Moses said: "Lord! Open my breast for me; (25) and

ease my task for me, (26) and loosen the knot from

my tongue (27) so that they may understand my

speech; (28)

CYCLIC VOLTAMMETRIC STUDY

OF SOME BIOLOGICALLY

ACTIVE DRUGS

By

ATYA HASSAN

B. Sc. (Hons), M. Sc. M. Phil.

This Thesis is submitted for the Fulfillment of the Degree of

DOCTOR OF PHILOSOPHY In Chemistry

DEPARTMENT OF CHEMISTRY

UNIVERSITY OF KARACHI

KARACHI-75270

PAKISTAN

2014

CERTIFICATE

It is certified that thesis entitled “Cyclic Voltammetric Study of Some Biologically Active

Drugs” by Atya Hassan fulfils the requirements for the award of Doctor of Philosophy (Ph.

D.) in Chemistry. To the best of my knowledge no part of the work has been submitted for

another degree or in any other institution.

Internal Examiner:

(Thesis Supervisor)

External Examiner:

Chairman:

Department of Chemistry

University of

Karachi, Karachi-75270

Dated:

ACKNOWLEDGEMENT

All praises to ALLAH, who gave me strength to accomplish this thesis and I always need

his blessing at every moment of my future life.

I am enormously grateful to my supervisor Prof. Dr. Syed Azhar Ali, for his excellent

supervision, guidance and support during my research work.

I am thankful to Prof. Dr. Iqbal Chaudhary (Director of H.E.J) and Prof. Dr. Mehboob

Muhammad for their guidance and providing facility for analysis throughout this study.

I am also grateful to the Department of Chemistry, University of Karachi for providing me

the professional environment during my study.

I express my sincere gratitude to Muhammad Zafar Iqbal, Ms. Asma Rauf, Ms. Saba Rauf,

Dr. Humera Anwar, Ms. Lubna Naz, Ambreen Abbas, Muhammad Sohail Ahmed,

Muhammad Rehan Alam, Muhammad Farhan, Farhanullah Khan and Zahid Khan who

helped me during my experimental work.

I am highly indebted to my family members specially my sisters Sufia Mehmood,

Farkhanda Jamal, Safia Hassan and my brothers Asif Hassan, Arif Hassan, Atif Hassan

and Asim Hassan for their moral support and continuous encouragement during my study.

Their tolerance, prayers and countless affection made me able to complete my thesis.

Dedicated

To

My Parents

ABSTRACT

This electrochemical study of three different biologically active compounds has been

conducted using cyclic voltammetry technique at gold electrode. Losartan Potassium is an

antihypertensive drug, while Gemifloxacin is antibacterial and Clarithromycin is primarily

bacteriostatic and also has antimicrobial effect. Cyclic voltammetric study has been

conducted by using (0.04M) Britton Robinson Buffer as supporting electrolyte with

different pH range. For Losartan Potassium pH range of B-R buffer was 8-11 while for

Clarithromycin and Gemifloxacin B-R buffer pH range (2-6) has been selected according

to the appropriate solubility of these pharmaceutical compounds. Voltammograms of all

three biologically active compounds have been recorded at six different scan rates of 20,

100, 200, 300, 400 and 500mV/s.

Different electrochemical parameters such as peak potential (Ep), peak current (Ip), transfer

coefficient (α), number of electron (nα), diffusion coefficient (D), and heterogeneous rate

constant (K0) were determined. Moreover, diagnostics tests have also been applied to define

the electrochemical properties of these compounds. Results indicate that Losartan

Potassium follows electrochemically irreversible reduction process with transfer of two

electrons involving adsorption controlled process on gold electrode. However,

electrochemical behavior of Gemifloxacin showed quasi reversible redox process with two

electron transfer and on the electrode surface some adsorption complications have been

observed. In case of Clarithromycin irreversible oxidation process with two electron transfer

has been identified and electrode processes were shown to be diffusion controlled.

These quantitative and qualitative investigations based on cyclic voltammetry technique

demonstrate that this method is very reliable, sensitive and appropriate for the

determination of electrochemical properties of different biological and pharmaceutical

compounds using gold electrode. Moreover, this technique can also be used for quality

control and pharmacokinetics studies of biologically active compounds.

CHAPTER #1

INTRODUCTION

CHAPTER #2

EXPERIMENTAL

CHAPTER #3

RESULT AND DISCUSSION

CHAPTER #4

TABLES AND FIGURES

CHAPTER #5

REFERENCES

PUBLICATION

TABLE OF CONTENTS

INTRODUCTION 1

1.1 Electrochemistry 1

1.2 Voltammetry 1

1.2.1 Cyclic voltammetry 3

1.2.2 Principal of Cyclic voltammetry 3

1.2.3 Cyclic voltammetry of electrochemical process 4

1.2.4 Study the reaction mechanism using cyclic voltammetery 6

1.2.4(a) Reversible Process 6

1.2.4(b) Irreversible process 9

1.2.4(c) Quasi – reversible reaction 10

1.2.5 Voltammetry as quantitative and qualitative tool 12

1.3 Electrode 13

1.3.1 Working electrode 13

1.3.2Reference Electrode 14

1.3.3 Auxiliary Electrode 14

1.4 Drugs 15

1.4.1 Antihypertensive drug 15

1.4.1.1. Classification of antihypertensive drugs 15

1.4.1.1(a) Diuretics 16

1.4.1.1(b)Beta blockers, alpha blockers drugs 16

1.4.1.1(c )Calcium channel blockers (CCBS) 16

1.4.1.1(d) Angiotensin converting enzyme inhibitors (ACEIS) 16

1.4.1.1(e) Angiotensin receptor blockers (ARBS) 16

1.4.1.1(f) Sympatholytic drugs 17

1.4.1.1(g)Direct arterial vasodilators 17

1.4.2 Antimicrobial Drugs 17

1.4.2.1Classification 17

1.4.2.1(a) Antibacterial 18

1.4. 2.1(b) Antifungal 18

1.4.2.1(c) Antiviral 18

1.5 Electrochemical studies of biologically active compounds 19

1.5.1 Losartan Potassium 19

1.5.1(a) Structure and physical properties 19

1.5.1(b) The mode of action 19

1.5.1(c) Pharmacological aspects 20

1.5.1(d)Adverse effects 20

1.5.1(e)Different techniques used to study the LP 20

1.5.2 Gemifloxacin (GFX) 20

1.5.2 (a) Structure and physical properties 20

1.5.2 (b)Therapeutic use of Gemifloxacin

21

1.5.2(c) Mode of action 21

1.5.2 (d)Adverse effects 22

1.5.2(e) Different techniques used for studies of Gemifloxacin 22

1.5.3 Clarithromycin (CAM) 22

1.5.3(a) Structure and physical properties 22

1.5.3(b) Therapeutic use of clarithromycin 23

1.5.3(c) The mode of actions 23

1.5.3(d) Adverse effect 24

1.5.3 (e) Different techniques used for studies of clarithromycin 24

1.6 Objective of the study 25

2. EXPERIMENTAL 26

2.1Chemicals 26

2.1 (a) Biologically active compounds 26

2.1(c) Buffer 26

2.2 Others chemicals 26

2.3 Apparatus 27

2.4 Instrumentations 27

2.5 Preparation of Stock Solutions 29

2.6 Standard Procedure 31

3. RESULTS AND DISCUSSIONS 32

3.1 Losartan Potassium 32

3.1.1 Suggested reaction mechanism 32

3.1.2 Diagnostic test for quasi reversible process 33

3.1.3 Diagnostic test for irreversible process 33

3.1.4 Effect of scan rate 34

3.1.5 Effect of concentration 35

3.1.6 Effect of pH 35

3.1.7 Transfer Coefficient 35

3.1.8 Heterogenous rate constant 35

3.1.9 Repeated cyclic voltammogram 36

3. Gemifloxacin 36

3.2.1 Proposed reaction mechanism 36

3.2.2 Diagnostic test for quasi reversible process 37

3.2.3 Effect of scan rate 37

3.2.4 Effect of pH 37

3.2.5 Effect of concentration 38

3.2.6 Heterogenous rate constant 38

3.2.7 Repeated cyclic voltammogram 39

3.3 Clarithromycin 39

3.3.1 Suggested reaction mechanism 39

3.3.2 Diagnostic test for irreversible process 40

3.3.3 Effect of scan rate 40

3.3.4Effect of pH 41

3.3.5Effect of concentration 41

3.3.6 Transfer Coefficient 41

3.3.7 Heterogenous rate constant 42

3.3.8 Repeated cyclic voltammogram 42

4. CONCLUSION 43

5. REFERENCE 132

6. PUBLICATION

1. INTRODUCTION

1.1 ELECTROCHEMISTRY

Electrochemistry is the field of chemistry and this term was used to explain the relation

