cyclic voltammetric study of some biologically...
TRANSCRIPT
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.