

-Department of Applied Sciences, Chemistry Section, Higher College of Technology, P O Box 74, AI-Khuwair-133. Muscat, Sultanate of Oman

Received OH September 2009; revised 03 December 2009: accepted JI December 2009

A simple and selective spectrophotometric method has been described for determination of Fe(ni) in synthetic mixture and soil samples. Method is based on chelation of ferric ion with piroxicam to produce at RT a coloured metal complex, which absorbs maximally at 495 nm. Beer's law is obeyed (cone, 2.79-25.13 mg ml"') with apparent molar absorptivity (5.88 x ICPI mol'cm ') and Sandell's sensitivity (0.019 mg/cmVO.(X)l absorbance unit). Proposed method was successfully applied in determination of Fe(III) in synthetic mixture and soil samples.

Keywords: Ferric sulphate, Metal chelate, Piroxicam, Spectrophotometry, Validation

Introduction Iron (Fe), one of the most important constituent

of biological systems as well as of metallurgical industry, reacts with di-antipyrene methane to form a red coloured complex, which absorbs maximally at 520 nm'. Trace amount of Fe (III) was determined by complexation with potassium thiocyanate and methyl violet in presence of polyvinyl alcohol". Colour reaction^ between Fe(IIl) and p-diethyl-amine phenyl fluorine (cone, 0.056-6.7 ixg / 25 ml) at pH 9.0 was utilized for determination of Fe(III). A number of kinetic spectrophotometric methods have been utilized for Fe (III) determination using methyl thymol blue at pH 'i.5\ azocarmine B and potassium periodate', chlorophosphonazo and potassium bromate\ methyl orange and potassium bromate^, [4,4'-bis(dimethyl-amino) diphenyl-methane], potassium bromate and potassium iodide*, orange G and potassium periodate', methylene blue and hydrogen peroxide'**. Various reagents (4-aminoantipyrene", 2-hydroxyl-5-methyl benzophenone oxime'^, 2-hydroxy-4-n-butoxy propiophenone oxime'^ 3- hydroxyl-3-phenyl-I-m-chlorophenyl triazene'"*, thioglycolic acid", 1,2-

Aulhoi lor correspondence Tel:+91-571-2703515 E-mail: lutfullah786@email,coin

dihydroxy-3,4-diketocyclo butene"", diformyl hydrazine'^ and 1,3- diphenyl -4-carboethoxy pyrazole-5-one'*) have been used for determination of Fe(IIl). Other spectrophotometric methods for determination of Fe(III) have been reviewed''*. Derivative spectrophotometric procedures have also been reported for delerniination of iron^"-'.

This study presents a simple and rapid spectrophotometric method via complexation with piroxicam for determination of Fe(lll) in synthetic mixture and soil samples.

Experimental Materials

All absorbance measurements were made on a Spectronic 20D'' spectrophotometer (Milton Roy Company, USA) with 1 cm matched glass cells. Absorption spectra were recorded on a Shimadzu UV-visible spectrophotometer (UV-160 A, Shimadzu Corporation, Kyoto, Japan) with matched quartz cells. Soil samples were collected from Department of Chemistry, AMU, Aligarh, India. All reagents used were of analyti­cal grade. 0.005 M Ferric sulphate (CAS, 10028-22-5; MW, 399.88; Fluka Chemie AG, Swilzerlandj solution was freshly prepared in distilled water. 6.038 x 10 ' .VI (0.20%)

136 J SCI IND RES VOL 69 FEBRUARY 2010

200 240 280320 360 400440480520560 600 640660

Wavelength, nm

Fig. I—Absorption spectra of: a) 0.2 ml of 5 10' M ferric sulphate in distilled water; b) 0.3 ml of 0.016% piroxicam in methanol; c) 0.45 ml of 0.005 M ferric sulphate in water + 2.5 ml of 0.16% piroxicam in methanol (Each solution is diluted up to the mark with ethanol in 10 ml standard volumetric flask)

Piroxicam (CAS: 36322-90-4; M.W.: 331.35, Sigma Chemical Company, St. Louis, USA) solution was prepared in methanol. The solution was stable up to 3 days.

Proposed Method (PM) for Determination of Fe(III) Into a series of standard volumetric flasks (10 ml),

aliquots (0.05-0.45 mi) of 5.0 x 10" M standard ferric sulphate solution were pipetted. Then to each volumetric flask, 6.038 10' M piroxicam (2.6 ml) was added and diluted up to the mark with ethanol. Contents of each flask were mixed well at RT (25 + 1°C) and absorbance was measured at 495 nm against reagent blank prepared similarly except Fe (III) within stability period (24 h). Concentration of Fe (III) was calculated either from a calibration curve or regression equation.

Determination ofFe(III) in Synthetic Mixture

Synthetic mixture of iron (III) was prepared by tak­ing 200 mg of ferric sulphate with 13.9 mg FeSO^.TH^O, 192.3 mg 3CdSO,.8H,0, 90.0 mg Pb(N03)„ 23.01 mg ZnSO^.VHp, 222.18 "mg MgS0^.7H,0 and 84.5 mg MnSO^.H,0 in 100 mi standard volumetric flask and diluted up to the mark with distilled water. Fe (111) was detemiined by PM.

Determination of Fe(III) in Soil Sample •Air-dried finely powdered soil sample (2 g) of AMU

••0.7

•0.6

-0.9 -4.8 -30 I -4.4 -4.2 -4.0 -3.6 -3.6 -3.4 -3

Log [Ferric sulphate] or [Piroxicam]

Fig. 2—Limiting logarithmic plot for stoichiometric ratio between Fe (III) and piroxicam: a) log A vs log [Fe'*]; and b) log A vs log [piroxicam]

locality was shaken with 0.5 M HCl (15 ml) for 24 h to extract iron^^ After filtration, volume of filtrate was made up to 25 ml with distilled water. Of this solution. 15 ml was percolated through column packed with Amberlitc IR 400 (Clform) because Fe (111) was found to be com­pletely adsorbed on resin in 4 M HCl. Column was washed with 2.5 M HCl to remove unadsorbed species and non targeted metal ions. Fe (111) was eluted with 0.05 M HCl (flow rate, 2 ml /min). Effluent was evaporated and residue was dissolved in 5 ml of distilled water. pH of solution was adjusted to 2.6 by addition ol ammonia solution and final volume of solution was maintained to 15 ml. Fe (III) was estimated by both PM and reference method (RM)".