of electrical and chemical effects in the late 19th and early 20th centuries [1, 2]. In

recent decades, progressive developments in the field of electrochemistry have

introduced various fast, sensitive, inexpensive and reliable analytical methods with

wide range of applications such as industrial electrolysis, electroplating, batteries, fuel

cells, electrochemical matching, bioelectrochemistry and biosensor [3-7]. Previously,

electroanalytical methods have been classified as Interfacial and bulk methods [8]. Bulk

methods, are based on phenomena that occur in the bulk of solution, while interface

methods demonstrate the phenomena which take place between electrode surface and

thin layer of solution. Furthermore, classification of electroanalytical methods are also

presented in (Fig. 1). Among the different techniques voltammetry, conductometry and

potentiometry are the most popular techniques with variety of applications. In this

chapter brief introduction of cyclic voltammetry (CV) technique and some biologically

active compounds are given.

1.2 VOLTAMMETRY

Voltammetry technique has been developed in 1922 from polarography technique by

the Czech chemist Jaroslav Heyrovsky [1, 8, 9]. Previously, this method had some

limitations which caused difficulties during routine analytical process. However, in

1960s and 1970s significant advances in theory, methodology and instrumentation of

this technique have drawn the attention of organic, inorganic, physical and biological

chemists for variety of purpose like oxidation reduction process in various media,

adsorption processes on surface, thermodynamics, reaction mechanism, qualitative and

quantitative determination of metal ions, inorganic anions, organic compounds [10] and

pharmaceutical compounds [11, 12]. There are different categories of voltammetric

methods which are mentioned below:

Figure:1 Brief summary of common electroanalytical Methods.

Cyclic voltammetry

Potential Step voltammetry

Linear Sweep voltammetry

Anodic Stripping voltammetry

Cathodic Stripping voltammetry

Adsorptive Stripping voltammetry

Alternative current voltammetry

Polarography

Rotated electrode voltammetry

Normal pulse polarography

1.2.1 Cyclic Voltammetry

Cyclic voltammetry is considered as sensitive, selective and versatile technique [12]. It

is used for the investigation of electrochemical behavior of a system as reported by

Randles in 1938 [13]. In this technique current flow between the working electrode and

a counter electrode and this current is measured under the control of a potentiostate. The

voltammogram is recoded on a recorder which determines the peak potentials (Ep) and

peak current density (IP) [2, 7]. It gives sufficient information about the thermodynamics

of redox reaction, kinetics of heterogeneous electron-transfer reactions, adsorption or

diffusion processes and coupled chemical reactions [14].

1.2.2 Principle of Cyclic Voltammetry

Working principal of cyclic voltammetry is based on sweeping of the electrode potential

which lies between two limits at a known scan rate. Working electrode is set at potential

(E1) at which no electrode reaction takes place. Potential is swept linearly at a rate (υ)

between two limiting potentials (E1) and (E2) during the measurement. The same scan

rate is usually selected for the forward and reverse rate. The corresponding current is

noted or recorded as a function of the varying potential and when the applied potential

is decreasing, cathodic scan represents negative sign while as the applied potential is

increasing anodic scan shows positive sign [17-20].The recorded voltammograms

provide useful information about the nature of reaction which can be used to explain

the kinetic parameters [21].

1.2.3 Cyclic Voltammetry of Electrochemical Process

Generally, when the potential is applied, concentration of oxidized (O) and reduced (R)

species on surface of electrode is different from the bulk of solution. To equalize the

concentration, analyte moves from bulk of solution to electrode surface through the

diffusion process [2, 22]. If electron transfer is fast (i.e. the electrode reaction is

reversible) then the current is determined by the rate of mass transfer of analyte to the

electrode. The difference in concentration between the solution near the electrode

surface and that far from it is determined by the value of the applied potential via the

Nernst equation [13, 14].

O + n e R

E = Eo + RT/nF ln [Oxi /Red] --------------------- (1.1)

E= Potential (V)

Eo= Standard electrode potential

(V) n = Number of electron

R= Gas constant (J/K/mol)

T= Absolute temperature (K)

F= Faraday constant (C/mol)

Oxi/Red= [Oxidize species]/ [reduce species]

By putting the values of R (8.314 J/K/mol), F (96500 C/mol) and temperature (298K),

equation (1.1) turns into:

E = Eo + (0.0591/n) log [Oxi /Red] ------------- (1.2)

The volume of solution in which concentration gradient develops is called as diffusion

layer [23]. The rate of diffusion flux can be expressed by the first Fick`s law [8, 13]

Ji = Di -------------- (1.3)

Where flux (J) of species (i) concentration (ci) in direction(x), concentration gradient

and Di is proportionality factor between flux and concentration gradient.

Thus, the flux is converted into current i by using Faraday's Law, which states that

potential is directly propotional to diffusion rate of oxidized species to the electrode

surface:

i= nFAJ ------------- (1.4)

Where, n = number

of electrons

F= Faraday’s constant (96500C/mol)

A = area of the electrode surface (cm2)

Ji = flux of the species i (mol/cm2/s)

In cyclic voltammetry, when potential is applied, the oxidized species moves toward the

electrode surface and causes the higher concentration gradient resulting more flux and

higher cathodic current. Continuous supply of more negative potential causes the

depletion of oxidized species which finally becomes zero. In contrast, when the

concentration gradient decreases, flux to the surface would become less and as a result

current will begin to decrease. On reversing the voltage scan depleted layer of the

oxidized species begins to raise so current decreases further. Consequently, anodic

current begins to dominate at specific region. In case of reduced species same profile is

observed [8].

1.2.4 Study of reaction mechanism using cyclic voltammetry

In 1964 Nicholson and Shain have conducted quantitative simulations of cyclic

voltammetry and introduced the field of electrochemistry in different way [24].

Depending on the shape of cyclic voltammograms three different electrochemical

reactions can be explained [8,13, 25].

Reversible process

Irreversible process

Quasi- reversible process

1.2.4 (a) Reversible Process

A cyclic voltammogram for reversible process can be observed when oxidized (O) and

reduced (R) species both are stable and the kinetics of the electron transfer process is

fast [17], So that potentials and scan rate of the electron transfer process on the surface

are in equilibrium (a relative term based on comparison of the rate of a forward and

backward electron transfer reaction with prevailing rate of diffusion of material to and

from the electrode surface) so, that the surface concentration follow the Nernst equation

[1, 13].

dco = Do d2co …………….(1.5)

dt dx2

dcR = DR d2cR ……....…...(1.6)

dt dx2

“O” initially presents in solution and if Do = DR = D, the initial and boundary condition

are.

∞

t = 0, x> 0, co = co and cR = 0

t > 0, x = ∞, co = c∞o and cR= 0

t > 0, x = 0, D dco + D dcR

dx dx

co nF (E – Eeө) = exp

cR x=0 RT

-I = nFD dCo

dx x = 0

For a sweep rate of υ

0 < t < λ E = E1 –υt

t > λ E = E1- 2υλ + υt

E = initial Potential λ = time at

which sweep is reversed

The peak current in a cyclic voltammogram having only one species is defined by

Randles Sevcik equation by [7, 13. 26].

