ELECTROCHEMISTRY OF NEW METAL COMPLEXES

DISSERTATION SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

^ Masttv of $l)iIo£{opI)p IN

CHEMISTRY

-..V;'^

BY

AHMAD ASIM

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2009

:$^t^^p6.

DS3933

i9e^cafm

To

Ti^fare^^

<^of, Sartaj TiiBassutn Department of Chemistry Aligarh Muslim University

Aligarh'202002, India.

Certificate

Phone No. 0571-2703893 E-mail: tsartaj62@yahoo.com

The work embodied in this dissertation entitled "Electrochemistry of New Metal

Complexes" is the result of original research carried out by

Mr. Ahmad Asim under my supervision and is suitable for the award of M.Phil. Degree.

C (Prof. Sartaj Tabassum)

Content

Page No.

Acknowledgement 3-4

Introduction 5-18

Present Work 19

Experimental 20

• Experimental Methods. 21-23

• Materials and Methods. 24

• Synthesis. 25-27

Results and discussion 28-46

Conclusion 47

References 48-52

Acknowledgement

Firstly, I bow in deep reverence to "Almighty Allah", the most beneficial and merciful

for bestowing and blessing me during the completion of this dissertation.

Reverently, I express my heartiest thanks and depth of gratitude to my esteemed

supervisor Prof. Sartaj Tabassum for his intelligent guidance, inexhaustible

encouragement and invaluable critical suggestions during the entire course of my

research work. In fact, his interest untiring and ever willing help, sympathy, meticulous

and talented guidance has been much greater than what I deserved. I shall treasure his

encouragement and motivation.

I express my indebtness to Dr. (Mrs.) Farukh Arjmand for her imstinted help to

enhance my dedication and useful suggestions, noble guidance, constructive criticism,

cooperative and generous help to sort out the complexities and to accomplish this task

successful.

I am extremely thankful to Prof Arunima Lai (Chairperson), Department of Chemistry,

A. M. U, Aligarh for providing all research facilities.

In this hectic world we live in, we very rarely take time to complement people for a job

well done. But I rarely want to say a special thanks to my senior lab colleagues for their

entiring help, positive outlook and constant encouragement. I extend my thanks to my

junior lab colleagues who assisted me in many ways.

I wish to express appreciation to my friends for their spontaneous help, constant

encouragement and moral support.

Last but not least, I want to convey my deepest gratitude and profound regards to my ever

loving and dearest parents, brother and sister whose love has always been a source of

energy for me and without whom I would not have been where I am today. They lit the

flame of learning in me and their affection, sacrifice, devotion and constant

encouragement help me to gain this success.

With all faith in the almighty, I place this work in the hands of my examiner with the

hope that he will bear with the shortcomings that might have crept into this dissertation

inadvertently.

Ahmad Asim

Introduction

Introduction

Cyclic voltammetry (CV) is the most popular electroanalytical technique utilized for the

study of electro active studies or electrochemical reactions. Its versatility combined with

ease of measurement has resulted in extensive use of CV in the various disciplines of

electrochemistry [1-5], inorganic chemistry, organic chemistry and bioinorganic

chemistry. A more recent interdisciplinary sub-area of the research work emerged from

interface of chemistry and chemical biology [6]. Cyclic voltammetry is often the first

experiment performed in an electrochemical study of a compound, a biological material

on an electrode surface [7]. An increasing number of inorganic chemists are using cyclic

voltammetry to evaluate the effect of ligand on the oxidation/reduction potential of the

central metal ion in complexes [8]. There has been tremendous advances in the field of

electrochemical studies from studying simple electron transfer in redox active species to

elucidation of structure of redox enzyme particularly metalloenzymes [9-10] and these

techniques have been instrumental road maps for interpreting their spectroscopic and

biochemical properties.

Cyclic voltammetry has been extensively utilized for studying simple redox active

molecules (the effect of DNA and RNA, biomolecule in peptide, amino acid and glucose)

[11-15]. This technique has gained impetus owing to the applications for inexpensive

determination of the structural effects in the areas of gene therapy, drug discovery,

medical diagnostics, environmental monitoring and forensics etc [16-22].

Fundamental concept of cyclic voltammetry [7, 1] consists of cycling the potential of an

electrode immersed in an unstirred solution, and measuring the resulting current. The

voltage applied to the working electrode is scaimed linearly from an initial value Ei to a

predetermined limit Ex, switching potential where the direction of the scan is reversed

(Figure 1 A).

(B)

(A)

o o

o UJ

0 . a. <

TUJE

Figure 1 A) Applied potential program. Exi andExiare sy^itching potentials.

B) A typical cyclic voltammogram oflmMK3Fe(CN)6 at a platinum electrode in aqueous 0. IMKCl solution at a scan rate of 100 m V/sec.

A typical voltammogram for ImM [Fe(CN)6]^' is depicted in (Figure IB) for a platinum

working electrode in aqueous O.IM KCl solution. As the potential is scanned in the

negative direction, the current rises to a peak and then decays in the regular manner. A

more detailed theoretical interpretation (reversible system) for the electron transfer rate

constant is given by Nemst equation and change in concentration that occurs in solution

adjacent to the electrode during electrolysis (Eq. 1).

E = E°-RTln nF

lEl M

(1)

v=o Where O is oxidized form and R is the reduced form.

The potential excitation signal exerts control of the ratio of Fe"\CN)6"^/ Fe"(CN)6'^ at the

electrode surface as described by the Nemst equation for a reversible system (Eq. 2).

E = E° Fe"(CN)6-^. Fe"'(CN)6-' + 0,059 log \ Fe"'(CN).-^l (2) 1 [Fe"(CN)6-^]

The peak current for a reversible system is described by the Randles-Sevcik equation for

the forward sweep of the first cycle [23-24].

ip = (2.69 X lOV^^AD'^Ct) '^

where ip is peak current (ampere), n is electron stoichiometry, A is electrode area (cm^),

D is diffusion coefficient (cm^/s), C is concentration (mol/cm^) and v is scan rate (V/s).