Procedure for Reference Method (RM)" Appropriate volumes of standard solution of Fe (III)

coiTesponding to 0.2-4.8 mg ml ' were transferred to a series of 25 ml volumetric flasks. To each flask, 1.0 ml of I NHCI, 1.0mlof0.1%4-aminoamipyreneand2.0mlof 0.1 % iminodibenzyl were added. The contents were mixed thoroughly and diluted to volume with ethanol. Absor­bance of resultant blue coloured complex was measured at 620 nm against corresponding reagent blank.

Validation PM was validated for specificity and selectivity,

accuracy and precision, robustness, linearity and evalua-

LUTFULLAH el ah SPECTROPHOTOMETRIC DETERN4INATI0N OF FEdll) VIA COMPLEXATION WITH PIROXICAM 1 37

0.6 o.r o.e 0.9 i.o Mole Iraclion of Fe (111)

Fig. 3—Job's plot of contiiuious variations for stoichiometric ratio between Fe(in) and piroxicani (1:2)

tion otbias. Specificity arid selectivity of PM was evalu­ated by determining concentration of Fe (HI) (22.34 mg ml ' ) in presence of various metal ions added in the form of FeSO^-VH^O, N i C I , 6 H p , AKNOjlj.gH^O, SCdSO^.SHjO, PbCNO,),, ZHSO^.TH^O, MgSO,.7H,0, CaCl,, MnSO^.H,0 and CuCl^. Accuracy and precision of PM was evaluated by replicate analysis (n = 5) of calibration standards at three concentration levels (5.59, 13.96 and 25.13 mg ml')- Five sample solutions of each concentration were analyzed within one day (intra day precision) and in five consecutive days (inter day precision).

Robustness of PM relative to each operational parameter was judged by analyzing content of Fe (III) in synthetic sample by observing influence of small variations of concentration of piroxicani. A synthetic mixture sample containing 25.13 mg ml ' of Fe (111) was assayed five times using PM. Mean recovery (%) and relative standard deviation were calculated by standard methods. Linearity was evaluated at nine concentration levels (2.79,5.59,8.38,11.17,13.96,16.76,19.55,22.34, and 25.13 mg ml ' ) . Each concentration level was analyzed repeatedly for five times. Absorbance obtained at each concentration was plotted against initial concentration of Fe (III) and linear regression equation was evaluated by statistical treatment of calibration data. Other regression characteristics were calculated using Origin Software. Limit of detection (LCD) and limit of quantitation (LOQ) were calculated as

where S is standard deviation of calibration line and b is slope.

Bias was evaluated by point and interval hypoth­esis tests^'. Where test method is compared with RM and considered to be acceptable if mean recovery is within + 2.0% of that of RM. Lower (0) and upper (8) acceptance limits were calculated as

where and are mean values based on n, and n, respectively. S is pooled standard deviation and t ^ is tabulated one-sided t-value, with n -)- n, freedom at 95% confidence level.

-2 degrees of

LOD = 3.3x ; and LOQ = 10 x —^ h

Results and Discussion Piroxicani was found to react with Tc (111) at

25±!°C re.sulting in formation of coloured complex. .Ab­sorption spectrum of Fe (lll)-piroxicam complex showed two absorption bands peaking at 430 nm and 495 nm (Fig. 1). Absorbance measurement at 495 nm as a func­tion of initial concentration of Fe (III) was utilized to develop a rapid, simple and selective spectrophotomet-ric method for determination of Fe (111),

Stoichiometry Stoichiometry of reaction between Fe (III) and

piroxicam was evaluated by limiting logarithmic method "", wherein two sets of experiments were performed. In first set, concentration of Fe (III) was varied keeping a constant concentration of piroxicam. while in second set. concentration of Fe (III) was kept coiistaiil and piroxicam concentration was varied. Log absoibancc versus log iferric sulphate] or [piroxicam] (Fig. 2) wa,-. plotted to evaluate slope of respective line. Slope was found to be unity in first case and 0.5 in second case; thus confirm­ing molar combining ratio of 1:2 between Fe (III) and piroxicam. Hence, one mole of Fe (III) was consumed by two moles of piroxicam. Job's method of continuous variation also indicated that one mole of Fe (III) reacted with 2 moles of piroxicam (Fig. 3). Formation constant of complex was calculated and found to be 6.12 x 10'", Chelating reaction of piroxicam with metal ions can be expected through three coordination sites (-OH, -CONH

and N functional groups). Therefore, reaction sequence of PM is given as in Fig. 4.

Optimization of Variables Optimization of variables was assessed bv lesting

reaction time, concenti'ation of piroxicam and solvents.

138 J SCI IND RES VOL 69 FEBRUARY 2010

Step 1

FejCSO^), 2Fe-'^ + 3SO4

Ethanol-HsO medium

r^TO y

N O O

O ^ . ^ ' •^3^^ / S

0

o ^ S ^ O C 3

Pink coloured metal chelate

Fig. 4—Reaction sequence of proposed method

Complex got stabilized immediately at 25 + TC after mixing analyte and reagent. Complex remained stable for 24 h. For optimizing concentration of piroxicam, ab-

ml' Fe (HI) with piioxicam (cone. 3.018 x 10""-1.691 x lO' M). Highest absorbance was obtained with 1.45 xlO"-' M piroxicam and remained constant up to 1.70

sorbanceof metal complex was investigated at 22.34 |ig x lO ' M piroxicam (Fig. 5). Therefore. 1.57 x lO ' M

LUTFULLAH ei ai. SPECTROPHOTOMETRIC DETERMINATION OF FEdll) VIA COMPLEXATION WITH P1ROXICAM139

<

1.2 •

1,1 •

1.0 •

0.9-

0.8

0.7 -

0.6 -

0.5

0.4

0.3

0.2

0.1

/ ^

/ ^

/ /

^

0.0 -I 1—-T 1 1 1 1 1 1 y r 1 1 1 1 r"

2 3 4 5 6 7 8 9 10 11 12 13 14 IS 16 17 18

[Piroxicanil-xlO^, M

Fig. 5—Effect ofconcentraiioiiofpiro.xicam on absoibance of metal complex at 22.34 mg m l ' Fe(ni)

piroxicam was used for the determination process. Ef­fect of solvents (ethanol, acetone, methanol, 1,4-dioxan and dimethylsulphoxide) was investigated on absorbance of colored complex, which showed maximum absorbance in ethanol (Fig. 6), indicating ethanol as best solvent

Validation Varying concentrations of each ion with fixed con­

centration of Fe (III) were taken and absorbance was recorded to know concentration of Fe (III). Tolerated amounts of each ion, which is concentration value tested that caused less than ± 2% absorbance alteration, has been found as follows: Fe-, 0.50; Cd^^ 84.31; Pb"-. 62.16; Zn 2 32.31; Mg-, 21.80 and Mii*\ 27.47 '/4g ml'. How­ever, metal ions (Ni-*, Al'*, Ca-*, and Cu-*) interfere in determination of Fe'*.

