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Chapter 1 General Introduction

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PPPPharmaceutical research has played a key role in the development of drug [1].

The process of drug development is basically an innovation of a drug molecule that has

the capabilities to control, check, cure and fight against a particular disease. In

pharmaceutical research, the scope of drug analysis, analytical investigation of bulk

materials, intermediates, drug products, drug formulations, impurities and biological

samples containing drugs and their metabolites is very important [2]. The pharmaceutical

and biomedical analysis is among the most important branches of applied analytical

chemistry. Analytical measurement procedures are in understanding the physical and

chemical stability of the drugs, identification and quantification of the drug molecule to

evaluate the toxicity profile and to distinguish them from the impurities.

In recent years, various analytical assay methods have been applied for the

determination of pharmaceuticals that include titrimetry, spectroscopic methods,

chromatographic methods, capillary electrophoresis and electroanalytical methods [3].

The application of electrochemical techniques in the analysis of pharmaceuticals has

increased greatly over the last few years. Interest in electrochemical techniques for

quantification of pharmaceuticals can be attributed to their high sensitivity and selectivity

with fast response speed [4-7]. These techniques have become an alternative to other

analytical methods which have complicated instrumentation, high cost and need time

consuming extraction procedures or some derivatisation processes.

Electrochemistry has many advantages making it an attractive choice for

pharmaceutical analysis [8, 9]. These techniques have introduced the most promising


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applications for the determination of various types of electroactive compounds including

their redox mechanisms in different matrices [10-16]. Many of the active constituents of

formulations, in contrast to excipients, can be readily oxidized or reduced [17]. Ozkan et

al. [18] have studied various applications of modern electroanalytical techniques in the

analysis of pharmaceuticals and other compounds of medicinal importance. Applications

of various electrodes and chemically modified electrodes for electroanalytical

measurements has increased in recent years due to their applicability to the determination

of active compounds that undergo redox reactions particularly in the field of clinical and

pharmaceutical analysis [19-26].

In the present work electrochemical behaviour of some pharmaceuticals has been

studied using various electroanalytical techniques at chemically modified electrodes and

suitable analytical methods for their detection have been developed.

1.1 Electroanalytical Methods

Electroanalytical techniques can be easily adopted to solve many problems of

pharmaceutical interest with a high degree of accuracy, precision, sensitivity and

selectivity. Electrochemical methods include areas of intensely active research from

nanotechnology through biology to energy storage/transformation in addition to their well

established analytical/sensing utility [27]. These methods encompasses a group of

quantitative analytical methods based upon the electrical properties of a solution of the

analyte provided that the analyte species exhibits electroactivity and can be detected using

the tools of electrochemistry [28].


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Many modern electrochemical techniques have been developed for fundamental

studies of electrochemical reactions and for the determination of different electrochemical

properties of electroactive species by measuring the potential or current in an

electrochemical cell [3, 29-32]. These methods can be classified into several categories

depending on which aspects of the cell are controlled and which are measured. The three

main categories are potentiometry, coulometry and voltammetry.

1.1.1Potentiometry

Potentiometric methods of analysis are based upon measurement of the potential

of electrochemical cell in the absence of appreciable current. In potentiometry, the

measuring setup consists of two electrodes, the indicator electrode and the reference

electrode. Both electrodes are half-cells. When placed in a solution together they produce

a certain potential. Depending on the construction of the half-cells, the potential of the

electrochemical cell (Ecell) produced is the sum of several individual potentials [31].

1.1.2 Coulometry

Coulometry uses applied current or potential to completely convert an analyte

from one oxidation state to another. In these experiments, the total current passed is

measured directly or indirectly to determine the number of electrons passed. Coulometric

methods are basically performed by measuring the quantity of electrical charge which is

required to convert an analyte quantitatively to a different oxidation state. The quantity of

electrical charge (Q) is generally expressed in terms of coulomb and is measured as

transported by a constant current (I) of one ampere in one second. Thus, the no of


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coulombs (Q) resulting from a constant current of I amperes operated for t seconds can be

calculated as per equation (i) [31],

Q =It (i)

Coulometric techniques can be subdivided into different categories on the basis of

measuring the quantity of charge. The major categories are potentiostatic coulometry

known as bulk electrolysis and amperometric coulometry or coulometric titrimetry. In

potentiostatic coulometry, the electrolysis current is recorded as a function of time. In

amperometric coulometry, electrons are added to the analyte that immediately react with

the analyte until an end point is reached. At that point, electrolysis is discontinued and the

amount of analyte is determined from the magnitude of current and time to complete the

titration.