Ip = - 2.7×105 n3/2 AD1/2Cυ1/2 ------------- (1.7)

Where

Ip = peak current (µA)

A = area of the electrode surface (cm2)

D = diffusion co-efficient of the electroactive specie

(cm2s-1) C = concentration (mol/cm3) υ = scan rate

(V/s)

Figure: 2 Voltammogram of reversible process.

Cyclic voltammogram of reversible process is evaluated by applying the following

diagnostic test at room temperature [7, 13].

Table-1.1: Diagnostic test for reversible process

S.No Diagnostic test for reversible process at 25oC.

1 ∆Ep = (Epa –Ep

c) = 59/n mV at all sweep rates

2 │Ep-Ep/2│ = 59/ n mV

3 Ipa/Ipc = 1 (at all sweep rates).

4 Ip α υ1/2 ( υ= sweep rate)

5 Ep is independent of υ

6 At potential beyond Ep, I-2 α t

Potential (V)

1.2.4 (b) Irreversible process

In an irreversible process the surface concentration of “O” species changes more slowly

with potential and when the surface concentration becomes zero, the concentration

profile for “O” is less steep. The flux to the surface become lower and no reverse peak

is obtained. The value of Epc is variable with scan and the rate of electron transfer

process at all scan rates is higher than mass transfer. The high potential sweep rate which

increases the peak separation leading the characteristic of the voltammogram of the

reversible process. In some cases of irreversible process reduction peak shifted

catholically [7, 13].

Epc = K – 2.3RT / 2αc nα F log υ ---------- (1.8)

In case of irreversible process the shape factor |Ep-E1/2| becomes different comparative

to reversible case. This is shown in equation.

│Ep-Ep/2│= 48 αcnα mV ----------- (1.9)

Potential (V)

Figure: 3 Voltammogram of irreversible process.

Following are the diagnostic tests justify the irreversible process [7, 13].

Table-1.2: Diagnostic test for irreversible process at 25oC

S.No Diagnostic test for irreversible process at 25OC

1. No reverse peak

2. Ipc α υ1/2

3. Epc shifts - 30/ αcnα mV for each decade increase in υ

4. │Ep-Ep/2│= 48 αcnα mV

1.2.4 (c) Quasi – reversible reaction

The voltammogram of quasi reversible process represents a large peak to peak

separation as compared to reversible process. In this process peak current is not

increased linearly to the scan rate and difference in peak potential (ΔEp) is greater than

59/nmV and it increases with increasing scan rate (υ). Moreover, in this process current

is controlled by

both mass and charge transfer kinetics [7] [13] [27].

Potential (V)

Figure: 3 Voltammogram of quasi reversible process.

Nicholson has work on measurement of electrode kinetics in CV. He has explained

Ψ = [(Do/Dr) /2 Ko [Л Do υ(nF/RT)]1/2 ……………. (3.14)

Here Ψ is dimensionless parameters. He has also given the values of Ψ for different

vaules of n (Epa-Epc)=n ∆Ep for 0.5 at 25oC

Table: 1.3 Variation of the difference between anodic and cathodic peak potentials with

the degree of reversibility, express as Ψ (=ΛЛ1/2, see equation 3.14), assuming = 0.5

[8]

Ψ n(Epa-Epc) mV/s Ψ n(Epa-Epc) mV/s

20 61 0.38 117

7 63 0.35 121

6 64 0.26 140

5 65 0.25 141

3 68 0.16 176

2 72 0.14 188

1 84 0.12 200

0.91 86 0.11 204

0.80 89 0.10 212

0.75 92 0.077 240

0.61 96 0.074 244

0.54 104 0.048 290

There are some criteria for the justification of quasi reversible process which is given

below [7], [13],

Table-1.4 Diagnostic test for quasi-reversible process at 25oC

S.No Diagnostic tests for quasi-reversible process at 25OC

1 │ Ip │ increases with υ1/2 but not proportional to it

2 │Ipa / Ip

C│ = 1 provided αc = αa = 0.5

3 ΔEp is greater than 59/ nmV and increases with increasing υ

4 Epc shift negatively with increasing υ.

1.2.5 Voltammetry as quantitative and qualitative tool

Cyclic voltammetry has become most popular technique and used in different discipline

of chemistry for variety of purpose. This method has been used to investigate the

quantitative determination of organic and inorganic compounds in aqueous and non

aqueous solutions. Voltammetry can also be employed for qualitative analysis by

determining the standard electrode potential for a specific redox reaction. As a result of

redox reaction current flows through the cell. This current is proportional to the

concentration of the redox species in the bulk solution. It implies that voltammetry is

also useful for quantitative analysis of test substance in the solution [7-9] based on

Ilkovic equation

id = 607 n C D1/2m 2/3 t1/6 ---------------(1.9)

Where n = Number of

electrons

C = Concentration (mmol)

D = Diffusion coefficient in (cm2 s-1)

m = Mass flow of Hg in (mg/sec)

t = Drop time (s)

1.3 ELECTRODES

In voltammetry technique electrodes have significant importance to investigate the

electrochemical phenomena. There are three electrodes which have been used in

voltammetric cell assembly [9, 10].

Working electrode

Reference electrode

Auxiliary electrode

1.3.1 Working Electrode

Working electrode is also called as indicator electrode at which reduction or oxidation

of analyte takes place and its potential varies with time [9]. This electrode is referred to

as microelectrode due to smaller surface area and it exhibits greater polarizing effect [7,

8]. Working electrode either made of solid material like platinum (Pt), silver (Ag), gold

(Au), and glassy carbon (GCE) as shown in (Fig. 2) or may be semi liquid like mercury

(Hg).

The solid (metallic) electrodes such as platinum (Pt), gold (Au) and modified glassy

carbon (GCE) have wide range of potential [11, 16, 17] low background current, rich

surface area, wide range of sensing application, high reactivity and conductivity. These

advantages have made solid electrodes more efficient as compared to mercury electrode

[18]. The solid electrodes are electrochemically modified electrodes [19] and have

effective application to investigate the dosage forms of biologically active compounds

or drugs. Moreover, these electrodes are also used for qualitative and quantitative

determination of pharmaceutical and biological samples [19].

1.3.2 Reference Electrode

In voltammetric technique, saturated calomel electrode (SCE) and silver-silver chloride

electrode (Ag/AgCl) are used as reference electrode. Potential of these reference

electrodes remains constant and used to observe the change in potential of the working

electrode [10-14].

1.3.3 Auxiliary Electrode

Auxiliary electrode is also known as counter electrode. It is usually made of inert

material such as platinum (Pt) and gold (Au). It has relatively large surface area. The

process which takes place in electrochemical cell is not affected due to this electrode

and it only provides the current required by working electrode [1,9]. In this experiment

platinum (Pt) wire has been used as counter electrode (Fig. 2)

Figure 5 Working and Counter Electrodes

1.4 DRUG

The drug is a chemical constituent which may have medicinal, intoxicating,

performance enhancing and other effects when taken or ingested by human body. Drugs

are used for the treatment of various physical and mental disorders including cure and

prevention [28,29]. It can be given either for a short duration or on a regular basis

depending on the nature of disease [28].

Drugs are classified into different groups according to their chemical characteristics,

structure and mechanism of action. A detail description of some major categories of

drugs which have been used in the present work is given below.

1.4.1 Antihypertensive Drugs

Term “hypertension” can be defined as high blood pressure and also called arterial

blood pressure. However, blood pressure is the pressure or force which pushes the blood

up against the wall of blood vessel. This increased blood pressure can cause disruption

or damage of organs and other several illnesses such as kidney failure, aneurysm and

heart attack [28, 29]. Hypertension may be of the two types. First, is essential

hypertension caused by a disorder of unknown origin affecting the blood pressure

regulating mechanisms and second type is associated with other diseases [30]. The

antihypertensive drug therapy for hypertension was made available in early 1950s. The

drugs related to hypertension have been classified into seven different groups according

to their mechanism of action.

1.4.1.1 Classification of Antihypertensive Drugs

Diuretics.

Beta adrenergic blockers.

Calcium channel blockers.

Angiotensin converting enzyme inhibitors.

Angiotensin receptor blockers.