Accordingly ip increases with M '''' and is directly proportional to the concentration. The

relationship to concentration is particularly important in analytical applications and in

studies of electrochemical mechanisms. The values of ipa and ipc should be identical for a

simple reversible (fast) couple. That is

Ipc'-Ipa = 1

Redox couple whose peaks shift farther apart with increasing scan rate are categorized as

quasi-reversible. There are some cases in which peaks are so widely separated that no

parts of the two peaks overlap on the potential axis at all. These are generally known as

totally irreversible system. A subset of this class are those reactions that yield products

that cannot be recycled electrochemically to give back the original reactants (for

example, those that involve extensive bond breaking and/or loss of substituents to

solution), these are chemically irreversible reactions and many yield no return peak at all.

Electrochemistry and coordination chemistry are closely related areas

pertaining to the subject of electron transfer in the metal centers of enzymes. Most of the

biological enzymes contain transition metals in relevant oxidation state (+1) and (+11)

[25].Copper complexes are more prevalent particularly coordinated to imine-nitrogen

atom in imidazole ring of histidine for example blue copper centers [26] and as a general

rule the redox potential for Cu'VCu' transitions are higher than that of Fe"/Fe'" pairs both

coordinated with physiological and non physiological ligands. Redox potential of

Cu"/Cu' system depends on the relative thermodynamic stabilities of the two oxidation

states in given ligand environment. The factor influencing the metal located redox

properties have been extensively studied [27-28]. Some copper complexes exhibit

superoxide dismutase (SOD) activities as well as other important biological activities like

9

dioxygen transport [29-30]. The essential part of Cu,Zn SOD enzymes is the ability of the

redox-active center copper to oxidize metastable superoxide ion in one state and reduce it

in other oxidation state as illustrated in equation (3).

2O2" + 2H*-* H2O2+O2 (3)

In typical Cu2Zn2SOD, Cu" is centered in distorted square planar structure coordinated to

four unsaturated nitrogen atoms or four imidazole radicals in which His-61 is bridged to

the zinc atom (Figure 2). The biomimetic activity for different compounds can be

evaluated from their electrochemical behavior and their spectroscopic characterstics.

Compounds with E1/2 values, for the Cu"/Cu' process, between those of the superoxide

system could present mimetic activity [31].

Asp \

o

\ / ^ i i — - ^ K i n i s •)

H,0

His .ri 118

'His \ I X > ^ I 65 I \ ^ ^

/ H i s ^ / His 44 I <. 46

Figure 2. The structure of SOD active centers.

10

A. Mendiola et al. have carried out voltammetric studies on Cu" complexes of

benzylbisthiosemicarbazone and established a relationship of their complexes with SOD

mimetic activity [32]. SOD mimetic activity of the tested complexes correlate with their

redox potential values for Cu'^/Cu' and it was observed that copper complexes with less

activity exhibit more negative potential (Table 1).

Table 1: Reduction potential values for the Ci/VCi/ process.

Compound E^ [V]

TTITH^CU] -0.575 2[(L'H6)2Cu(NO:,)2] -0.600 JlL 'HjCu,] -0.600 4((L'Hj2CuBr2] -0.575 5[L2Cu2(EtOH)2] 0.050 elL^HjCuEtOHlNOj -0.050 siL^HjCuEtOHBr] +0.025 9 ( L 2 H 4 C U C 1 2 ( H 2 0 ) J -0.050

The complexes show higher SOD-mimetic activity and present E° values close to that of

Cu,Zn SOD [33]. From these studies, it is obvious that structure and conformation of

ligand has profound influence on the redox potential of the central atom in coordinated

compounds.

Similarly, cyclic voltammetry was used to determine the redox properties of Cu"

diothiocarbamate complexes (DTCAA) where AA is Gly, Ala, Ser, Asn, Glu to

evaluate the influence on the dismutation of O2" (Figure 3) [34]. A typical cyclic

voltammogram of the dithiocarbamate complexes can influence the dismutation of O2".

11

The Cu'VCu' redox process is quasi-reversible with very similar characterstic to those

earlier reported for dialkyldithiocarbamate complexes of Cu".

0.1 nVK

••• I I I I I 02 0.4 0.« 0.* 1.0

E/VivtM/MtAoainOMSO)

Figure 3. A typical voltammogram ofCu(II)-DTCAA complex in DMSO.

K. Anuas et al have described Cu , Sn ^ and S " on indium tin oxide (ITO) glass

substrate from aqueous solutions employing cyclic voltammetry technique [35] to study

this deposition phenomenon in an effort to develop new materials for energy conversion

and other photochemical applications. The experiment outlined demonstrates the

determination of the peak current and peak potential, diffusion coefficient,

electrochemical reversibility, scan rate and scan cycle.

12

Insulin secretion by pancreatic P-cells is mainly induced by circulating glucose [36]. The

glucose metabolism of the P-cells causes several alterations that result in an increase in

the intercellular ionic calcium concentrations. In several tissues, under physiological

conditions, glucose oxidation results in the generation of hydrogen peroxide and oxygen

free radicals such as OH* [37]. ROS (Reactive oxygen species) are known to interact

with nucleic acids, proteins and the lipid bilayer of cell membrane, leading to cell damage

[38]. It has been earlier demonstrated that a high glucose concentration causes over

expression of Cu,Zn superoxide dismutase (Cu,Zn-SOD) and catalases in cultural human

endothelial cells [39]. The activity of SOD however, increased with glucose

concentration and this increment was closely co-related with the rate of insulin secretion.

Keeping this rationale in mind, we have investigated the effect of glucose on the copper

bimetallic complex which may act as biomimetic SOD model.

J. Labuda et al have studied the redox behavior of the [Cu(TAAB)]Cl2 complex, the bis

(ethylenediamine)bis(salicylate) Cu" complex, Cu(sal)2(en)2, the Cu" complex of N-

salicyldeneglutamate Schiff base ligand and pyridine, Cu(sal-D,L-glu)py, and three N-

salicylidene-L-ethylglutamato Cu" complexes, Cu(sal-L-etglu)X, where X is imidazole,

thiourea, or water molecule as an equatorial ligand (Figure 4), in aqueous and DMSO

media and have evaluated their redox behavior by cyclic voltammetry.

13

O) ~1

^>t^ [o-o] (CiKrfOHjCOCn,

Cu(sm3(aa>i

H

Ci<MM).L-gM)W Cu(MW^«IOl»»X. X - HjO. Imld. fu

Figure 4. The proposed structure of some SOD-like biomimetic copper complexes.