Accuracy and precision were evaluated within same day and on five consecutive days. Recovery and RSD (%) were found between 100.04-100.08%; 0.22-0.74% for intra day and 99.93-100.11 %; 0.28-0.81% for inter day precision, respectively (Table 1). Robustness of PM was established by deliberately changing concentration of piroxicam as volume of 6.038 x 10' M piroxicam, 2.6 ml (+ 0.2 ml). Under optimized conditions, Fe (III) solution [25.13 mg ml' Fe (III)] was analyzed and mean recovery (100.04%) and RSD (0.22%) indicated robustness of PM.

Solveni

Fig. 6—Effect of solvent on absorbance of coloured complex. (Fe(III)J = 22.34 mg m l '

<

|Fe(lllVl.Lig nil '

Fig. 7—Calibration curve for determination of Fe(lll)

CaUbration curve was constructed by plotting ab­sorbance against initial concentration of Fe (III) (Fig. 7). Beer's law was obeyed (cone. 2.79-25.13 ig ml ') with apparent molar absorptivity (5.88 x 10' 1 rnol 'cm ')and Sandell's sensitivity (0.019 mg/cm^/0.001 absorbance unit). Calibration data were fitted to the equation, A = a -I- bC, where A is absorbance at 495 nm, C is concentra­tion in mg ml"' b is slope and a is intercept of calibration. Calibration data (n = 9) were treated statistically. High value of correlation coefficient (0.9999j indicated excel­lent linearity (Table 2). Values oft (calculaicd as. i = a /

140 JSCIINDRES VOL 69 FEBRUARY 2010

Table I—Test of accuracy and precision of proposed method

Parameters

Concentration taken, | i g m l '

Concentration found", ng m l '

Standard deviation', !,ig ml '

Recovery, ' r

RSD, "if

Intra

5.59

5.59

0.041

100.07

0.74

'Mean for 5 independent determinations

day assay

13.96

13.97

0.043

IU0.08

0.30

25.13

25.14

0.055

100.04

0.22

Inter

5.59

5.586

0.045

99.93

O.Kl

day assay

13.96

13.974

0.049

100,1 1

0.35

25.13

25,13

0,070

100,00

0,28

Table 2—Optical and regression characteristics of proposed method

Parameters

Wavelength, nm

Beer's law limit, |j,g ml'

Molar absorptivity, 1 mol'cm"'

Sandell's sensitivity

Linear regression equation

±tS a

Correlation coefficient (r)

Variance (S ,-) of calibration line

Detection limit, |ig ml'

Quantitation limit, (tg ml '

Proposed method

495

2.79-25.13

5.88 X 10'

0.019 (ig/cmV 0.001 absorbance unit

A = 5.55x 10-^+1.52 C

2.814 X 10-'

1.791 X 10-

0.9999

2.690 X 10"

0.10

0.31

±t Sim: and ±t S^ are confidence limits for intercept and slope, respectively.

Table 3—Evaluation of bias; Comparison and applicability of proposed method with reference method for delerniinatioii of Ff(IlI) from synthetic mixture sample at 95% confidence level

Samples Proposed method Reference method t-value'' F value''

Synthetic 100.01

mixture

Recovery' RSD Recovery" RSD

% % % % 0.91 99.87 0.83 0.253 1.200

e,'

0.999 1.003

"Mean for 5 independent analyses; ''Theoretical r (v= 8) and F-values (v= 4, 4) at 95 % confidence level are 2.306 and 6.39, respectively; 'A bias, based on recovery experiments, of ± 2% is acceptable

S -'') found to be 1.27, which did not exceed theoretical t-value (2.365) at 95% confidence level, indicate that in­tercept for PM i.s not significantly different from zero.

Applicability of PM for determination of Fe (III) in synthetic mixture and soil samples has been tested. Re­sults of PM were statistically compared with those of RM" using point and interval hypothesis tests. Paired t-and F-values at 95% confidence level were found to be less than tabulated t- (2.036 at t) = 8) and F-values (6.39

at\) = 4,4)at95% confidence leveF'', thus coiifiirning no significant difference between performance of PM and RM (Table 3). Thus, bias evaluated by interval hypoth­esis test by means of 0 ^ and Q^ were in tlie langc of 0.98-1.02. Peiformance of PM was also judged by analy­sis of soils taken from different locations. Concentration of Fe''* in soil samples was found as follows: Soil 1 — PM, 8,379 & RM, 8.382 mg ml '; and Soil 2—PM, 8.382 & RM, 8.375iTig ml V Thus, PM is suitable for routine

LUTFULLAH et at SPECTROPHOTOMETRIC DETERMINATION OF FE(III) VIA COMPLEXATION WITH PIROXICAM 141

analysis of Fe(lii) in real samples of soil. Speed of analysis of PM is as good as tiiat of RM.

Conclusions Proposed spectrophotometric method is found

simple, selective and accurate for determination of Fe (III) The method has advantage of using a commonly available solvent (ethano! with the use of one reagent, piroxicam). Proposed method, which has avoided use of acid, buffer solution and heating of reaction mixture, can be used as an alternate method for routine quality control analysis of Fe(III) in soil samples.

A cknowledgements Authors are grateful to AMU, Aligarh, India and

Ministry of ManPower (Higher College of Technology) Muscat, Sultanate of Oman for facilities.