Sympatholytics and adrenergic blockers.

Direct arterial vasodilators.

1.4.1.1(a) Diuretics:

Diuretic drugs are used for the treatment of hypertension which lowers the blood

pressure by depleting the body sodium stores. Initially they reduce the blood pressure

by reducing blood volume and cardiac output. It can also be given in combination with

other antihypertensive drugs to produce synergetic effect and increase the effectiveness.

Hydrochlorothiazide or chlortalidone are the thiazide diuretics used for the treatment of

hypertension [31].

1.4.1.1(b) Beta blockers, Alpha blockers drugs

These drugs are also used for the treatment of hypertension and act on sympathetic

nervous system to controls blood pressure. It lowers the blood pressure by relaxing

blood vessels and as well as decreasing the rate and force of contraction of the heart.

[32].

1.4. 1.1(c) Calcium channel blockers (CCBs)

These drugs are very effective for the treatment of hypertension. They cause relaxation

blood vessels directly. They are used sometimes as first line therapy and more often as

second or third line therapy with diuretics or ACEIs or ARBs. They are effective in

preventing stroke [34]. However, high dose of short acting calcium blockers increased

risk of myocardial infection (rupture).

1.4.1.1(d) Angiotensin converting enzyme inhibitors (ACEIs)

ACE inhibitor is also an antihypertensive drug and used for the treatment of

hypertension (elevated blood pressure) and congestive heart failure. This drug inhibits

the production of angiotensin II and relaxes the blood vessels. It is given specially to

those patients who have diabetic mellitus and chronic kidney disease (CKD) [34].

1.4.1.1 (e) Angiotensin receptor blockers (ARBs)

ARBs block the actions of angiotensin II in the tissues. It is given with thiazides and

loop diuretics due to its effectiveness and beneficial interactions. It is effective in

preventing stroke and do not cause an irritant cough or the rare danger of swelling of

the lips, tongue and throat.They may have an additional action to diminish the

progression of Alzheimer’s disease in those patients having early dementia [32,33,34].

1.4.1.1(f) Sympatholytic drugs

Sympatholytic drugs act in the brain and decrease the drive to the sympathetic nerves.

The effects are similar to beta blockers. However, often rather worse, spectrum of

adverse effects due to their action in the brain. [32,33,34].

1.4.1.1(g) Direct arterial vasodilators

Vasodilator act by causing the relaxation of smooth muscles. It increases the plasma

rennin concentration resulting in sodium and water retention and lowers the

hypertension. Hydralazine and minoxidil are the vasodilator used for the treatment of

hypertension [33, 34].

1.4.2 Antimicrobial Drugs

Antimicrobial drugs are used for the prevention and cure of microbial infections. The

development of antimicrobial drugs began in the late 1800's with Paul Erlich, a German

scientist, who discovered that arsenic compounds were an effective treatment for

syphilis. In 1928, Sir Alexander Fleming accidentally stumbled upon the discovery of

the wonder drug, penicillin. As he was inspecting a plate of Staphylococcus aureus

contaminated with the mold penicillium. He noticed that the mold had inhibited the

growth of the bacterial colonies. He isolated the compound later which is called

penicillin [32,36].

1.4.2.1 Classification of Antimicrobial Drugs

Antimicrobial drugs are classified in three different types.

a) Antibacterial

b) Antifungal

c) Antiviral

Antimicrobial drugs can also be classified on the basis of their spectrum of activity.

A narrow or broad spectrum explains the effectiveness of antimicrobial drugs.

Broadspectrum drugs are effective against many types of microbes and tend to have

higher toxicity to the host. However, narrow-spectrum drugs are effective against a

limited group of microbes and exhibit lower toxicity to the host [35].

1.4.2.1 (a) Antibacterial drugs

Antibacterial or antibiotics are commonly used for the treatment of bacterial infections.

The side effects of antibiotics in human and animals are very little. However, number

of gut flora can be suppress due to long term use of some antibiotics and causes the bad

impact on health [36, 37]

1.4.2.1(b) Antifungal drugs

Antifungal are generally used to prevent the growth of fungi. These drugs are given in

case of different infections like athlete's foot, thrush and ringworm. It is effective to kill

off the fungal organism with but due to the similarity fungal and human cells at the

molecular level, it is difficult for an antifungal drug to find a target to attack and

consequently, these drugs have side effects which can be life-threatening if the drug is

not used properly [38].

1.4.2.1(c) Antiviral drugs

Antiviral drugs are used specifically for treating the viral infections. Specific antiviral

are used for specific viruses and relatively harmless to the host. Therefore, it can be

commonly used to treat infections [39].

1.5 ELECTROCHEMICAL STUDIES OF

BIOLOGICALLY ACTIVE COMPOUND

1.5.1 Losartan Potassium

1.5.1 (a) Structure and Physical Properties

Losartan Potassium is a white crystalline fine powder, compound soluble in aqueous

medium. It is soluble in other organic solvents like methanol, ethanol, chloroform, ethyl

acetate and n-hexane [32, 33, 35-38, 40]. The I.U.P.A.C name of losartan potassium is

monopotassium salt of [2- butyl -4- chloro -1-[2,-(1H-Tetrazol-5-yl) 1, 1-biphenyl}-4yl]

methyl-1H-imidazole-5-yl) methanol [14], [37, 41-45]. The Molecular mass of Losartan

potassium is 461.01 g/mol and its decay time is 1.5 to 2 hr. It is metabolized in liver

[46].

Figure.6 Structure of Losartan Potassium

1.5.1(b) The mode of action

Administration of Losartan Potassium causes reduction of total peripheral opposition

and cardiac venous return. As a result angiotensin II releases aldoestron and the pressure

of blood become reduced. This drug also gives renal protection for the type II diabetic

patients with proteinuria and stroke prevention [47].

1.5.1(c) Pharmacological aspects:

Losartan Potassium belongs to the class I antihypertensive drug and angiotensin II

receptor antagonist [45]. Clinical use of Losartan Potassium is similar to angiotensin

converting enzyme inhibitors (ACEI). In contrast to ACEIs, Losartan Potassium has the

advantage of not causing cough and angioedema [48]. It is effectively used for the cure

of hypertension and cardiac disease either singly or in combination with diuretics [30].

If blood pressure is not reduced singly with Losartan Potassium, it is used with low

dosage of hydrochlorothiazide [44].

1.5.1(d) Adverse effects

Adverse effect of Losartan Potassium is similar to ACE inhibitors. It has fetotoxic

effects and should not be used for treating hypertension in pregnant women [44].

1.5.1(e) Different techniques used to study the Losartan Potassium:

Literature review reveals that different techniques such as UV spectrophotometric

method [30, 50], HPLC method [42]. Reverse phase HPLC method [14, 43], cathodic

adsorptive stripping voltammetry [41, 45], techniques have been used for the

identification, simultaneous determination and validation of Losartan potassium due to

its pharmacological effect as reported an effective antihypertensive drugs and used for

the treatment of moderate or severe hypertension and heart disease either singly or

sometimes with diuretics [29, 32, 35, 45, 47, 48, 51-57].

1.5.2 Gemifloxacin (GFX) 1.5.2 (a) Structure and Physical Properties

This biologically active compound is yellow, crystalline and soluble in water. The

molecular mass is 389.381gm and I.UP.A.C name of Gemifloxacin (GFX) is [(R, S)-7-

[4Z)-3-(aminomethyl)-4-(methoxyimino)-1-pyrolidinyl]-1-Cyclopropyl-6-fluoro-1,4

dihydro-4-oxo-1,8-napthyridin-3-carboxalic acid mesylate [58- 68].

Figure.7 Structure of Gemifloxacin

1.5.2 (b) Therapeutic use

Gemifloxacin (GFX) is a kind of flouroquinolones which belongs to the class of

antibacterial drugs [67-71, 99] with enhanced affinity towards bacterial isomerase IV

[72]. After the approval of FDA (Food drug administration) of infections of the

respiratory and genitourinary [68, 69], GFX can also be used for treatment of

pneumonia and acute bacterial exacerbation of chronic bronchitis. This compound has

a wide-range of therapeutic effect against gram positive and gram negative bacteria [59-

61, 74]. It is in particularly active against penicillin microlide and quinolone resistant

streptococcus pneumonia [75-77]. Moreover, GFX has also potent activity against the

other major pathogens involved in respiratory tract infections including haemophilus,

influenza, and moraxella and catarrhalus. [78]. However, it is also used for treatment of

urinary tract infection and bronchitis [79].