1.1.2.1 Chronocoulometry

In a chronocoulometric experiment, the total charge (Q) that passes for a certain

time t following a potential step is measured as function of time. For a diffusion

controlled process, the obtained charge Q is given by the integrated Cottrell equation (ii)

[29],

Q = 2nFACD1/2

t1/2π

-1/2 (ii)

Where A is the effective surface area of the working electrode, C is the

concentration of the analyte, n defines the no of electrons involved in the electrode

process, D is the diffusion coefficient of the analyte and other symbols have their usual

meanings.


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1.1.3 Voltammetry

Voltammetry comprises of electroanalytical methods that are based upon the

measurement of current as a function of applied potential [31]. These techniques are

distinct analytical tools for the determination of many inorganic and organic substances

which exhibit electroactivity. These techniques are also used for nanoanalytical purposes

including fundamental studies of oxidation and reduction processes in various media,

adsorption processes on surfaces and electron transfer mechanisms at chemically

modified electrode surfaces. Voltammetry is an electrolysis process on a microscale,

using a microelectrode in which the potential of the microelectrode is varied and the

resulting current is recorded as a function of applied potential. The recorded current-

potential curve is commonly known as voltammogram. When an analyte is present that

can be electrochemically oxidized or reduced, a current is recorded when the applied

potential becomes sufficiently negative for reductions and positive for oxidations. If the

analyte solution is sufficiently dilute, the current reaches a limiting value which is

proportional to analyte concentration. When an analyte is reduced or oxidized reversibly,

its half wave potential is very close to its standard potential for the redox reaction

whereas mechanism of redox process at the working electrode needs an increased applied

potential in the form of activation overpotential for the electrolysis to happen irreversibly.

In voltammetry, the applied potential controls the concentrations of the redox

moity at the electrode surface which have been described by Nernst equation. Nernst

quantitatively established a relation between potential and concentration. For a diffusion

controlled reversible electrochemical reaction, aOx + ne -

bRed, the reduction


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potential E forces the specific concentrations of reduced and oxidized species at the

electrode surface to ratio in accordance with Nernst equation (iii) [3, 31],

(iii)

Where, is standard reduction potential for the redox couple, R is the molar gas

constant (8.314 J mol-1

K-1

), T is the absolute temperature (K), n is the no of electrons

transferred, F corresponds to Faraday constant (96,485 C/equiv). The current during

electrolysis is determined by the rate of transport of the analyte (Y) from the outer edge of

the diffusion layer to the electrode surface. As per Nernst equation, a flow of continuous

current is required to maintain the surface concentration of the electrode surface as the

product of the electrolysis diffuses away from the surface. This current quantitatively

defines the rate of transfer of analyte Y to the electrode surface and given by, δcY / δx

where x is the distance in centimeters from the electrode surface. It can be shown that the

current is given by the expression (iv) [32],

I = nFAD (δcY / δx) (iv)

Where I is the current in ampere, n is the number of moles of electrons per mole

of analyte, F is the faradays constant, A is the effective surface area in cm2, D is the

diffusion coefficient of the analyte in cm2 s

-1 and cY is the concentration of Y in mol cm

-3.

The actual value of this current is affected by many other factors like size, shape and

material of the electrode, the solution resistance, cell volume and no of electrons being

involved in the redox process.


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1.2 Types of Voltammetric Techniques

1.2.1 Polarography

Polarography is a particular type of voltammetry that was discovered by the

Czechoslovakian chemist Jaroslav Heyrovsky in the early 1920s. It differs from other

classes of voltammetry by the specific use of dropping mercury electrode (DME) as

working electrode. Polarographic methods are based upon the measurements of current as

a function of potential applied to dropping mercury electrode.

1.2.2 Linear Sweep Voltammetry

Linear sweep voltammetry (LSV) is the earliest and simplest form of voltammetry

in which the potential of the working electrode is increased or decreased linearly with

time. The current is then recorded to give a voltammogram, which is a plot of current as a

function of potential applied to the working electrode [29, 31]. Oxidation or reduction of

the analyte is recorded as a peak in the current signal at the potential at which the analyte

is reduced or oxidized. LSV has been used by many workers to determine the

electroactivity of various compounds. Various pharmaceuticals like albendazole,

rebeprazole, fenbendazole have been successfully quantified by linear sweep

voltammertry [8, 15, 33-36].