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21 Reddy V K, Reddy S M, Reddy R P & Reddy T S, First order derivative spectrophotometric determination of copper(n) and iron(ni) individually and simultaneously, Chem Anal (Warsaw). 46(2001)687-695.

22 Stalikas C D, Pilidis G A & Tzouwara-Kaiayanni S M, Use of a sequential extraction scheme with data normalisation to assess the metal distribution in agricultural soils inigaled by lake water, Sci Total Enviton. 236 (1999) 7-18.

23 Hartmann C, Smeyers-Verbeke J, Pinninckx W, Heyden Y V, Vankeerberghen P & Massarl D L, Anal Chem. 67 (1995) 4491 -4499.

24 Rose ], Advanced Physico-chemical Experinwiiis (Pitman, Lon­don, UK) 1964, 67.

25 Nalimov V V, The Application of Mathematical Statistics to Chemical Analysis, (PergmonPress, Oxford) 1963, 167-189.

26 Mendham J, Denney R C, Barnes J D & Thomas M, Statistics: Introduction to Chemometrics, in Vogel's Textbook of Quantita­tive Chemical Analysis, 6"' edn (Pearson Education, Singapore) 2002, 137.

622 Journal of the Chinese Chemical Society, 2010, 57, 622-631

Communication

UV Spectrophotometric Determination of Cu(ll) in Synthetic Mixture and

Water Samples

Lutfullah;'* Saurabh Sharma," Nafisur Rahman," Syed Najmul Hejaz Azmi,*" Basliir Iqbal,'' Maisa Ismail Bilal Bait Amburk'' and Zuweina Masoud Hilal Ai Barwani^

"Department of Chemistry, Aligarh Muslim University, Aligarh-202002, Utlar Pradesh, India ^ Department of Applied Sciences, Chemistry Section, Higher College of Technology. P. O. Box 74,

Al-Khuwair-133, Muscat, Sultanate of Oman

A new and novel UV spectrophotometric method has been developed for the determination Cu(II) in synthetic mixture and water samples. The method is based on complex formation of Cu(II) with cefixime immediately in 1,4-dioxan-distilled water medium at room temperature. The complex showed maximum absorption wavelength at 336 nm. Beer's law is obeyed in the concentration range of 1.015-8.122 \ig mL' (Linear regression: A = 1.59 x lO"'' + 1.305 x 10'' C) with apparent molar absorptivity (8.29 x 10^ L mol'cm"') and Sandell's sensitivity (0.008 ng/cmVO.OOl absorbance unit). The limits of detection and quantitation for the developed method are 3.19 x 10" and 9.65 x 10" ng mL"', respectively. Interferences due to Mn(ll), Ca(ll), Mg(II), Cd(ll), Al(lll), Pb(n), Zn(ll), Fe(Il), Fe(IIl), Ni(ll), Cr(lll), Hg(Il) and As(IlI) was investigated. Nine cations do not interfere in the determination of Cu(U). Proposed method was successfully applied to the determination of Cu(ll) in synthetic mixture, sea and well water .samples.

Keywords: UV Spectrophotometry; Validation; Cu(ll); Cefixime; Cu(ll)-cerixime complex;

Sea and well water samples.

INTRODUCTION Copper has received considerable attention owing to

its technological and biological significance. It is an essen­tial constituent of about thirty enzymes and glycoproteins. It is required for the synthesis of hemoglobin and for some biological processes.'" The concentration of copper plays a very important role which decides its vitality and toxicity for many biological systems.^'' It has been reported that the toxic copper species are Cu(OH)*, Cu2(OH)2^* and CuCOj. Therefore, Copper ions present in various aqueous systems are considered to be the most toxic of dissolved copper spe­cies.'" The excess concentration of copper in water sys­tems is harmful to human beings, affects the self-purifica­tion of bulk water and disturbs the microbiological treat­ment of waste water,'' Thus, the determination of trace amounts of Cu(ll) is becoming increasingly important be­cause of the increased interest in environmental pollution.' In chemical analysis, metal chelation followed by solvent extraction and spectrophotometric determination is consid­ered as the most preferred mode of analysis for a number of metal ions in different matrices.* Several spectrophotomet­

ric methods have been reported in which the solvent extrac­tion step is replaced by the use of surfactant.'"'^ Micellar media are mainly used to simplify the system and replacing the extraction step. Many spectrophotometric methods have been developed for the determination of Cu(ll) based on reaction with reagents such as l-nitroso-2-naphthol,''' 1 -phenyl-1,2-propanedione-2-oxime thioseinicarbazone,' 2-hydroxy-l-naphthaldehyde benzoylhydrazone,'* N-eth-yl-2-naphthylamine," ethyl violet,'* mixture of 1,10-phen-anthroline and neocuproine,' 1,5-diphenyl carbazone,^" 2-ketobutyric acid thiosemicarbazone,"' pyruvic acid thio-semicarbazone,"^ 2-acetyl thiophene-4-phenyl-3-thiosemi-carbazone, ' 4-vanillidene amino-3-methyl-5-mercapto-1,2,4-triazole," dimethyl glyoxime," variamine blue."' Most of the reported spectrophotometric methods are lime consuming, employing many reagents to develop the col­our and extraction of copper complex into organic solvent. Spectrophotometry is the good tool for determining metal ion concentration in leafy vegetables, pharmaceuticals, natural water and soil samples due to its low cost, simplic­ity and adaptability. Analytical methods based on spectro-

* Corresponding author. E-mail: lutfuiiah786@gniail.com

Quantitative Analysis olXu([l) / Chin. Chem. Soc. Vol. 57, No. 4A. 2010 623

photometry"""' were published in reputed journals. There­fore, it is decided to exploit this technique to develop an op­timized and validated UV spectrophotometric method for the determination of Cu(II) in synthetic mixture and water samples. The present UV spectrophotometric method is based on the complex formation of Cu(II) with cefixime in 1,4-dioxan-distined water medium at room temperature (25 + 1 °C). The formed complex showed maximum ab-sorbance at 336 nm. The reaction conditions are optimized and validated as per International Conference on Harmoni-sation (USA).'"

EXPERIMENTAL Apparatus

All spectral and absorbance measurements were made on a Shimadzu UV-visible 160 A spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with 1 cm matched quartz cells.