The reactivity of copper compounds toward oxygen and superoxide anion is of great

interest and previous literature survey [40] reveals that SOD-like activity of various

Schiff base Cu" complexes as well as with macrocyclic schiffs base ligands; it was

further observed that half wave potential values for Cu'VCu^ redox couple in aqueous

solution and reaction of Cu' complexes with molecular oxygen and O2" electrogenerated

in DMSO were in good agreement with SOD-like activity of the complexes.

In both aqueous and DMSO solution, the cyclic voltammograms of the copper complexes

obtained in absence of O2 exhibit a pair of peaks (Figure 5) corresponding to one electron

Cu'VCu' reduction and subsequent CuVCu" oxidation. The peak potential Ep, was slightly

dependent on the potential scan rate: with the decreasing scan the Epci become more

positive and Epai values more negative. At a scan rate of 0.200 Vs"', the peak to peak

separation, AEp, obtained at the hanging mercury electrode (HMDE) ranges from 50 to

14

150 mV and the anodic to cathodic peak current ratio is near to one. It indicates a quasi-

reversible redox process localized on copper centre.

A 2

C 1 C 2

-0.4 - 0 . 2 0 ,0 0-2

E / V vs. Ag/AgCI

Figure 5. Cyclic voltammograms at HMDEfor the reduction ofCu(sal-D-L-glu)py in 0.005 Mphosphate bufferpH 7.4 at a scan rate of 0.050 Vs''.

Table 2 summaries the half wave potential data calculated as the arithmetic mean of the

Epci and Epai values. The E° values lie between the potentials of the reduction of O2 to O2

(-0.40 V vs SCE at pH 7) and O2" to H2O2 (+0.65 V vs SCE) as it is expected for

compounds with a SOD-like activity in dependence of O2' concentration [31, 41]. In

DMSO, the corresponding E1/2 values shifted negatively by ca. 0.4 V. This shift indicates

an interaction of the electrodonor solvent molecule with central metal atom of the

15

complex particle. The presence of such free coordination site at Cu" represents a

fulfillment of another known requirement on the SOD-like active copper complex.

Table 2: Redox potential data at HMDE in aqueous solution, Vvs Ag/AgCl, 0.05 M phosphate buffer pH 7.4, ionic strength 0.1 Mwith NaCl04 at a scan rate 0.2 Vs'.

Complex

CuCTAAB)-* CirfsaDilen)^ Cu(sal-D4.-glu)py Cu(sal*L-«tglu)H20 CiKsal-L-etgluMmid Cu(sal*L-etg}u)tu

^ . K ,

-0.03 -0.14 -0.19 -0.20 -0.23 -0.26

^pal

0.03 • -0.04

-0.12 -0.12 -0.06 -0.12

^1/2

0.00 -0.09 -0.16 -0.16 -0.15 -0 .19

^pa2

—

—

0.05 0.04

—

—

The human serum albumin (HSA) is a large globular protein present in blood plasma with

various functions involved in transport, distribution, metabolism of various endogenous

ligands such as fatty acids, bilrubin, amino acids, metals like Cu", Zn", Co" etc which is

widely applicable in pharmaceutical industries [42-45]. The crystailographic analysis

revealed that HSA consist of 585 amino acid residue monomer, containing three

homologous a-helical domains (192, 187, 199) and a single tryptophan (Trp214) (Figure

6) [46-48].

Solubility of hydrophobic drugs in plasma is increased by HSA and HSA plays a

dominant role in drug disposition and efficiency, thus modulate their delivery to the cell

in vivo and in vitro [46]. An extensive research in the area of medicinal inorganic

chemistry for the ailment of many chronic diseases constitute metal based drugs which

16

HydrophUie, Cop

Ly«l95

Hydrophobic Pocket

Figure 6. The environment of the Lysigg binding sites in HSA as obtained directly from X-ray refinement. Distances in A.

involves principle of interaction of metal based drugs with HSA, capable of affecting the

drug distribution and biotransformation. Binding drugs to HSA become necessary in

present environment because free (non-protein bound) drug can easily be diffused to

tissues due to its active nature and this principle involves in pharmacology [47]. An

approach should be there to design a compound which will have low affinity for HSA,

can act as potent drug and can be used in lower dosage to improve in-vivo tolerance [49].

Cyclic voltammetry technique was employed to investigate the binding of Cu' complex

derived from oxamide diiminodiacetic and triethylene tetramine to HSA [50]. The

electrochemical behavior of Cu complex with HSA was monitored by cyclic

17

voltammetry in a phosphate buffer. The Ep values -0.730 and -0.560 V respectively, were

obtained at a scan rate of 0.1 Vs'V The interaction of Cu" complex with HSA, was

studied at the same scan rate, which reveals weak binding as the E° values do not shift

considerably (Figure 7). The cyclic voltammogram of the Cu" complex bound to HSA

was recorded at different pH's (6.5 to 7.4) (Figure 8)

Figure 7. Cyclic voltammogram of Cu!' complex bound with HSA in phosphate buffer at 30°C at scan rate ofO. 1 Vs'.

095

085

075 1

OGS

-

•

•

1.

•

1

60 as 70 PH

74

Figure 8. pH dependent plot E° value ofCi/' complex bound with HSA at a scan rate of 0.1 Vs'.

18

Present work

In this work, we have carried out the cyclic voltammetry of two copper complexes

[Ci6H38N809Sn2Cu2Cl8] 1 and [C28H44N8O6CU2CI4] 2 which were synthesized in our

laboratory previously by the procedure described in the synthesis part. The interaction of

copper complex 1 with bioanalytes viz glucose, amino acids (glycine, L-valine) and HSA

was investigated by exploring cyclic voltammetry as it is the versatile technique to

validate the binding studies of the redox active species. Its versatility combined with ease

of measurement has resulted in extensive use of CV in the various disciplines of

electrochemistry, inorganic chemistry, organic chemistry and bioinorganic chemistry.

The effect of glucose was studied owing to the fact that copper complexes are well

known for in vivo/in vitro SOD like activity and in many complexes SOD activity has

been related to insulin secretion and glucose concentration dependence. The comparison

of other important bioanalytes was studied to evaluate the influence of these

biomolecules on the coordination behavior of the central metal ion. All the parameters

Epc, Epa, AEp, E° Ipc/Ipa were evaluated in the free complexes and also in the presence of

the bioanalytes.