1.2.3 Pulse Techniques

By the end of 1960, LSV methods were modified to increase the sensitivity, speed

and particularly detection limits and pulse techniques were evolved. The idea behind all


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pulse voltammetric methods is to measure the current at a time when the difference

between the desired faradaic curve and charging current is large following a potential

step. The charging current decreases exponentially with time and faradaic current decays

as a function of 1/ (time)1/2

suggesting that the rate of decay of the charging current is

faster than the faradaic current. In pulse methods the pulse amplitude defines the potential

pulse, pulse width defines the duration of the potential pulse and sample period is the time

at the end of the pulse during which the current is measured.

1.2.3.1 Normal Pulse Voltammetry

Normal pulse technique in voltammetry is the one in which current is measured

near the end of each pulse. It is carried out in unstirred solution at either dropping

mercury electrode or at solid electrodes with a series of potential pulses of increasing

amplitude. The duration of each pulse is generally 1 to100 milliseconds and pulse interval

varies between 0.1 to 5 s. The measured limiting current (Il) is given by the following

Cottrell equation (vii) [29],

Il = nFACD1/2

t1/2π

-1/2 (vii)

1.2.3.2 Differential Pulse Voltammetry

Differential pulse voltammetry (DPV) is a derivative of linear sweep voltammetry

or staircase voltammetry. This technique uses a series of regular voltage pulses

superimposed on the potential linear sweep or stairsteps. The current is measured

immediately before each potential change and the current difference is plotted as a


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function of potential. By sampling the current just before the potential is changed, the

effect of the charging current can be decreased. The potential between the working

electrode and the reference electrode is changed as a pulse from an initial potential to an

interlevel potential and remains at the interlevel potential for about 5 to 100 milliseconds,

then it changes to the final potential, which is different from the initial potential. The

pulse is repeated, changing the final potential, and a constant difference is kept between

the initial and the interlevel potential. The value of the current between the working

electrode and auxiliary electrode before and after the pulse are sampled and their

differences ∆i [(i2-i1)] are plotted versus potential (E). In these measurements, only

faradaic current is extracted.

1.2.3.3 Square Wave Voltammetry

Squarewave voltammetry (SWV) is a further improvement of staircase

voltammetry which is itself a derivative of linear sweep voltammetry. In linear sweep

voltammetry, the current at a working electrode is measured while the potential between

the working electrode and a reference electrode is swept linearly in time while in

squarewave voltammetry, a squarewave is superimposed on the potential staircase sweep.

Oxidation or reduction of species is observed as a peak or trough in the current signal at

the potential at which the species begins to be oxidized or reduced. The differential

current is then plotted as a function of potential, and the reduction or oxidation of species

is measured as a peak or trough. Due to minimized charging current, SWV offers much


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wider range and much lower detection limits generally on the order of nanomolar

concentrations.

In square wave voltammetry, the difference in current the difference of current is

larger than either forward or reverse currents, so that the height of the peak usually

becomes measurable and thus increasing the accuracy. The forward current i2, reverse

current i1, or difference current ∆i [(i2-i1)] can be used as the response in this technique.

The net current has only very small charging current contributions, and in typical

experiments the total faradaic charge is much less than equivalent to a monolayer of

material. SWV is a powerful electrochemical technique that can be applied in both

electrokinetic and quantitative determination of redox couples strongly immobilized at the

electrode surface.

DPV and SWV have been frequently used for the electrochemical characterization

of various types of compounds. Different pulse voltammetric methods were developed for

the determination of trace amounts of electroactive compounds in pharmaceuticals and

biological fluids [37-77].

1.2.4 Cyclic Voltammetry

In Cyclic Voltammetry (CV), the current response of the working electrode in an

unstirred solution is a triangular wave form. CV has been an important tool for

fundamental and diagnostic studies that provides quantitative as well as quantitative

information about electrochemical processes and their intermediate products. The

effectiveness of CV results from its capability for rapidly observing the redox behaviour


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over a wide potential range. For a reversible electrode reaction, anodic and cathodic peak

currents are approximately equal in absolute value but opposite in sign and difference in

peak potential is 0.0592/n where n is the no of electrons involved in half reactions. CV

has been widely used for studying various pharmaceuticals [78-83], biological matrices

[84] and raw materials [85, 86].

1.2.5 Stripping Voltammetry

In stripping analysis, the analyte is first deposited on the working electrode from a

stirred solution and electrolysis is carried out for a certain period of time. After an

accurately merasured period, the electrolysis is discontinued, the stirring is stopped and

deposited analyte is redissolved or stripped from the working electrode. In stripping

analysis, the quantitative results depond not only upon control of electrode potential but

also upon electrode size, length of deposition and stirring rate of the solution. The

concentration of the analyte at the working electrode surface is much greater than the bulk

solution. As a result of pre-concentration step, stripping methods are able to yield the

lowest detection limits of all voltammetric procedures. Stripping voltammetric methods

have been greatly employed for trace analysis of anions and cations. A variety of

pharmaceuticals have been determined electrochemically employing stripping

voltammetric techniques with low detection limits [87-92].