IR spectra were recorded on a Perkin-Elmer FTIR 1650 spectrophotometer in wave number region 4000-400 crn' using KBr pellet technique.

An Elico LI 120 pH meter (Hyderabad, India) was used for pH measurement. Reagents and standards

All reagents used were of analytical reagent grade. • 3.195 X 10" M copper sulphate (M.W.: 159.61,

Suprachem products Ltd, England) solution (203.06 ppm Cu) was prepared by dissol ving 0.051 gin 100 mL distilled water.

• 2.205 X 10' M (0.1 %) cefixime (CAS; 79350-37-1, M.W.: 453.45, Sigma Chemical Company. St. Louis, USA) solution was freshly prepared in methanol. The solution was stable up to 12 h.

• Buffer solutions ranging from 3.72-5.57 were pre­pared by mixing varying volumes of 0.2 M acetic acid (9-1 mL) and 0.2 M sodium acetate (1-9 mL) in 10 mL standard volumetric flask."

Sea and well water samples were collected from Mus­cat, Oman. Procedure for the Determination of Cu(II)

Into a series of 10 mL standard volumetric flask, aliquots (0.05-0.4 mL) of 3.195 x lO"' M standard copper sulphate aqueous solution corresponding to 1.015-8.122 l-ig mL"' were pipetted. To each flask, 1.8 mL of cefixime (0.1%) solution was added and diluted up to the mark with 1,4-dioxan. The contents of the flask were mixed well and the absorbance was nieasured at 336 nm against reagent

blank prepared similarly except Cu(!l) within stability pe­riod (6 h). The amount of Cu(Il) was obtained either from the calibration graph or the regression equation. Determination of Cu(II) in Syntlietic Mixture Sample

Synthetic mixture of Cu(II) sulphate sample solution was prepared by taking 51 mg of copper sulphate with 200 mg" MgS04, 7.28 mg HgCl,, 200 mg CrjOj, 290 mg Al2(S04)3.16H20, 51 mg ZnS04.7H20, 51 mg MnSOj. HjO, 51 mg CaCb, 51 mg Pb(N03)2 and 72.8 mg Fe:(S04)j in 100 mL standard volumetric flask and diluted up to the mark with distilled water. The amount of Cu(ll) was deter­mined by the proposed procedure. Determination of Cu(ll) in Water Samples

100 mL of sea and well water samples were collected and transferred into cleaned polyethylene bottles. The sea and the well water samples were filtered through a Milli-pore 0.45 \im pore size membrane and analyzed within 6 h of collection. Aliquot (0.5 mL) of sea and well water sam­ples was pipetted into a 10 mL standard volumetric flask with 1.8 mL of cefixime (0.1%) solution. The contents of each flask were mixed well at room temperature and di­luted up to the mark with .1,4-dioxan. The absorbance of each solution was recorded at 336 nm against the reagent blank prepared similarly except Cu(II). The amount of Cu(II) in sea and well water samples was obtained either from the calibration graph or the regression equation. The amount of Cu(II) was also estimated by the reference method." Procedure for Reference Method''"

Aliquots (0.025-0.25 mL) of standard copper sul­phate (1.598 X 10"' M) solution corresponding to 0.5-5 ug mL" were pipetted into a series of 50 mL separating fun­nels. To each funnel, 3.0 mL of universal buffer mixture (100 mLofa solution of mixed acids, being 0.04 M HjP04, 0.04 M acetic acid and 0.04 M boric acid; neutralized 56.76 mL of 0.2 M NaOH and made up to 200 mL) of pH 7.5,3.0 mL of 1.98 X 10"'M l,5-diphenyl-l,4-pentadien-3-oxime and 1.0 mL of magnesium sulphate were added and mixed well. The contents of the separating funnel were shaken vigorously with 5 mL of chloroform for 30 s and then al­lowed to separate the two layers. The absorbance of the or­ganic layer was recorded at 503 nm. The amount of Cu(II) was obtained either from the calibration graph or the re­gression equation. Determination of stoichiometry

The stoichiometry of the reaction was studied by Job's method of continuous \ariations." for this purpose.

624 J. Chin. Chem. Soc, Vol. 57. No. 4A, 2010 Liitfuliah ct al.

different volumes (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.35, 1.5,

1.8, 2.0 niL) of 2.205 x lO"'M copper sulphate was added

with different volumes (2.0. 1.8, 1.6, 1.4, 1.0,0.8,0.65,0.5,

0.2, 0 ml.) of 2.205 x 10"' M cefixime and diluted with

1,4-dio.xan in 10 niL standard volumetric flask. The

absorbance was recorded at 336 nm and plotted against the

mole fraction of copper sulphate.

Validation

The present method has been validated for specific­

ity, precision, accuracy, linearity, robustness and evalua­

tion of bias.

The specificity of the proposed method was evaluated

by determining 7.107 |ig mL'' Cu(ll) in the presence of

metal ions such as Mn(II), Ca(II), Mg(n), Cd(II), Al(III),

Pb(II), Zn(II), Fe(II), Fe(III), Ni(II), Cr(III), Hg(II) and

As(IIl) added in the form of MnS04.H20, CaCh, MgS04,

SCdSOi.SHiO, Al2(S04)3.16H2Q, Pb(N03)2, ZnS04.7H20,

FeS04.7H20, Fe2(S04)3, Ni(N03)2.6H20, CT2O3, HgCb,

and AS2O3.

The precision of the proposed method was evaluated

by intra-day and inter-day precisions. Standard Cu(ll) solu­

tion at three concentration levels (2.031, 4.061 and 8.122

Hg mL'') was assessed with five replicates (n = 5) for each

of three working sample concentrations in a single day

(intra-day precision) and over five days (inter-day preci­

sion).

The accuracy of the proposed method was deter­

mined by standard addition method. For this purpose, 0.15

mL synthetic mixture sample solution corresponding to

3.046 ng mL' Cu(Il) was spiked with 0,0.05,0.1,0.15 and

0.2 mL standard Cu(ll) solution corresponding to 0, 1.015,

2.031, 3.046 and 4.061 ng mL' Cu(ll).

The robustness of proposed method was assessed by

analyzing 5 (ig mL'' Cu(II) in synthetic mixture sample by

varying the volume of cefixime (1.8 ± 0.2 mL) at room tem­

perature (25 ± 1 °C).