19

Experimental

20

EXPERIMENTAL METHODS

1. Cyclic voltammetry

Cyclic voltanunetry involves the measurement of current-voltage curves under diffusion

controlled, mass transfer conditions at a stationary electrode, utilizing symmetrical

triangular scan rates ranging from a few millivolts per second to hundred volts per

second. The triangle returns at the same speed and permits the display of a complete

polarogram with cathodic (reduction) and anodic (oxidation) waveforms one above the

other. Two seconds or less is required to record a complete polarogram [51].

Consider the reaction

O +ne • R (1)

Assuming semi-infinite linear difftision and a solution containing initially only species O.

With the electron held at a potential Ei where no electrode reaction occurs. The potential

is swept linearly at v V/sec so that the potential at any time is

E(t) - Ei-vt

or Epeak = E°-0.0285

The rate of electron transfer is so rapid at the electrode surface that species O and R

immediately adjust to the ratio according to the Nemst equation, which is as follows.,

Co (0,t) = Co* - [nFA(7iDo)"2]-' J I(7t)(t-T)-''' dx (2)

21

I = nFACo*(7tDoCT) ' X (a t) (3)

Redox (electron-transfer) reactions of metal complexes can be investigated by cyclic

voltammetry. An electrode is immersed in a solution of the complex and voltage is swept

while current flow is monitored. No current flows until oxidation or reduction occurs.

After the voltage is swept over a set range in one direction, the direction is reversed and

swept back to the original potential. The cycle may be repeated as often as desired.

Figure 9 shows the cyclic voltammograms (CV) for a reversible one-electron redox

reaction such as,

CpFe(CO)LMe • CpFe(CO)LMe^ + e

Sweeping the potential in an increasing direction oxidize the complex as the anodic

current la flows; reversible reduction of CpFe(CO)LMe^ generates cathodic current Ic on

the reverse sweep. The magnitude of the current is proportional to the concentration of

the species being oxidized or reduced.

The measured parameters of interest on these cyclic voltammograms are Ipc/Ipa the ratio

of peak currents, Epa - Epc the separation of peak potentials and the formal electrode

potential E°. For a Nemstian wave with stable product, the ratio Ipc/Ipa = 1 regardless of

scan rate, E and diffusion coefficient, when Ipa is measured from the decaying current as

a base line. The difference between Epa and Epc (AEp) is a usefiil diagnostic test of a

Nemstian reaction. Although AEp is slightly a function of E°, it is always close to

2.3RT/ nF.

22

f x r

t £

Switching time, X

Figure 9. (A) Cyclic potential sweep (B) Resulting cyclic voltammogram

The technique yields information about reaction reversibihties and also offers a rapid

means of analysis for suitable systems. The method is particularly valuable to study

interaction of metal ions to glucose as it provides a useful compliment to other methods

of investigation, such as UV/vis spectroscopy. Cyclic voltammetric studies were

accomplished on a CH Instrument Electrochemical analyzer using a three-electrode

configuration comprised of a Pt wire as the auxiliary electrode, a platinum disk as the

working electrode and Ag/AgCl as the reference electrode. Supporting electrolyte for the

experiments was 0.4 M KNO3. Electrochemical measurements were made under nitrogen

atmosphere. All electrochemical data were collected at 298 K and are imcorrected for

junction potentials.

23

Experimental

Materials and Methods

All the reagents were of best commercial grade and used without further purification.

Triethylenetetramine, Diethyioxalate (Fluka), Pthalic anhydride (Merck), Tris base

(Merck), l,8-diamino-3,6-diazaoctane (Fluka), Tin tetrachloride (Loba Chemei), copper

(II) chloride (Qualigens) were used as received. Glycine and L-valine was purchased

fi-om Merck. Glucose and human serum albumin (HSA) was purchased fi"om Sigma

Chemical Co. All the experiment involving interaction of complex with glucose was

performed in Tris buffer and pH was maintained at 5 and 6. Cyclic voltammetry was

carried out at CH instrument electrochemical analyzer. All voltammetric experiments

were performed in single compartmental cell of volume 10-15 ml containing a three-

electrode system comprised of a Pt-disk working electrode, Pt-wire as auxiliary electrode

and an Ag/AgCl electrode as reference electrode. The supporting electrolyte was 0.4 M

KNO3 in milli-Q water. Deaerated solutions were used by purging N2 gas for 15 minutes

prior to measurements.

24

Syntheses

Synthesis of the 1,4,7,10,13,16,19,22 octaaza 2,3,14,15-

tetraoxocyclohexadecane tin(IV) Cu(II)chloride [Ci6H38N809Sn2Cu2Cl8]

1

This complex 1 was synthesized by the earlier reported method [52] as described below:

To a solution of [Ci6H3oN805Sn2Cl4] (0.793g, Immol) in 10ml of methanol was added

CUCI2.2H2O (0.342g, 2mmol) in 5ml of methanol dropwise. The resulting solution was

stirred for Ihr; a green coloured precipitate formed was filtered and thoroughly washed

with methanol and dried in vacuo. Yield: 63%. m.p. 205 °C. Anal. Calc. for

CeHssNgOpSnsCuaClg: C, 16.9; H, 3.35; N, 9.88. Found: C, 17.33; H, 3.48; N, 9.91.

Selected IR in KBr (v/cm"'): 3283 v(N-H), 1631 v(C=0), 435 v(Cu-O). ESI (m/zy. 1133

[Ci6H38N809Sn2CU2Cl8]^.

25

Synthesis of Bis [aqua 1, 8-(l, 2-dicarboxainide benzene) 3, 6-

diazaoctane copper (II)] tetrachloride [CisIiMNgOeCuiCU] 2

This complex 2 was synthesized by the earlier reported method [53] as described below:

1, 8-diamino-3, 6-dia2aoctane (2.9 ml, 20 mmol) was added drop wise to the ethanolic

solution of CuCl2,2H20 (3.4 g, 2 mmol) in 1:1 molar ratio. The resulting deep blue

solution, Xmax ~ 536 run corresponding to formation of copper(II) l,8-diamino-3,6-

diazaoctane dichloride complex was stirred at 25 °C for 30 min and subsequently pthallic

anhydride (2.9g, 20 mmol) dissolved in 25ml ethanol was added to it. The reaction

mixture was refluxed for ca. Ihr and a light blue solid product is obtained. The product

was filtered, washed with ethanol and dried in vacuo. Yield:, (85%), m.p. 195°, Anal.