1.5.2 (c) The mode of action

This drug enters into bacterial cells through passive diffusion process and rapidly

inhibits the bacterial DNA replication by inhibiting the bacterial DNA enzyme gyrase.

This enzyme helps in super coiling of DNA to compact the chromosomes into the

bacterial cell. The activity of enzyme topoisomerase IV can also be prevented in the

gram +ve bacteria [59-61, 74] by this drug which is responsible for the separation of

replicated DNA chromosomes [80].

1.5.2 (d) Adverse effects

Gemifloxacin is well tolerated with most side effects being mild and severe effects being

rarely. When other antibiotic drugs used with Gemifloxacin, it produced some serious

adverse effects. These effects are produced by over dosing of the flouroquinolones

including CNS toxicity, cardiovascular toxicity, tendon/ articular toxicity, and renal

failure [81].

1.5.2(e) Different techniques used to study the Gemifloxacin (GFX)

The pharmacological and other analytical aspects of Gemifloxacin (GFX) have been

studied by various analytical methods. These include high performance

chromatography [82], liquid chromatography resolution microchip electrophoresis

method [79-85], direct liquid Chromatographic separation [76], tandem mass

spectroscopy (LS, MS), spectrophotometric methods [86] and reversed phase

chromatography [87-93]. A fluorometric method [94] was also reported for the

determination of GFX in plasma, simultaneous determination of GFX and diuretics in

bulk and in human serum by RPHPLC [95]. Recently, volatmmetric determination

using screen print carbon [67], sensor/ biosensor electrode [96, 97], and multiwall

carbon nanotubes modified glassy carbon electrode [98] have also been used to explain

the pharmaceutical, electrochemical and biological role of GFX [81].

1.5.3 Clarithromycin (CAM)

1.5.3 (a) Structure and Physical Properties

Clarithromycin (CAM) is semi synthetic antimicrobial 14 –membered macrolide

compound exhibiting a broad in vitro antibacterial spectrum [99]. It is a white crystalline

solid and partially soluble in water. Its molecular formula is C38H69NO13 and molecular

weight is 747.96 gm. Its common name is Clarithromycin (CAM). Its IUPAC name is

(3R, 4S, 5S, 6R, 7R, 9R, 11S, 12R, 13S, 1S)-6-{[(2S, 3R, 4S, 6R) -4-

(dimethylamino)3hydroxy-6-methyloxan-2-yl]oxy}-14ethyl, 12, 13dihydroxy-4{[(2R,

4S, 5S, 6S)-5hydroxy-4-methoxy-4, 6-dimethyloxan-2-yi]oxy}-7-methoxy-

3,5,7,9,11,13-hexamethyi1-oxacyclotetradeceane,10 di-one [100].

Figure.8 Structure of Clarithromycin

1.5.3 (b) Therapeutic use of Clarithromycin (CAM)

Clarithromycin (CAM) is partially synthetic macrolide antibiotic [99-101]. It has good

stability in gastric acid and isolated from erythromycin [99,102]. It acts as anti-infective,

gastrointestinal and antibacterial agent. Recently, it is commonly used for therapy [103]

with various clinical benefits such as better oral bioavailability, with broad spectrum

activity, higher tissue concentration and improved tolerability. The combining effect of

Clarithromycin with a variety of other drugs for the treatment and preventation of

disseminated mycobectrium avium-intracellular complex (MAC) infection in patients

with Immune deficiency syndrome (AIDS) is also under investigation [22, 104-106].

1.5.3 (c) The mode of action

Clarithromycin exerts an antibacterial action by binding to the 50S ribosomal subunit

of susceptible organisms which inhibits protein synthesis through translocation of

aminoacyl transfer – RNA. It is poor inducer of messenger -RNA so, it is unable to

activate the methylase enzyme. It thereby retains activity against inducible bacteria [99,

107].

1.5.3 (d) Adverse effects

In case of Clathromycin no toxicity was found during clinical trials. This antimicrobial

drug has proven to be well tolerated. The common adverse effects have been mild- to-

moderate such as genital infection (GI) and irritation [99].

1.5.3(e) Different techniques used to study the Clarithromycin (CAM)

Reported investigations have indicated that variety of techniques related to qualitative

and quantitative determination of Clarithromycin (CAM) has been used. For instance

UV- spectroscopy [108,109], electrochemical methods [110, 111], sensitive liquid

chromatography (SLC) technique were used for the analysis of Clarithromycin (CAM)

in human serum [112]. In addition, high performance liquid chromatography (HPLC)

with electrochemical detection (ED) has also been applied for CAM. Recently

determination of electrochemical behavior of Clarithromycin by single- sweep

oscillopolarography [113] and electrochemical activity of (CAM) were reported at gold

electrode with 0.05 M NaHCO3 [114].

1.6 OBJECTIVES OF THE STUDY

Aim of this work is to focus on the application of cyclic voltammetry technique for

the determination of chemical nature of different biologically active drugs.

For the electrochemical investigation of biologically active compound, gold as test

electrode has been selected to understand its applicability as non toxic test electrode

as compared to the other mostly used electrodes like a Hg dropping electrode and

carbon paste electrode.

Determination of electrochemical parameters such as peak potential (Ep) ,half peak

potential (Ep1/2), Peak Current (Ip) , transfer coefficient ( ) , diffusion coefficient(D),

number of electron transfer (n) and heterogeneous rate constant (K0). These

investigations would also be the helpful to evaluate the pharmacological effect of

other different biologically active compounds in vivo and vitro studies.

2. EXPERIMENTAL

In the present study electrochemical investigation of some selected biologically active

compounds has been carried out by cyclic voltammetry (CV). Details of chemicals and

methodology are given below:

2.1 CHEMICALS

The purity and source of the reagents used in the present research work are as under.

Analytical grade reagents were used in the present research work.

2.1(a) Biologically active compounds

In the present research wok active ingredients of Losartan Potassium (Novance,

pharmaceutical grade), Gemifloxacin (M.S.D pharmaceutical grade) and

Clarithromycin (M.S.D Pharmaceutical grade) were used.

2.1(b) Buffer

Britton Robinson buffer was also used as supporting electrolyte for the electrochemical

studies of biologically active drugs. Different pH range of Britton Robinson (B-R

Buffer) buffer was prepared in laboratory by using the following reagents. Boric Acid

(E. Merck), Acetic Acid (BDH Analar) and Phosphoric acid (E. Merck) were used for

the preparation of buffer.

2.2 OTHERS CHEMICAL

2.2(a) Sodium Hydroxide

Standard solution of Sodium hydroxide (NaOH) (E. Merck) was used to maintain the

pH of B-R buffer (supporting electrolyte). The standardization of sodium hydroxide

was carried out by Oxalic acid (H2C2O4.2H2O) (E. Merck) in which Phenolphthalein

(E.

Merck) was used as indicator.

2.2(b) Chromic acid:

Chromic acid (H2CrO4) solution was prepared as described in literature [10] used for

the cleaning of apparatus and counter electrode (platinum electrode) surface. The

glasswares were rinsed with chromic acid solution and thoroughly washed with distilled

water several times and kept in oven at 120oC for drying.

2.2(c) Potassium manganate

Potassium manganate (KMnO4) (Merck Extra Pure) was used for the re- distillation of

distilled water.

2.2(d) Argon gas

Argon gas (Pakistan Oxygen Ltd. 99.99% pure) was used in degassing to achieve an

inert atmospheric condition or removal of dissolved oxygen.

2.3 APPARATUS:

2.3(a) Glass ware

A-grade Pyrex glassware (Pyrex France) such as volumetric flasks, pipettes, measuring

cylinders, cell vial glass and beakers were used in this experiment.