Linearity of the proposed method was assessed at

seven concentration levels (1.015, 2.031, 3.046, 4.061,

6.092, 7.107 and 8.122 |.ig inL"'). Each concentration level

was independently analyzed repeatedly for five times. The

instrumental response i.e. absorbance obtained at each con­

centration was plotted against initial concentration of

Cu(Il). The linear regression equation was evaluated by

least square treatment of the calibration data. The other sta­

tistical parameters for the proposed method were calcu­

lated using Origin Software. Limits of detection and quan­

titation were calculated.'*''

Point and interval hypothesis tests have been utilized

to evaluate the bias of the proposed method and the refer­

ence method.'' The proposed method is compared with tiie

reference method and considered to be acceptable it nieaii

recovery of the proposed method i.s \\ ithin + 2.0"IJ ofihat ol

the reference method. The lower (OL) and the upper (0; ) ae-

ceptance limits can be calculated by the following quad­

ratic equation:

_ p tah

*2 '^p'ub +ixl-Slt,^:/n,) = 0

where JC, and x.^ are mean values at ni and nj measurements,

respectively. Sp is the pooled standard deviation and t,„(, is

the tabulated one-sided t-value at 95% confidence level.

RESULTS AND DISCUSSION

The aqueous solution of copper sulphate was maxi­

mally absorbed at 206 nm. The methanolic solution of

cefixime was peaking at 210 and 290 nm. When the two so­

lutions were mixed together, a red shift in the wavelength is

observed due to the compiexation reaction orCu(ll) and

cefixime. Thus, a complex was obtained immediately in

1,4-dioxan-distilled water medium with /.„,.,, = 336 nm.

The UV spectra for copper sulphate, cefixime and Cu(II)-

cefixinie complex are shown in Fig. 1. It is clear from the

graph that the maximum absorbance wavelength of the

Cu(II)-cefixime complex is different to that of cefixime.

Thus, the absorbance measurement at 336 nm as a function

of initial concentration of Cu(II) is exploited to develop a

new and novel UV spectrophotometric method for the de­

termination of Cu(ll) in synthetic mixture and water sam­

ples. The reaction was carried out at room temperature and

the complex was stable up to 6 h.

Stoichiometry

The stoichiometric ratio between Cu(ll) and cefixime

was evaluated by Job's method of continuous variations.

Varied volumes of equimolar (2.205 x 10"' M) solutions of

Cu(ll) and cefixime were taken, keeping the total volume

of Cu(ll) and cefixime constant at 2 mL in 10 mL standard

volumetric flask and diluted up to the mark with 1,4-diox-

an. The absorbance of each set is recorded and plotted

against the mole fraction of Cu(Il) (Fig. 2). This is due to

the compiexation reaction of Cu(II) with cefixime at 336

nm. The plot of absorbance versus mole fraction of Cu(ll)

has confirmed that I mol of Cu(ll) reacted with 1 mol of

Quanlilalive Analysis orCu(Il) .; Chill. Chcin. Soc-. Vol 57, \y;. 4A. 2lill) 625

Fig. 1

210 225 240 255 270 286 300 316 330 345 380 375 390 405

Wavelength, nm

Absorption spectra of (a) 2 mL of 3.195 x lO-* M copper sulphate in distilled water (b) 0.05 mL of 0.1% cefixirae in methanol and (c) 0.4 mL of 3.195 x 10"' M copper sulphate in dis­tilled water + 1.8 mL of 0.1% cefixime in meth­anol. Solutions a, b and c are diluted up to mark with distilled water, methanol and 1,4-dJoxan in 10 mL standard volumetric flask.

0,2 0-3 0,4 0 5 0,6 0.7 0.8 0,9 1,0

Mole traction of copper sulphate

Fig. 2. Job'splotof continuous variations for stoichio­metric ratio between Cu(ll) and cefixime (2.205 X 10^ M each).

cefixime. The resulting Cu(ll)-cefixime complex remained stable for about 6 h. Thus, the stoichiometry of the complex is established and found to be 1:1.

The I.R. spectra of free cefixime and Cu(ll)-cef]xime complex are shown in Fig. 3 a and b, respectively. Cefixime has -NH2, -COOH, -CONH and C=0 lactam groups which are the potential sites for coordination with metal ions. Comparison of IR spectrum of the complex with those of free cefixime indicates that the lactam (C=0) band appears at 1766 cm"' in the free cefixime while the complex shows this band again at 1766 cm ' suggesting that no coordina­tion occurs with copper ion. The amide carbonyl band. (C=0)-NH in the free cefixime appears at 1674 cm ' with a weak shoulder at 1635 cm"' while the Cu(ll)-cefi\ime com­plex shows this band at 1676 cm' with a promiiieni peak at 1631 cm"' indicating the coordination of cefixime with Cu(ll) through nitrogen. The asymmetrical and symmetri­cal stretching bands of carboxylate groups change from 1533 cm"' to 1543 cm ' and 1373 to 1379 cm', respectively due to the coordination. The Cu-N stretching vibration oc­curs at 428 cm"'.' ' A tentative mechanism for the com-plexation between Cu(II) and cefixime is given in Scheme I.

Scheme I

Slal

Cu(SOJ

SICD2

Cir* SO -"

^ ' ) NOCHiCOM HjN.,,S „ '^ .

i-=CH^ " ^ T I . H -

Cffr.Ktmc

Step 3

C=CH,

COOH

CuiI!)-ccrtsiiy:C complex

626 ./. Chin. Cliem. Soc, Vol. 57. No. 4A. 2010 Liilfullah el al.

j a n ^ : , EH

m^ 1/ n

4S § * *

7 5 0 SOO

iiiiii B *lliis"N

/ 8

II^IIH^

m A. t

^ i

Fig. 3. IR spectra of (a) free cefixime and (b) Cu(II)-cefixime complex.

The apparent formation constant (Kf) for the complex formation between Cu(n) and cefixime is calculated using the following expression":

K. (^„..,/^„,.)C

[c,-^ (A ^ C][C, -

\ "V J 4

'-C\

where Aobs and Acxip are observed and extrapolated absorb-ance values for the complex, respectively. CM and C^ are the initial concentration of Cu(ll) and cefixime in mol L"', respectively. C is the limiting concentration. Thus, K.,- for the complex is found to be 1.723 x 10*'. The apparent Gibbs free energy (AG°) is calculated using AG° = -2.303 RT log Kt and found to be -35.59 kj mol"' confirming the feasibil­ity of the reaction.