Calc. for C28H44N8O6CU2CI4 C 39.3, H 5.1, N 13.1, Cu(II) 14.7; found; C 39.26, H 5.13,

N 13.08. Selected IR in KBr (v/cm^ ) : 3,152 u(C-N of carboxamide moiety), 1,713

u(C=0), 1,398 u(C-N), 2877 ^(CHj), 2,933 u(CH), 416 u(Cu-N), ESI-MS: m/z

[C28H4oN8Cu2]Cl4 821,; Cu(II) 14.6%.

26

H2O-CU-0H2

I Sn SnT IH2O

CI V ^ CI

H2O-CU-0H2 / \

Cl CI

H N ">A4f N-H

H -N N.

H ^ C , .^

Cu—OHg

/ \ N—

H

CL

Figure l(S. Proposed structure of complexes 1 [Ci6H38N809Sn2Cu2Clg]

2 [C28H44N8O6CU2CI4]

27

Results and Discussion

28

Results and discussion

Cyclic voltammetry of copper complexes :

There is an inherent resemblance between the electrochemical and biological reactions

due to the presence of redox-active metal centers (Fe"/Fe"'/Fe'^, CuVCu") in the

numerous metalloproteins and enzymes involved in different life processes; it can be

assumed that the redox mechanism taking place at the electrode surface at different

concentrations and in the body share similar principles. The obtained results from the

redox property of complexes and biomolecules might have profound effect on the

understanding of their in vivo redox behavior [54]. In general, pH is one of the strong

variables that strongly influence the shape of the voltammograms and thus it is important

to investigate the effect of pH on the redox behavior of the complex on a representative

biomolecule like glucose. In this context, the effect of pH on peak current and formal

potential (E°) were investigated by cyclic voltammetry in tris buffer at different pH's.

The copper complexes exhibit a well defined quasi-reversible wave attributed to

the redox couple Cu'VCu' in the potential range of +2.4 to -1.2V [23,55]. The cyclic

voltammogram of complex 1 recorded in Immol solution in 0.4 M KNO3 exhibited

reduction peak at -0.598V and in a subsequent anodic scan, the anodic peak appeared at -

0.179V, respectively (Figure 11). The separation of anodic and cathodic peak potentials

is -0.419 V. The formal potential E° was taken as the average of Epa and Epc and was

found to be -0.388 V. The ratio of cathodic and anodic peak current Ipc/Ipa was found to

29

be 0.118. The cyclic voltammogram of complex 2 (Figure 12) reveals one electron redox

process involving the Cu'VCu' couple, as judged from the peak potential separation (AEp)

and formal potential E° of 110 mV and 230 mV, respectively (Table 4). The voltammetric

response remains unchanged at different scan rates, which features that the initial

complexes are regenerated during the potential cycle. These results were attributed to

quasi-reversible one electron transfer, corresponding to Cu'VCu' redox couples of

complex 1 and complex 2, respectively. The anodic to cathodic current ratios for these

couples were consistently less than unity in cyclic voltammograms at a restricted

potential range [56].

I

8.0

6.0 ^

4 .0 ^

2.0

O

- 2 . 0 :

- 4 . 0 :

-e.o :

-so -

- 1 0 . 0

- ^ ^

- I 1 • . — I • 1 1 • r-

- 0 . 3 -0 .6 -0 .9 -1 .2

PotemtisJ/V

Figure 11. Cyclic voltammogram of complex I at a scan rate of 0.3 Vs'' in H2O.

30

u u u «

< w

i 0

-w -13

•£0

•U •&0

• • • ' • • • • ' • • • • ' • • • ' • • • • '

/ / / / / / / y / y

1 \^ /

v_^ ' ' ' ' 1 ' ' • ' 1 • ' ' • 1 • • ' ' t • ' • ' 1 ' • • • 1 ' • • •

PotentUI/V

-/ Figure 12. Cyclic voltammogram of complex 2 at a scan rate of 0.3 Vs' in H2O.

%:.0 -

6 . 0 -

^.0 '-

«S 2.0 -

3 oi 1 - °:

-6 .0 :

-10.0 -

h

^ ^

-

-o.:: -0.3 -o<> - 1 2

Figure 13. Cyclic voltammogram of complex 1 at different scan rates (0.1, -1 0. 2, 0.3 Vs") in H2O.

31

Effect of glucose on redox behavior of complex 1 :

The effect of glucose on redox potentials of the complex 1 was examined by cyclic

voltammetry which depicts a pair of well defined redox peaks which undergo significant

shift in peak potential, current ratio as shown in Figure 14 (Table 3).0n addition of

glucose to complex 1, there is a remarkable shift in formal potential values, E°= -0.41 V,

the ratio of anodic peak current to cathodic peak current (Ipc/Ipa) = 0.15 and AEp = -0.40

V in comparison to the free unboimd complex is due to reducing behavior of the glucose

and SOD mimic nature of the complex 1.

«.o

-0.3 -o.e -0.9 -1.2

Figure 14. Cyclic voltammogram of copper complex 1 in the presence of glucose at a scan rate ofOJVs'' in H2O.

32

Table 3: Cyclic voltammetric data of complex 1 in the presence of glucose.

Concentration of

GIucose(10 ) M

0.2

0.4

0.6

0.8

1.0

EpcCV)

-0.61

-0.54

-0.52

-0.50

-0.48

Ep.(V)

-0.21

-0.23

-0.26

-0.29

-0.31

AEp(V)

-0.40

-0.31

-0.26

-0.21

-0.17

E°(V)

-0.41

-0.38

-0.39

-0.39

-0.39

Ipc/ipa

0.15

0.43

0.50

0.38

0.18

Table 4: Cyclic voltammetric data of complex 2 in the presence of glucose.

Concentration of

GIucose(10-^ M

0.5

1.0

1.5

2.0

2.5

EpcOO

-0.45

-0.42

-0.40

-0.39

-0.41

Ep.(V)

-0.34

-0.38

-0.57

-0.73

-0.93

AEp(V)

-0.90

-0.40

-0.17

-0.24

-0.54

E°(V)

-0.44

-0.40

-0.48

-0.71

-0.67

Ipc'lpa

0.27

0.43

0.38

0.55

0.78

33

-0.3 -0.6 -0.9 P o t e n t f a l / V

-1.2

Figure 15. Cyclic voltammogram of complex 1 at different concentrations of glucose (0.2, 0.4, 0.6, 0.8, 1.0 x ICT^) M.

-0.411

Jtsa

•«M

1-0.56

.O.S>-

.O.M.