2.4 INSTRUMENTATION

2.4 (a) Cyclic voltammetry (CHI-700c)

Cyclic voltammetry (CHI-700c series) computerized electrochemical instrument was

used during this study. This instrument has three major components

Electrochemical cell

Potentiostate

Recorder

The electrochemical cell is the place where, an electrochemical process takes place. The

three different electrodes, such as working electrode (test electrode), calomel electrode

(reference electrode) and auxiliary electrode (counter electrode) were dipped in the

electrochemical cell in the presence of analyte. The two holes are present at the top of

the electrochemical cell. One is connected with the purging tube and other is linked with

blanket tube. The purposes of these tubes are to provide inert atmosphere during

electrochemical analysis and to avoid the interference of dissolved oxygen. The cell vial

is filled with appropriate volume of test solution and connected with the electrode

assembly which is attached with potentiostate and voltammograms were recorded in the

recorder [115].

2.4(b) Selection of electrodes

In the present research work gold (Au), platinum (Pt) and saturated calomel electrode

(Hg2|Hg2Cl2) were used as working, counter and reference electrode respectively.

2.4(c) Polishing of gold electrode

The surface of the gold electrode becomes dull due to applying scan repeatedly.

Therefore, it is necessary to re-polish the surface of the gold electrode prior to the further

experiment.

2.4(d) pH-meter

A pH meter (Jenvay –3510) was used to adjust pH of the solution.

2.4(e) Electrical balance

Electrical balance (Mettler College 150, Germon) was used for weighing the analyte for

their appropriate concentration.

2.4(f) Conductometer

Conductivity meter (Romania) HANNA (HI-8633) was used to record the conductivity

of distilled water.

2.4(i) Electrical oven

An electric oven (Model - Memmert TV 60U, 760151) 854, Schwabach, was used to

dry the glassware.

2.4(j) Sonicator

Sonicator, Elma Ultrasonic (LC- 30 H) was used for the dissolution of analytes at high

frequency of sound and for removal of trapped O2 gas.

2.4(k) Magnetic stirrer

Magnetic stirrer (Germon) IKA- Combimag RCH was used in the present research

work.

2.4(l) Distillation apparatus

BIBBY (model W 14S, Bibby, England) was used for the distillation of double distilled

water.

2.5 PREPARATION OF THE STOCK SOLUTION

2.5.1 Preparation of buffer

Britton Robinson buffer of different pH range (2-11) which was freshly prepared by

using stock solution of the following reagents.

2.5.1(a) Acetic acid

A stock solution of acetic acid (0.04 M) was prepared by taking 1.176 ml in 500dm3

volumetric flask. This flask was filled up to the mark with double distilled water.

2.5.1(b) Boric acid

A stock solution of boric acid (0.04 M) was prepared by weighing 12gm of boric acid

and dissolved in 500dm3 volumetric flask which was filled up to the mark with double

distilled water.

2.5-1(c) Phosphoric acid

A stock solution of phosphoric acid (0.04 M) was prepared by taking 1.4 ml of

(14.57M ) in 500 dm3 volumetric flask which was filled up to the mark with double

distilled water. 2.5.1.1 Procedure for the preparation of Britton Robinson buffer

(B-R buffer)

Equal volume of 0.04M acetic Acid, boric Acid and phosphoric acid were mixed in 500

ml volumetric flask and shaked well. Desire pH of this mixture or buffer solution was

adjusted by pH meter using standard 0.2M NaOH solution.

2.5.1.2 Preparation of the stock solutions of biologically active compounds

1. Stock solution of Losartan Potassium (4mM) was prepared by dissolving

0.1844 gm of Losartan Potassium in 100ml volumetric flask with B-R buffer

(used as supporting electrolyte) at 30±1oC. Same procedure was repeated for the

preparation of the solution with different pH of the B-R buffer solutions.

2. Stock solution of Gemifloxacin (4mM) was prepared by dissolving 0.1557gm

in 100 ml volumetric flask which was filled up to the mark with B-R buffer at

30±1 oC. Same procedure was repeated for the preparation of the solution with

different pH of the B-R buffer solutions.

3. A stock solution of Clarithromycin (4mM) was prepared by dissolving 0.298gm

in 100 ml volumetric flask which was filled with 100ml of B-R buffer (used as

supporting electrolyte) at 30±1 oC, Same procedure was repeated for the

preparation of the solution with different pH of the B-R-buffer solutions.

2.6 STANDARD PROCEDURE:

2.6-1 Determination of base line:

Residual or back ground currents were estimated in the determination of base line in

each supporting electrolyte to minimize the effect of non faradic contribution in total

current. This was done before each cyclic voltammogram taken either in quantitative or

qualitative studies. For this purpose the supporting electrolyte was taken in

electrochemical cell and the three electrode assembly was placed. Argon (99.99%) gas

was purged for 20 minutes to have inert atmosphere. The solution was stirred for three

minutes after that the cell stand was lifted up to keep the cell in thermostat for constant

temperature. After several adjustments related to current sensitivity, initial and final

potential, voltage scan rate and then potential was applied. Finally, voltammogram was

recorded on recorder as base line.

2.6-2 Determination of cyclic voltammogram of sample

After recording the base line in B-R buffer used as supporting electrolyte, the

electrochemical cell assembly was rinsed thrice with the solution of these analyte being

prepared in the same supporting electrolyte. After this 10cm3 of analyte was transferred

to the cell and ensured the removal of air bubble from the surface of gold electrode and

adjusted the electrode assembly in the cell. This procedure was similar as followed for

the base line determination. Voltammograms were recorded at different scan rates like

20,100,200,300,400 and 500 mV/s. Same procedure was repeated with other biological

active compounds.

3. RESULTS AND DISCUSSIONS

Present study has been conducted to explain the electrochemical properties of three

biologically active compounds by using CV technique. Generally, various electrochemical

parameters such as peak potential (Ep) and peak currents (Ip) were recorded. Other

parameters like diffusion coefficient (D), transfer coefficient (α) and nature of reaction were

also determined for these compounds.

3.1 LOSARTAN POTASSIUM (LP)

Cyclic voltammograms of Losartan Potassium have been recorded in (0.04M) B-R buffer

within pH range (8 to 11). This pH range has been selected because of appropriate solubility

of analyte in basic medium. Voltammograms were recorded at 20, 100, 200, 300, 400 and

500mV/s scan rates by using gold electrode vs. calomel electrode in supporting electrolyte

at 30±1oC.

The voltammogram of Losartan Potassium represented two cathodic waves at all six applied

scan rates within the range of pH 8-10 at 0 to -1.0 V potential (Fig. 1-2). which indicates

the sample is being reduced with transfer of two electrons at the surface of gold electrode.

3.1.1 Suggested reaction mechanism

The structure of Losartan Potassium molecule contains imidazole and tetrazolyl groups.

The imidazole group can be reduced in organic media at ca.-1.77 V while tetrazolyl group

can be reduced in alkaline media [67]. As the CV profiles of Losartan Potassium were

recorded in alkaline medium (pH 8 to 10) which showed two cathodic waves, indicating

the occurrence of reduction process due to addition of two electrons with the removal of

two protons at C═N in the tetrazolyl group which is shown in equation (3.10) as described

in the reported work [40, 119].

+2 e + 2 H + ------ (3.10 )

Suggested Reaction Mechanism of Losartan Potassium

The absence of anodic peak and presence of two cathodic peaks within the range of pH 810

at each scan rate demonstrate that the reaction may be irreversible. Therefore, the diagnostic

tests were performed for Losartan Potassium to verify the nature of electrochemical process.

3.1.2 Diagnostic test for quasi-reversible process

In case of Losartan Potassium peak current (Ip) is proportional to square root of scan rate

(υ1/2) and at pH 8-10 no reverse peak was observed (Fig. 13-16). However, at pH 11 a

reverse peak was observed although the ratio of Ipa/Ipc was not equal to 1 and peak

separation ∆Ep value is less than 59mV/n which is against the quasi reversible criteria.

