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
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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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
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[24j J. Rose. Advanced Physico-chemical Experiments; Pitman, London. UK (1964)
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[25] V. V. Nalimov. The Application of Mathematical Statistics to Chemical
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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).
J •^.JilO'tiS
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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.
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207.
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[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.
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[21] L. E. Attah, Indian J. Chem. Tech. 16 (2009) 351.
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[25] U. Muralikrishna, A. Shivaramakrishna, Asian J. Chem. A 13 (2001) 289.
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(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.
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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
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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
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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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114] M. i:. Khalifa. M. A. H. Hafez, Talanta 47 (1998) 547.
[15] E. Kavlentis, Microchem. J. 38 (1988) 188.
[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.
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o CO
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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: [email protected]
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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