.0.62

0.4 0.6 0.8

Coac of Glucose(M)

0.4 0.6 0.«

CoaccatratiMl of CIUCIMC(M)

Figure 16. Plots for cathodic (Epc) and anodic peak potential (Epa) vs. cone, of glucose (M)for complex 1.

34

Effect of glucose on redox behavior of complex 1 at different pH :

The redox behavior of the Cu" complex bound to glucose was studied by cyclic

voltammetric measurements recorded at different pH. The cyclic voltammogram of

complex 1 at pH 5 exhibits one quasi-reversible redox couple corresponding to the

Cu'VCu' with Epc -0.54 V and Epa -0.27 V, respectively at a scan rate of 0.3 Vs'' (Figure

17). For this couple, the difference between cathodic and anodic potential AEp is -0.27 V

and the Ipc/Ipa value is less than one which is attributed to one electron redox couple

Cu"/Cu'. At different scan rates, there is no major change in Ep and Em values, indicating

that Ep is independent of scan rate.

IE

1

10.0

8.0

6 .0

4.0

2.0

0

-2.0

-4.0

-6.0

-^.0

10.0 T • • T

O -0.3 -0.6 -0.9 P o t e n t t a d / V

-1 .2

Figure 17. Cyclic voltammogram of complex 1 at different concentrations of glucose atpH5.

35

-0.51

? -0.S2H

-0.63

-0.54-

0.4 0.6 0.1 CMC«rGI«c«M(M)

a2 0.4 0.6 a s CaBcorGiiicaM(M)

1.0

Figure 18. Plots for cathodic (Epc) and anodic peak potential (EpJ vs. cone, of glucose (M) at pH 5 for complex 1.

Table 5: Cyclic voltammetric data of complex 1 in the presence of glucose at pH 5.

Concentration of

Glucose(10-') M

0.2

0.4

0.6

0.8

1.0

Epc(V)

-0.54

-0.53

-0.51

-0.50

-0.49

Ep.(V)

-0.27

-0.29

-0.27

-0.28

-0.26

AEp(V)

-0.27

-0.24

-0.23

-0.22

-0.23

E^oo

-0.40

-0.41

-0.39

-0.39

-0.37

Ipc/ipa

0.18

0.24

0.25

0.23

0.29

36

At pH 6, CV of complex 1 exhibits a quasi-reversible one electron wave attributed to

Cu"/Cu' redox couples of illustrating that the redox peak is due to electrochemical

reaction of metal complex with glucose. However, the change of pH from 5 to 6 produce

no significant difference in the interaction. A pH gradient exists in the both normal and

pathophysiological conditions. For example the extra-tumor environment has a slightly

more acidic pH than blood and normal tissue [57-58].

-0 .3 -0.6 -0 .9 Potential / V

-1 .2

Figure 19. Cyclic voltammogram of complex 1 at different concentrations of glucose atpH6

37

I 0» I

Ca«>fGtacme(M) CtocmCnliaa orGI«coM<M)

Figure 20. Plots for cathodic (Ep^) and anodic peak potential (Epa) vs. cone, of glucose (M) atpH 6 for complex 1.

Table 6: Cyclic voltammetric data of complex 1 in the presence of glucose atpH6.

Concentration of

Glucose(10^ M

0.2

0.4

0.6

0.8

1.0

Ep.(V)

-0.50

-0.48

-0.49

-0.49

-0.49

Ep.(V)

-0.31

-0.28

-0.28

-0.29

-0.27

AEp(V)

-0.19

-0.20

-0.20

-0.20

-0.22

E^CV)

-0.40

-0.38

-0.38

-0.39

-0.38

Ipc/Ip.

0.86

0.23

0.22

0.19

0.18

38

Effect of HSA on redox behavior of complex 1 :

The cyclic voltammogram of complex 1 in presence of HSA (human serum albumin)

exhibits a quasi-reversible wave attributed to Cu"/Cu' redox couple, depicting a large

reduction wave due to Cu(II) + e" —» Cu(l) while the oxidation wave was quite small. At

different scan rates, there is no major changes in Epc, Epa and E° values, clearly indicating

that Epc, Epa are independent of scan rate. The Epc and Epa values of complex 1 obtained at

a scan rate of 0.3 Vs"' is -0.54 V and -0.28 V, respectively, resulting in significant

decrease of reduction peak potential and increase of oxidation peak potential as shown in

Figure 21 which is due to slow diffusion of an equilibrium mixture of free and HSA

boimd complex to the electrode surface [23, 59]. The observed shift in the Epa and Epc

value indicate that both Cu(II) and Cu(I) forms bind to HSA. Figure 22 depicts cyclic

voltammogram of complex 1 at different concentration of HSA (Table 7).

39

s o

I -4.0 :

' - 6 .0 -

-e.o

-10 .0

• ' •

• — • — I — • — • — I — • — • — I — •

> - 0 . 3 - 0 . 6 - 0 . 9 P o t e s i t i a a / ^V

-1 .2

Figure 21. Cyclic voltammogram of complex 1 in the presence ofHSA at a scan rate of 0.3Vs' H2O.

_ I -0.4 -0.$ -1 .2

P o t e n t i a l / V

Figure 22. Cyclic voltammogram of complex 1 at different concentrations ofHSA.

40

0^ (.1 OJ

C « i K o r B S A ( M ) C«nc<irHSA(M)

Figure 23. Plots for cathodic (Epc) and anodic peak potential (EpJ vs. cone, of HSA (M)for complex 1.

Table 7: Cyclic voltammetric data of complex 1 in the presence of HSA.

Concentration of

HSA(10-^) M

0.2

0.4

0.6

0.8

1.0

Epc(V)

-0.54

-0.53

-0.51

-0.51

-0.50

Ep.(V)

-0.28

-0.28

-0.29

-0.29

-0.31

AEp(V)

-0.26

-0.25

-0.21

-0.22

-0.19

£"(¥)

-0.41

-0.40

-0.40

-0.40

-0.40

Ipc/Ip.

0.24

0.60

0.94

0.92

0.75

41

Effect of glycine on redox behavior of complex 1 :

The cyclic voltammogram of complex 1 in the presence of glycine reveals one electron

quasi-reversible wave attributed to the redox couple of Cu"/Cu' with Epc, Epa at -0.53 V

and -0.32 V at a scan rate of 0.3 Vs"' (Figure 24). The result indicated that both anodic

and cathodic peaks shifted and a new peak emerged on the addition of glycine which

suggests the formation of metal- glycine complex [60]. The ratio of anodic peak current

to cathodic peak current Ipc/Ipa is 0.90 and AEp is -0.21 V. There is a continuous decrease

in cathodic peak potential which suggests that Cu" form prevails at higher concentration

of glycine (Figure 25 and Table 8).