1.2.5.1 Adsorptive Stripping Voltammetry

Adsorptive stripping voltammetric (AdSV) technique is a well established and fast

growing area with a number of possible applications in the analysis of pharmaceutical and

biological compounds with very low detection limits [93-98]. In AdSV, most commonly a


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hanging mercury drop electrode is immersed in a strirred solution of the analyte for a

limited period of time. Deposition of the analyte occurs by physical adsorption at the

electrode surface rather than electrolytic deposition as in cathodic stripping and anodic

stripping techniques. After sufficient analyte has accumulated, the stirring is stoped and

the deposited analyte is determined by linear scan or pulse voltammetric techniques. The

sensitivity is significantly enhanced by adsorption of the drug on the electrode surface

[99, 100] and after careful choice of the operating parameters extremely low detection

limits can be achieved.

1.3 Working Electrodes

Working electrode is a part of three electrode system along with reference and

counter electrode in an electrochemical system at which the redox reaction takes place.

For electrochemical purposes, selection of working electrodes depends upon their

electrical conductivity, surface area, catalytic properties, cost and availability.

Futhermore, it also depends upon the reduction or oxidation potential of the analyte and

the background current over the potential range required for the measurement. Carbon

based electrodes have been widely used in voltammetric studies as working electrodes for

a variety of reasons, including low cost, availability, stability, low chemical interferences,

ability to easily modify the morphology of carbon along with their high mechanical

strength and inhibition of water electrolysis properties [27]. There are a number of

carbon-based electrodes including glassy carbon (GC), polycrystalline boron doped

diamond (pBDD), carbon paste electrodes (CPE), pyrolytic graphite electrodes (PGE),

carbon nanotubes (CNTs) and most recently graphene. Mercury electrodes are also quite


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common in electrochemical systems as working electrodes like hanging mercury drop

electrode (HMDE), dropping mercury electrode (DME) and static mercury drop

electrodes (SMDE).

1.3.1 Electrochemical Sensors: Chemically Modified Working Electrodes

In recent years, there has been a great deal of interest in the development of

various types of electrochemical sensors that exhibit increased sensitivity and selectivity.

The enhanced measurement capabilities of sensors are achieved by chemical modification

of the electrode surface to produce chemically modified electrodes (CMEs). These are

electrodes at which chemical species have been deliberately immobilised to produce

desirable properties. Increased selectivity, sensitivity and antifouling properties may be

achieved by applying an appropriate surface coating. Additionally, the rate of

heterogeneous electron transfer at CMEs is enhanced relative to the unmodified surface.

CMEs can be classified into two main types, namely, chemical sensors and biosensors

[101]. Biosensors are a special group of CMEs which incorporate a biological element

such as a substrate specific enzyme. Chemical sensors, which as the name suggests,

utilize chemical modifiers to achieve desirable properties. CMEs comprise a relatively

modern approach to electrode systems that finds utility in a wide spectrum of basic

electrochemical investigations including the relationship of heterogeneous electron

transfer, chemical reactivity to electrode surface chemistry and electrostatic phenomena at

electrode surfaces [102]. Compared with other electrode concepts in electrochemistry, the

distinguishing feature of a CMEs is that a generally quite thin film of a selected material


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is coated on the electrode surface to endow the electrode with the chemical,

electrochemical, electrical, transport, and other desirable properties.

The electrochemical detection can be enhanced by the use of conductive particles.

A variety of nanomaterials have been used to modify conventional detection electrodes.

These nanoparticles increase the electrode area and enhance the electron transfer between

the surface and redox centers in analytes and act as catalysts to increase the efficiency of

electrochemical reactions [103]. Significant chemically modified electrodes have been

prepared and widely reported for sensitive detection of compounds. Electrodes modified

by nanomaterials are emerging platform for determination of various redox processes.

Jain et al. [104-106] have reported various types of CMEs for studying a variety of redox

reactions.

1.4 Nanomaterials

The term nanotechnology is employed to describe the synthesis of particles or

assemblies with structural features in between those of atoms and bulk materials with

atleast one dimension in the nanometer range. Properties of marterials with nanometric

dimensions are significantly different from those of atoms as well as of bulk materials.