Optimization of Variables The optimization of variables was investigated by

testing reaction time, concentration of cefixime, solvents and sodium acetate-acetic acid buffer solutions of different pH.

The effect of reaction time on the absorbance of Cu(II)-cefixime complex and its stability was investigated. The Cu(II)-cefixime complex got stabilized immediately at 25 + 1 °C after mixing cefixime and 1,4-dioxan as solvent. Complex remained stable for 6 h.

The volume of 0.1% cefixime was optimized for method development. The absorbance of the metal com­plex was investigated at 8.122 |.ig mL"' CudD with volume of cefixime in the range 0,4-2.0 mL. It is clear from Fig. 4 that the maximum absorbance was obtained with 1.6 mL cefixime. Above this volume up to 2.0 mL of 0.1 % cefixime, the absorbance remained unchanged. Therefore, 1.8 mL of

Quanlilalivc Analysis ofCudl) .. Chill. Chciii Soc, i'tii. -V, \'t, ?.l, ..'nil! 627

0.1% cefixime was used in further measurement orCu(ll) ill synthetic mixture and water samples.

The effect of solvents such as methanol, acetone, di-iiiethylsulphoxide (DMSO), acetonitrile, ethanol, 1,4-di-oxan and distilled water was investigated at 8.122 ng mL"' Cu(ll) on the absorbance of Cu(II)-cefixime complex. The reaction mixture was turbid in ethanol, acetone and dis­tilled water. The absorbance for Cu(II)-cefixime complex in other solvents is shown in Fig. 5. It is clear from the fig­ure that the highest absorbance was obtained in 1,4-dioxan. Therefore, 1,4-dioxan was the best solvent for dilution of the reaction mixture of Cu(ll)-cefixime complex in deter­mination process of Cu(II) in synthetic mixture and water samples.

The pH of the complex was measured and found to be 4.68. The effect of pH on the absorbance of the complex was investigated using sodium acetate-HCl (range 3.72-5.57) buffer solutions. A constant absorbance was obtained in the pH range of 3.72-5.57, but the absorbance vaJue in this pH range was found to be less than that obtained with­out buffer solution when dilution is made with 1,4-dioxan. Therefore, all absorbance measurements of the complex were made in 1,4-dioxan solvent without the involvement of buffer solution.

Validation Varying concentrations of metal ions such as Mn(II),

Ca(ll), Mg(ll), Cd(ll), Al(lll), Pb(ir). Zn(ll). 1 edl), l-e(m), Ni(ll), Cr(III), Hg(ll) and As(Ill) with 7.107 (.ig m l ' Cii(ll) were taken and the absorbance was recorded to know the concentration of Cu(ll). The tolerated amount of each metal ion in ng m f ' was determined (Table 11, when the absorbance value did not exceed + 2% on addition of cat­ions. However, metal ions such as Ni(ll), Cd(ll), Fe(ll)and As(IlI) interfere in determination of Cu(II).

The intra-day and inter day precisions were evaluated by determining the concentration of Cu(II) at lower, middle and upper concentration levels for five repeated times within the same day and on five consecutive days, respec­tively (Table 2). It can be seen from the tabic that percent­age recovery and RSD (intra day and inter da\ precisions) were in the ranges of 99.93-100.08% and 0.14-0.67%. re­spectively. It is evident from the table that percentage re­covery and RSD values were precise and can be used to de­termine Cu(ll) in synthetic mixture and water samples.

Standard addition method is one of the methods to show the accuracy of the proposed method just by the addi­tion of the number of standardized aliquots to a veal sample in order to raise its concentration by a known amount. Thus, the accuracy of the proposed method was investi­gated by performing recovery experiments through stan­dard addition method. The absorbance for each solution is recorded at 336 nm and plotted as shown in Fig. 6. The re-

0.2 OA 0 6 0.8 1.0 12 14 1,6 1,8 2.0 2.2

Volume of 0.1% cefixime. ml

Fig. 4. Effect of the volume of 0.1% cefixime.

DMSO Acetonitrile Mettianol 1,4-dioxan

Solvent

Fig. 5. Effect of solvent on the absorbance of Cu((l) cefixime complex, [Cu(H)] = 8,122 ng niL'

628 ./. Chin. Cliem Soc, Vol. 57. No. 4A. 2010 LuirLillah et al.

Table I. Effect of metal ions on the determination of 7.107 (.ig niL"' Cu(II)

Metal ions

Mg"* Hg"* Cr'-Al" Zn-' Mn-" Ca'* Pb-' Fe'^

Added as

MgSO^ HgCI, Cr,0,

A1,(S04),.16H:0 ZnSOj.71-1:0 MnSOjUjO

CaCI,

Pb(NO,)2 Fe2(S04)3

Tolerance limit (ng mL ')

15.44 1.88

26.25 4.36 3.48 4.y7 5.50

12.76 3.56

suits of analyses are summarized in Table 3. It is clear from the table and the graph that the linearity of the regression line for synthetic mixture sample was good. As can be seen from the figure that the concentration of Cu(II) in synthetic mixture sample is given by intercept/slope. The ratio of the intercept and the slope of the regression line is subjected to error (SXE), thus SXE is calculated from the following ex­pression

S... =-b''^(x,~Jf

and found to be 0.02 ^g mL"'. The confidence limit for the concentration of Cu(n) in synthetic inixture sample is cal-

Concentration, ng mL

Fig. 6. Determination of Cu(ll) in synthetic mi.xture sample by standard addition method

culated by.tt ± ;S^t at n - 2 degrees of freedom and found to be 3.049 ± 0.062. The most attractive feature of the pro­posed method using standard addition method is its rckuivc freedom from various non targeted cations.

Table 2. Precision of the proposed method

Parameters

Concentration taken, (ig mL"' Concentration found, ng mL'' Standard deviation", pg mL"' Recovery, % Relative standard deviation, %

Intra day assay

2.031 2.023 0.012 99.93 0.57

4.061 4.062 0.011 100.02 0.28

8.122 8.125 0.011 100.04 0.14

Inter day assay

2.031 2.033 0.014 100.08 0.67

4.061 8.122 4.059 8.120 0.012 0.014 99.95 99.98 0.31 0.17

' Mean for five independent analysis. "" Confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).