-0.3 -0.6 -0.& -1 .2 Potentisa / V

Figure 24. Cyclic voltammogram of complex 1 in the presence of glycine at a scan rateof0.3Vs''inH2O.

42

O -0.3 -0.6 -0.9 P o t e n t i a l / V

-1.2

Figure 25. Cyclic voltammogram of complex 1 at different concentrations of glycine.

4.52

4.M-

0.4 0.6 0.* CaiicarGiyciK(M)

1.0

4.290

4.295-

4.300

4.305

4.310

4.315

4.320-

0.2 0.4 0.6 0.8 CoacorClydB<(M)

Figure 26. Plots for cathodic (Epc) and anodic peak potential (EpJ vs. cone, of glycine (M)for complex 1.

43

Table 8: Cyclic voltammetric data of complex 1 in the presence of glycine.

Concentration of

Glycine(10'^) M

0.2

0.4

0.6

0.8

1.0

Epc(V)

-0.53

-0.50

-0.51

-0.49

-0.51

Ep.(V)

-0.32

-0.30

-0.29

-0.30

-0.31

AEp(V)

-0.21

-0.20

-0.22

-0.19

-0.20

E^CV)

-0.42

-0.40

-0.40

-0.39

-0.41

Ipc/Ip.

0.90

0.70

0.25

0.17

0.28

Effect of L-valine on redox behavior of copper complex 1 :

The effect of L-valine on complex 1 (Figure 27) resulted in one electron quasi-reversible

Cu /Cu wave. On increasing the concentration of L-valine, the cathodic peak current

shifts towards more positive potential due to the interaction of L-valine with metal

(Figure 28 and Table 9) [13, 61-62].

44

- 0 . 3 - 0 . 6 - 0 . 9 -1 P o t e n t l s a / ^^

.2

Figure 27. Cyclic voltammogram of complex 1 in the presence ofL-valine at a scan rate of0.3Vs'H2O.

I l l

-0.4 -0.$ P o t e n t l s a / V

-1.2

Figure 28. Cyclic voltammogram of complex I at different concentrations ofL-valine.

45

-> - 0 ^ - t. -0.J10

-0.320

— r ' r • 1 • 1 ' 1 • 1 • 1 —

0.4 0.5 0.6 0.7 0.1 0.0 1.0

C«iK>rUvaliae(M)

0.4 0.« 0.8

Cone of L-valin«(M)

Figure 29. Plots for cathodic (Ep<) and anodic peak potential (EpJ vs. cone, of L-valine (M)for complex 1.

Table 9: Cyclic voltammetric data of complex 1 in the presence of L-valine.

Concentration of

L-vaIine(10-^ M

0.2

0.4

0.6

0.8

1.0

Epc(V)

-0.50

-0.52

-0.50

-0.48

-0.50

Ep.(V)

-0.32

-0.30

-0.30

-0.31

-0.31

AEp(V)

-0.18

-0.20

-0.19

-0.16

-0.17

E°(V)

-0.41

-0.41

-0.40

-0.39

-0.40

Ipc/Ip.

0.71

0.76

0.23

0.16

0.93

46

Conclusion:

Cyclic voltammetric study of the copper complexes was done to ascertain the reactivity

of the copper complexes in the presence of biomolecules. The values of the CV

parameters with different bio-molecules viz- glucose, glycine, HSA, L-valine were

calculated in different environments. On comparison of the experimental results of these

biomolecules, we observed that glucose is playing a different role and has influenced the

reduction potential of the copper metal ion substantially. In the light of our results it has

been concluded that both the copper complexes seem to be SOD mimics and show

dependence on glucose concentrations. It may be correlated to the high activity or fast

reduction of the copper in presence of glucose due to SOD mimic nature of the copper

complexes [l,4,7,10,13,16,19,22-octaaza-2,3,14,15 tetraoxocyclo Hexadecane Sn'^ Cu"

chloride [Ci6H38N809Sn2Cu2Cl8] (complex 1).

47

References

[I] G. A. Mabbott, J. Chem. Educ, 60, 697 (1983)

[2] J. B. Headridge, ''Electrochemical Technique for Inorganic Chemists,"

Academic press, London and New York (1969)

[3] M. J. Powers, T. J. Meyer, J. Amer. Chem. Soc, 102,1289 (1980)

[4] J. M. Bobbit, J. P. Wills, J. Org. Chem., 45,1978 (1980)

[5] D. R. Henton., R. L. McCreery, J. S. Swenton, J. Org Chem., 45, 369 (1980)

[6] J. K. Barton, K. D. Karlin, Curr. Opin. Chem. Biol., 5,165 (2001)

[7] P. T. Kissinger, J. Chem. Educ, 60, 702 (1983)

[8] J. A. Streeky, D. G. Pillsburg, D. H. Busch, Inorg Chem., 19, 3148 (1980)

[9] C. Leger, P. Bertrand, Chem. Rev., 108,2379 (2008)

[10] W. Zhang, G. Li, Anal. Sci., 20,603 (2004)

[II] T. C. Michael, M. Rodriguez, A. J. Bard, J. Am. Chem, Soc, 111, 8901 (1989)

[12] M. P. Youngblood, D. W. Margerum, J. Coord Chem., 11,103 (1981)

[13] M. F. Cerd4 E. Mendez, L. Malacrida, C. F. Zinola, C. Meli^, M. E. Martins, A.

N. Castro Luna, C. Kremer, J. Colloid Interface Sci., 249, 366 (2002)

[14] V.Cuculic, I. Pizeta, Croat. Chem. Acta, 79,41 (2006)

[15] A. Habrioux, E. Sibert, K. Servat, W. Vogel, K. B. Kokoh, N. Alonso-Vante., J.