3.1.3 Diagnostic test for irreversibility

CV profiles of Losartan Potassium showed no reverse peak in B-R buffer in 8-10 pH ranges

(Fig.1,2). However, a weak reverse peak at high scan rates (300, 400 and 500mV/s) has

been observed at 11 pH (Fig-3) because of very slow electron transfer process occuring

electrochemically irreversible because surface equilibrium was not maintained generally by

insufficient rate of electron transfer [8,13]. Moreover, by following all the criteria of

irreversible system (Table-1.2) such as Ip υ1/2, shift of Ep

(peak potential) cathodically by 30/αn mV for 10 fold increase in V(volt) and │EpEp1/2│=

48 αcnα mV demonstrate that the Losartan Potassium exhibits totally irreversible electron

transfer process.

3.1.4 Effect of scan rate

The CVs profile represent first cathodic peak current currents Ipc1 (R2=0.99) and second

cathodic peak current Ipc2 (R2= 0.98) have strong linear relationship with square root of scan

rates (υ1/2) in B-R buffer (Fig 13-16). The slope values deviate from theoretical value of

0.5 for ideal diffused species confirming the exsistance of adsoption component. [40].

The first and second cathodic peak potientials (Epc1and Ep

c2) increase with log of scan rates

(log υ) as shown in (Fig. 30) and this behaviour was consistent with electrochemical nature

of the reaction in which electrode reaction is coupled with an irrversible follow- up chemical

step [118].

The plot between Ep and log of scan rate (log υ) can be represented as

Epc1= 0.075logυ + 0.387

Epc2= 0.060logυ + 0.769

In case of irrversible process peak potiential is defined as following equation accoding to

Laviron[118].

Ep=E0+ [2.303RT/ nF] log [RTK0/ nF] + [2.303RT/ nF] logυ…… (3.11)

is transfer coefficient, K0 is standard heterogeneous rate constant, n is the number

of electron, υ is scan rate and E0 is formal redox potential. Other symbols have their usual

meanings. The value of n can easily calculated from slope value of Epc vs. log υ. In this

work slope of first plot is 0.075, therefore n is 0.06 and for second plot slope is 0.06 and

n is 0.098. Furthermore, the value of was calculated according to Bard and Faulkner by

using following equation [118]

……..(3.12)

The above equation was used to calculate the value of (0.91) and (0.68) for first cathodic

peak and second peak respectively. Thus, the number of electron for first peak is

1.1~1 and for second peak 0.69~1 has been observed which showed the transfer of two

electrons in reduction process of Losartan Potassium.

3.1.5 Effect of concentration

The cathodic peak current (Ipc) showed linear relationship with concentration (Fig 21-24)

of Losartan Potassium ranges from (1×10-3 to 3×10-3 M) and hence obeys Randles-Sevick

equation. This implies that the process is rate limiting step taking place during electrode

reaction [117].

3.1.6 Effect of pH

The decreasing trend in peak current (Ipc) has been observed as the pH is increased (Fig.

29). This trend represents that pH may affect the solubility of Losartan Potassium and a

contribution of proton which may involved in reduction process causing decrease in the

peak current [117, 116]

3.1.7 Transfer coefficents (α)

In case of Losartan Potassium transfer coefficient (α) calculated by using equation (1.9)

are given in (Table 1-9) which lie within the range of 0.5 to 1 as defined for totally

irreversible reaction [13]. The value of (α) is also dependent on the potential difference

|Ep-Ep1⁄2| in case of irreversible systems [13].

3.18 Heterogenous rate constant

The value of K0 can be determined by using the value of E0. The value of E0 can be obtained

from the intercept of Ep versus υ curve by extrapolating to the vertical axis at υ = 0 [121].

Reinmuth also reported an alternative simple expression [120]

Ip = Co K0 --------------(3.13)

nFA

Where K0 is standard heterogeneous rate constant, Co is concentration and other symbol has

their usual meaning. The value of heterogeneous constant for Losartan potassium were

shown in (Table 8)

3.1.9 The repeated cyclic voltammogram

The repeated cyclic voltammograms of Losartan Potassium were recorded at 100 mV/s scan

rate in the presence of alkaline medium of B-R buffer range (8-11) used as supporting

electolytes shown in (Fig. 8-11). A descrease in peak current as a result of sucessive cycling

indicates slow or weak adsorption or desorption of analyte at the surface of gold electrode

[117]

3.2 GEMIFLOXACIN (GFX)

Electrochemical study of Gemifloxacin has also been carried out with cyclic voltammetery

technique. The voltammograms for Gemifloxacin (3mM) were recorded by using gold

electrode vs. calomel electrode in B-R buffer (0.04M) within pH range (2 to 6). The selected

potential ranges were 1.0 to - 0.8V and the applied potential scan rate ranges were 20, 100,

200, 300, 400 and 500mV/s. The CV profiles showed two cathodic peaks and one revese

andoic weak peak at potential range of 1.0 to - 0.8V in B-R buffer (Fig. 31-34).

3.2.1 Proposed reaction mechanism

2e + 2H

As the cyclic voltammograms of Gemifloxacin exhibit the reduction process by showing

two well defined cathodic peaks which may occur with the transfer of two electrons or two

protons. The most susceptible position for reduction process is –O=N group as represented

in above mentioned proposed reaction mechanism of Gemifloxacin.

3.2.2 Diagonestic test for quasi reversibile

The recorded voltammograms of Gemifloxacin demonstrating the redox process by

showing one reverse anodic peak and two cathodic peaks (Fig. 31-34). The second cathodic

peak (Ipc2) shows irreversibility while the first cathodic peak (Ipc

1) and its corresponding

anodic peak (Ipa1) indicate quasi reversibility. The estimated parameters for Gemifloxacin

also shows the quasi reversible process by follwing the diagonstic certeria (Table 37)

1. Increase in Ipa with υ1/2 but not propotional to it.

- +

2. |Ipa/ Ipc| =1 provided ac= aa=0.5

3. Shifting of Epc negatively with increasing scan rate

4. ∆Ep is greater than 59/nmV and increases with υ

3.2.3 Effect of scan rate

In case of Gemifloxacin, peak current (Ip) increases with increase of scan rates.

Moreover, the plot of peak current (Ipc1), (Ip

c2) and (Ipa1) vs. square root of scan rate (υ1/2)

showed linear relationship but not proportional (Fig. 46, 47, 49). Moreover, peak current

Ipc1, Ipc

2 and Ipa1 verus square root of scan rates (υ1/2) gives slope values less than theoretical

value 0.5 for diffused species indicating that the electrode surface has some adsorption

complications [73]

3.2.4 Effect of pH

The voltammogram of Gemifloxacin clearly showed two cathodic peaks and one anodic

peak at pH 2.-3 (Fig 31, 32). However, at pH 4 and 5 andodic peak was gradually

suppressed (Fig 33, 34) and at pH 6 anodic peak was disappered (Fig 35). Moreover, an

increase in peak current (Ip) and peak potiential (Ep) were also observed with the increase

in the pH (Fig 51,52). Therefore, it can be concluded that pH may affect solublity of

compound and electrochemical process particularly oxidation process.

The value of n was estimated by using formal potential (E0) was determined by the midpoint

potential (Emid) between the Epa1 and Epc

1 [121]. The E0 then plotted as a function of pH of

the solution (Fig 55).

Eo = -0.310 pH + 6.250 (R2=0.71)

E0 = E0 pH=0 – (2.303mRT/2F) pH where m is number of protons = number of electron [122].

Thus, n = 1.04 ≅1 at first peak.

The second cathodic peak showed no corresponding anodic peak and the number of electron

(n=1.2~1) was estimated by using the value of n(0.90) which was estimated by using slope

(0.06) of Epc2 versus logυ(Fig 56) then value of (0.6) calculated by using equation(3.12)

was used to estimate the number of electron transferred on second cathodic peak [118].

3.2.5 Effect of concentraction

A linear dependence of peak current(Ipc) and Peak potential(Epc) on the concentraction

(1×10-3-3×10-3M) of Gemifloxacin was observed in B-R buffer(Fig 53). This linear

behaviour suggests the diffusion is rate limiting process [117].