Table 3

Sample

3.046

Test of accuracy in synthetic mixture sample by standard addition method

Concentration (^g mL"') Linear regression parameters

Standard Added Nominal Error (S,i;) Intercept Slope r"

0,1.015,2.031, 3.049 0.020 0.3982 0.1306 Oy-JW 3.046.4.061

Recovery'' ("/,.)

KM). It)

' Coefficient of correlation. '' Mean for five independent analyses.

Quantitative Analysis orCii(I[) ./. Chill. Cheiii. Soc. Vol. No. 4.1 2010 629

The robustness of the proposed method was estab­lished by deliberately changing the volume of 2.205 x lO'"' M cefixime, 1.8 mL (± 0.2 mL) for the determination of Cu(ll). The synthetic mixture sample solution containing 5.0 ng mL'' Cu(Il) was analyzed five times repeatedly by the proposed method. Percentage recovery and RSD were found to be 99.84% and 0.15%, respectively, indicating ro­bustness of the proposed method.

Under the optimized experimental conditions, the calibration graph by considering 7 independent concentra­tion levels of Cu(Il) was constructed by plotting the ab-sorbance against initial concentration of Cu(II) (Fig. 7). Beer's law is obeyed in the concentration ranges of 1.015-8.122 Jig mL'' with apparent molar absorptivity of 8.29 x 10'' L mof'cm"' and Sandell's sensitivity (0.008 (jg/cmV 0.001 absorbance unit). Linear regression equation is ob­tained by statistical treatment of the calibration data (n = 7) which is fitted with the straight line equation in the form of A = a + bC, where A is absorbance at 336 nm, C is concen­tration in (ig mL"', b is slope and a is intercept of calibra­tion. High value of correlation coefficient (0.9999) indi­cated excellent linearity (Table 4). The experimental inter­cept of the calibration line was tested for significance of de­viation from the theoretical intercept, i.e. zero. For this jus­tification, t-value calculated from relation, t = a /S /* found

Table 4. Optical and regression characteristus nl ihc proposed method

Parameters

Beer's law limit (^g mL ') Molar absorptivity (L mor'cm"') Sandell's sensitivity

Linear regression equation"

Sa ±tSa Sb ±tSb Correlation coefficient (r) Variance (So^) LOD (^g mL"') LOQ(ngmL')

Analytical data

336 nm 1.015-8.122 8.293 X 10' 0.008 ng/cnr/O.OOl absorbance unit A= 1.59 X 10 '+ 1.305 y. 1 0 ' C 9.850 X 10' 2.533 X 10" 1.919 X 10* 4.933 X 10^ 0.9999 1.588 X 10"'' 0.032 0.095

Fig-

1 2 3 4 5 6 7 8

[Cu(lt)], ng mL"'

7 Calibration curve for the determination of Cu(II) in synthetic mixture and water samples.

' With respect to .A = a + bC, where C is the concemraiion in [ig niL"' and A is absorbance.

± tS;i and + tSb are the confidence limits for intercepi and slope, respectively.

to be 1.61, did not exceed the tabulated t-value (2.571, v = 5) at 95% confidence level. This indicated that the intercept in the calibration equation of the proposed method is not significantly different from zero. Thus, the proposed method is free from procedural error.

The applicability of the proposed method for the de­termination of Cu(II) in synthetic mixture sample has been tested. Results of the proposed method were statistically compared with those of reference method^^ using point and interval hypothesis tests. The paired t- and the F-values at 95% confidence level were calculated and found to be less than the tabulated t- (2.036 at u = 8) and F- (6.39 at u = 4,4) values at 95% confidence level."''' thus confirming no sig­nificant difference between the performance of the pro­posed method and the reference performance of the pro­posed method and the reference method (Table 5). Thus. the bias calculated by interval hypothesis test in the form of lower limit (0L) and upper limit (Gy) were in the range of 0.98-1.02.

The performance of proposed method is also tested in sea and well water samples collected from Muscat, Oman. Results of analysis were found in good agreement with ref­erence method (Table 6). Thus, the proposed method is suitable for routine analysis of Cu(II) in real samples of wa­ter. The speed of analysis and less number of reagents uti-

630 ./ Chin. Cliem. Sac, Vol. 57. No. 4A, 2010 LLiirullah et al.

Table 5. Point and interval hypotliesis tests for the determination of Cu(II) in synthetic mixture sample for the evaluation of bias of the proposed method with that of the reference method at 95% confidence level

Proposed method Reference method

Recovery ^ ^ ^ . , „ , , Recovery „ „ „ , , „ , , Paired r-value f-value'^ 0^' d.

99,84 0.15 99.91 0.15 0.721 I.OI 0.998 1.003

' Mean for 5 independent analyses, '' Theoretical ( (= 8) and f-values (= 4, 4) at 9i% confidence level are 2.306 and ().39,

respectively. " A bias, based on recovery experiments, of ± 2% is acceptable.

Table 6. Determination of Cu(ll) in sea and well water samples by the proposed UV spectrophotometric method and the reference method

Sample Concentration of Cu(II) in ng mL''

Proposed method Reference method

Sea water Well water

2.164 1.022

2.163 1.021

lized in the proposed method are the main advantages of the

proposed method as compared to reference method.

CONCLUSIONS

The proposed method is a direct UV-spectrophoto-

metric method as compared to reference method (extiactive

spectrophotometric method). The proposed method is a

simple and accurate for the determination of Cu(II) in syn­

thetic iTiixture and water samples. The method has advan­

tage of using a cominonly available solvent i.e. 1,4-dioxan

with the use of one reagent, i.e. cefixime. The proposed

method has avoided the use of acid, buffer solution and

heating of reaction mixture, can be used as an alternate

method for routine quality control analysis of Cu(l 1) in soil,

vegetable and pharmaceutical samples.

ACKNOWLEDGEMENT

The authors are grateful to Aligarh Muslim Univer­

sity, Aligarh, India and Ministry of ManPower (Higher

College of Technology, Muscat) Sultanate of Oman for

facilities.

Received March 17, 2010.

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