Phys. Chem. B.,\l\, 10329(2007)

[16] S. P. A. Fodor, R. P. Rava, X. C. Huang, A. C. Pease, C. P. Holmes, C. L. Adams,

Nature., 364, 555 (1993)

48

[17] D. Noble, Anal. Chem., 67, 201A (1995)

[18] W. Bains, Chem. Br., Ill (1995)

[19] C. A. Scholin, M. C Villac, K. R. Buck, J. M. Krupp, D. A. Powers, G. A. Fryxell,

F. P. Chavez, Nat. Toxins., 2,152 (1994)

[20] M. Holodniy, L. Mole, D. Margolis, J. Moss, H. Dong, E. Boyer, M. Urdea,

J. Kolberg, S. Eastman, J. Virol, 69,3510 (1995)

[21] K. M. Millan, S. R. Mikkelsen, Anal. Chem., 65,2317 (1993)

[22] K. M. Millan, A. Saraullo, S. R. Mikkelsen, «a/. Chem., 66, 2943 (1994)

[23] A. J. Bard, L. R. Faulkner, "Electrochemical Methods: Fundamentals and

Applications", Wiley, New York (1980)

[24] P. T. Kissinger, W. R. Heineman (Editors)., "Laboratory Techniques in

Electroanalytical Chemistry", Dekker New York (1996)

[25] W. Kaim, B. Schwederski., "Bioinorganic Chemistry: Inorganic Elements in

Chemistry of life", John Wiley and Sons, New York (2001)

[26] N. Kitajima, K. Fujisawa, M. Tanaka, Y. Moro-oka, J. Am. Chem. Soc, 114,9232

(1992)

[27] A. A. Isse, A. Gennaro, E. Vianello, J. Chem. Soc. Dalton Trans., 2091 (1993)

[28] H. Yokoi, A. W. Addison, Inorg Chem. 16,1341 (1977)

[29] M. C. Apella, R. Totaro, E. J. Baran, Biol. Trace Elem. Res., 37, 293 (1993)

[30] P. Chautemps, G. Gellon, B. Morin, J. L. Pierre, C. Prevent, S. M. Refaif, C. G.

Beguin, A. E. Marzouki, G. Serratrice, E. Saint-Aman, Bull. Soc. Chim. Fr., 131,

49

434 (1994)

[31] D. T. Sawyer, J. S. Valentine, Ace. Chem. Res., 14, 393 (1981)

[32] M. A. Mendiola, Z. Durackova, M- T. Sevilla, A. Valent, Bioelectrochem Bioenerg,

48,109 (1999)

[33] J. A. Tainer, D. E. Getzoff, J.S. Richardson, D. C. Richardson, Nature, 306,284

(1983)

[34] R. Cao, N. Travieso, A. Fragoso, R. Villalonga, A. Diaz, M. E. Martinez, J. Alpizar,

D. X. West, J. Inorg. Biochem, 66, lU (1997)

[35] K. Anaur, W. T. Tan, M. S. Atan, K. Dzulkefly, S. M. Ho, H. M. Jeias, N.

Saravanan, Pacif. J. Sci Tech., 8,252 (2007)

[36] W. J. Malaisse, A. Sener, A. Herchuelz, J. C. Hutton, Metabolism., 28,373 (1979)

[37] S. P. wolff, R. T. Dean, Biochem. J., 245,243 (1987)

[38] B. Halliwell, J. M. C. Gutteridge, '^Free radical in biology and medicine", Oxford,

UK: Clarendon (1995)

[39] A. Ceriello, P. Russo, P. Amstad, P. Cerretti, Diabetes., 45,471 (1996)

[40] J. Labdua, L. Fenikova, Z. DuradKova, Bioelectrochem Bioenerg, 44, 31 (1997)

[41] G. Czapski, S. Goldstein, Free. Rad. Res. Commm., 4, 225 (1988)

[42] G. C. Roberts, Curr. Opin. Biotechnol, 10,42 (1999)

[43] R. E. Hubbard, Curr. Opin. Biotechnol, 8, 696 (1997)

[44] W. N. Hunter, Parasitology., 114, 31 (1995)

[45] M. L. Kleinberg, L. A. Wanke, Am. J. Health Syst. Pharm., 52, 1323 (1995)

50

[46] Y. J. Hu, W. Li, Y. Liu, J. X. Dong, S. S. Qu, J. Phar. BioMed. Anal, 39, 740

(2005)

[47] R. E. Olson, D. D. Christ, Ann. Rep. Med. Chem., 31, 327 (1996)

[48] C. D. KanaKis, P. A. Tarantilis, M. G. Polissiu, S. Diamantoglou, H. A.

TajmirRiahi, J. Mol. Struct., 798,69 (2006)

[49] H. Mao, P. J. Hajduk, R. Craig, R. Bell, T. Borre, S. W. Fesik, J. Am. Chem. Soc,

123,10429(2001)

[50] F. Arjmand, B. Mohani, Bioinorg. Chem. Appl., 2, 3 (2004)

[51] A. J. Bard, L. R. Faulkner, "ElectrochemicalMethods", Wiley, New York, 219

(1980)

[52] Irshad-ul-Haq Bhat, Ph.D Thesis, Chapter IV, 68 (2008)

[53] T. Sartaj, M. Suvigya, Biometals., 21, 299 (2008)

[54] J. P Hart. " Electroanalysis of Biologically Important Compounds ", Ellis Horwood

Ltd., England (1990)

[55] Z. D. Yu, H. Liu, M. Wang, S. L. Xiao, M. Yang, X. H. Bu, J. Inorg Biochem., 90,

79(2002)

[56] J. Lu, T. B. Lu, H. Li, Q. Zhang, L. W. Ji, Trans. Met. Chem., 27, 686 (2002)

[57] K. Engin, D. B. Leeper, J. R. Cater, A. J. Thistlethwaite, L. Tupchong, J. D.

Mcfarlane, J. Hypertherm., 11,211 (1995)

[58] A. S. E. Ojugo, P. M. Meeshley, D. J. O. Mclntyre, C. McCoy, M. Stubbs, M. O.

Leach, I. R. Hudson, J. R. Griffiths, NMR Biomed., 12,495 (1999)

51

[59] J. Liu, T. Zhang, T. Qu, L. Zhou, H. Zhang, Q. Li, J. Inorg. Biochem., 91,269

(2002)

[60] P. Luo, F. Zhang, R. P. Baldwin, Anal. Chem., 63, 1702 (1991)

[61] F. Huerta, E. Morallon, J. L. Vazquez, A. Aldaz, J. Electroanal. Chem., 38,475

(1999)

[62] E. J. Underwood," Trace Elements in Human and Nutritions", 4*, Academic press.

New York (1977)

52