3.2.6 Heterogenous rate constant

The heterogeneous rate constant K0 was obtained using the methodology described by

Nicholson [8, 13]. Through a working curve between n (∆Epc-Ep

a) and Ψ the dimensionless

kinetic parameter, Ψ were obtained by using a linearization of the Nicholson approach.

Finally, the obtained Ψ value and other parameters which were previously described and

used to calculate K0 are represented in (Table 38)

K0 1/2 ……………. (3.14)

3.2.7 The repeated cyclic voltammogram

The repeated cyclic voltammograms of Gemifloxacin were recorded at a scan rate

(100mV/s) in the presence of supporting electolyte (Fig 41-45). The decrease in peak

current(Ip) has been observed as a result of sucessive cycling. The descreased peak

current(Ip) indicates a slow or weak adsorption or desorption of the analyte at gold test

electrode as reported in literature [117] .

3.3 CLARITHROMYCIN (CAM)

In the present study electrochemical behavior of Clarithromycin was examined by using B-

R buffer as supporting electrolyte within the range of pH 2-6. The cyclic voltammograms

of Clarithromycin (3mM) were recorded at gold test electrode between the ranges of the

(0 to +1.6V) potential window and scan rates were (20, 100, 200, 300, 400 and 500mV/s).

The voltammogram of Clarithromycin represents one cathodic peak and two anodic peaks.

(Fig 57-59). The anodic peaks were observed in potential range of 1.0- 1.6V/s and cathodic

peak was appeared in the range of 0.2-0.6 V/s. In previous liteturer appearence of cathodic

peak in this range of potiential has also been reported due to reduction of gold electdrode

surface [118, 121]. Therefore, it can be concluded that the Clarithromycin undergoes

irreversible oxidation prosses and as result anodic peak appeared, while the observed

cathodic peak is due to reduction of gold electrode surface which was confirm with blank

shown in (Fig. 79).

As the voltammogram of Clarithromycin showed two anodic peak (Fig. 57-59) which

indicate the occurrence of oxidation process most prbably due to two exposed OH- groups

as shown in structure of Clarithromycin. Due to the removal of hydrogen, “OH” groups

have been oxidized and negative charged has appeared as O- after the oxidation in

Clarthromycin.

3.3.2 Diagonestic Criteria for irreversible system.

To verify the irrversible transfer mechanism, diagonistic test for irrversibility has also been

applied. Clarithromycin showed peak current is propotional to the square root of scan rate.

(Fig. 69-71) and Epc shifts -30/αCnα mV for each decade increase in scan rate (υ) as describe

the third criteria of irreversibility while the difference of Ep- Ep/2 is against the reported

criterion for total irreversibility [8, 13] and it varies from 20 to 500mV. However, by

following the maximum point of irreversible diagnostic test, the electro oxidation process

of Clarithromycin considered as irreversible.

3.3.3 Effect of scan rate

The Effect of scan rate (υ) on peak current (Ipa1 and Ipa

2) has also been studies for

Clarithromycin. The plot of peak current Ipa1(R2=0.99) and and Ipa

2 (R2=0.99) showed

strong correlation with square root of scan rates (υ1/2) as shown in (Fig. 69-71). The plot of

log of peak current Ipa1 and, Ipa

2 verus log of scan rates (log υ) gives slope values 0.55 and

3.3. 1 S tuggested reaction mechanism:

- 2 e - , - 2 H

+

0.57 respectivly, which closed to the theoritical value 0.5 for diffusion controlled process

rather than adsorption [121].

The peak potientials (Epa1and Epa

2) were showed linear corelation with log of scan rates (log

υ) as shown in (Fig. 78) and this behaviour was consistent with electrochemical nature of

the reaction in which electrode reaction is coupled with an irrversible follow- up chemical

step [118].

The linear relation between Epa and log of scan rate (log υ) can be represented by following

equations.

Epa1 = 0.182logυ + 1.011

Epa2 = 0.085 logυ + 0.949

In case of irrversible process (Ep) peak potiential is defined by Laviron and expressed in

equation (.3.11)

The values of n was calculated from slope values of Ep vs log υ. Slope of first plot slope

is 0.18 and n is 1.08 and for second plot is 0.08, therefore n is 1.07

Moreover, the value of calculated according to Bard and Faulkner by using (3.12).

The value of (0.71) and (0.6) for first peak and second peak respectively. Thus, the

number of electron for first peak is 1.4~1 and for second peak 1.3~1 have been observed

which showed the transfer of two electrons in electro oxidation process of the

Clarithromycin.

3.3.4 Effect of pH

The voltammograms of Clarithromycin at different pH showed variations in anodic peak.

At pH (2.5, 3 and 3.5) two andodic peak were noted( Fig. 57-59). At pH 4 and pH 5 only

one anodic peak have been observed (Fig. 3.3, 3.4-5). This variablity in peaks and peak

currents (Fig. 60-72) shows the solublity of Clarithromycin is affacted by the pH and as

result variation in voltammogramms have been observed.

3.3.5 Effect of concentraction

The peak current (Ipa) was directly proportional to concentration of analyte (1×10-3- 3×10-

3 M) at pH 2.5 (Fig. 69) This linear dependence of peak current (Ipa) on concentraction of

Clarithromycin represents diffusion as rate limiting step as explained by the A. Bard and

Faulkner [2] .

3.3.6 Transffer coefficents

The values of charge transfer cofficients (α) were calculated by using the value of the

potential difference Ep-Ep1⁄2 as described before. This charge transfer coefficients is given

in (Table 39-48), lies within the range of 0.5 to 1 as defined for totally irreversible reaction

[13]. The value of (α) is also dependent on the potential difference |Ep-Ep1⁄2| in case of

irreversible systems [13].

3.3.7 Heterogenous rate constant

The value of k0 can be determined by using the value of E0. The value of E0 can were

obtained from the intercept of Ep versus υ curve by extrapolating to the vertical axis at υ =

0 [121]. Reinmuth reported an alternative simple expression shown in equation 3.13.

The value of heterogenous rate constant for Clarithromycin were shown in (Table 49).

3.3.8 The repeated cyclic voltammogram

A gradual decrease in peak height (current) (Ipc) as a result of repeated voltammograms at

100 mV/s in R-B buffer (Fig. 64-68) indicates slow or weak adsorption or desorption of

analyte at gold test electrode as reported in literature [117].

CONCLUSION

The investigation of electrochemical properties of three biologically active compounds has

been carried out by cyclic voltammetery technique to investigate the different parameters

such as peak current (Ip), peak potential (Ep), Diffusion coefficient (D), transfer coefficient

(α) which are used to reveal the nature of electrochemical process , number of electron

transferred (n) and type of reactions.

According to the present study Losartan Potassium, an antihypertensive drug showed

irreversible electron transfer process by following the maximum criteria of irreversible

reaction system with two electrons transfer mechanism. Moreover, this reaction process

indicates adsorption controlled reduction process. The influence of pH, concentration and

scan rates in electrochemical reaction has also been observed.

Other drug Gemifloxacin is an antibacterial, represented quasi reversible reaction system

by following the maximum criteria of quasi reversible diagnostics test. It shows adsorption

controlled process on surface of electrode and involvement of pH and concentration in

electrochemical process has also been studied.

Clarithromycin is primarily bacteriostatic and as well as have antimicrobial effect. It shows

irreversible oxidation process in B-R buffer as compare to Gemifloxacin. In case of

Clarithromycin during oxidation process two electrons were transferred while the

electrochemical process is diffusion controlled. Effects of concentration and pH and scan

rates on electrochemical process have also been noted.

These parameters determined in this study by CV technique would be helpful for

formulation or evaluation drug dosage with the consideration of physio-chemical

parameters such as pH and concentration. This method is suitable for quality control

laboratories as well as pharmacokinetic studies. Moreover, this technique is suitable

alternative due to easy handling, less time consuming and cheaper as compare to other

techniques such as HPLC or chromatography.