We have different types of nanoparticles, nanowires, nanocrystals and clustures (quantum

dots), nanotubes, nanoporous solids and their assemblies with some remarkable and novel

optical, mechanical, electrical, structural and magnetic properties with a no of

applications in solar cells, semiconductor devices, electrochemical, photochemical,

catalytic and other aspects [107]. Recently, besides the eshtablished techniques of

electron microscopy, diffraction methods and spectroscopic tools, scanning probe


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microscopy have provided powerful means for studying nanostructures. The immediate

objectives of nanomaterial chemistry are to explore and generate a variety of new classes

of high performance materials with desired properties and discovering better tools for

studying nanostructures. The use of nanomaterials has become an increased area of

research in electrochemical sensors. The incorporation of these nanomaterials in

conjunction with one another to form novel composites is particularly interesting, as

many of these materials have been found to have synergistic effects. Such interactions

depend not only on the fabrication method but also on the size and specific geometry of

the nanoparticles. These characteristics combined with the ability to form hydrogen

bonds, dispersion forces, dative bonds, and hydrophobic interactions can affect the

stability and selectivity of nanomaterials [108]. Consequently, the distinctive properties of

nanomaterials have sparked interest in analytical chemistry and have been used to

develop innovative applications in sensor designing. Nanomaterial based electroanalytical

techniques show enormous potentials to construct sensors and platforms for chemical

sensing and biosensing of organic compounds [109-112]. Within these sensors, the active

sensing material on the electrode acts as a catalyst and catalyze the reaction of the

biochemical/chemical compounds to obtain output signals by different electroanalytical

techniques like cyclic voltammetry, square wave voltammetry, differential pulse

voltammetry, chronocoulometry and this combination give rise to a class of sensors

which are called electrochemical sensors [113, 114]. Because of the property of

nanomaterials that they can catalyze redox processes of molecules of analytical interest

due to their high conductivity, large surface area and good surface chemistry, they are


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extensively applied in sensor designing [108, 115, 116]. A fairly broad spectrum of

nanomaterials has been used for analytical sensing [117-134]. The selection and

development of an active sensing material is still a challenge for construction and

fabrication of nanomaterial based sensors for sensing application.

There are various types of nanomaterials available that are extensively used as

chemical sensors like carbon nanotubes, graphene and various metal oxides.

Figure 1.1: a) Single walled CNTs, b) Multi walled CNTs, c) graphene, d) metal oxides

1.4.1 Carbon Nanotubes

Carbon nanotubes (CNTs) have become the subject of intense researches in the

last few decades because of their unique properties and the promising applications in any

aspect of nanotechnology. CNTs are electrochemically inert materials similar to other

carbon-based materials used in electrochemistry, i.e. glassy carbon, graphite, and


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diamond [135]. Because of their unique one-dimensional nanostructures, CNTs display

fascinating electronic and optical properties that are distinct from other carbonaceous

materials and nanoparticles of other types. Basically, there are two groups of carbon

nanotubes, multiwall (MWCNTs) and single-wall (SWCNTs) carbon nanotubes.

MWCNTs can be visualized as concentric and closed graphite tubules with multiple

layers of graphite sheets that define a hole typically from 2 to 25 nm separated by a

distance of approximately 0.34 nm. SWCNTs consist of a graphite sheet rolled seamlessly

defining a cylinder of 1–2 nm diameters. They offer unique advantages including

enhanced electronic properties and rapid electrode kinetics. CNTs are most widely

employed for the construction of various detection devices, such as gas sensors,

electrochemical detectors and biosensors with immobilized biomolecules. Their

application in voltammetric methods is especially favourable and also employed for

sorption of different analytes and in electrochemical stripping methods [136, 137]. The

use of CNTs as analytical tools and construction of nanodevices and nanosensors based

on CNTs are other exciting areas of development for modern analytical science.

Applications based on CNTs driven electrocatalytic effects, the construction of new

hybrid materials with polymers or other nanomaterials and the increasing use of modified

CNTs for electroanalytical applications have also become an area of considerable interest

in modern electrochemistry [120].

Electrocatalysis of analytes at CNTs based sensors have been widely reported by

using various types of electroanalytical techniques like square wave voltammetry, cyclic

voltammetry, differential pulse voltammetry and chronocoulommetry. Goyal et al. [89]


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used an edge-plane pyrolytic graphite electrode (EPPGE) modified with single-walled

CNTs (SWCNTs) as a sensor to determine triamcinolone, a doping substance abused by

athletes. Swamy et al. [138] used carbon-fiber microelectrodes modified with SWCNTs

(SWCNTs/CFMEs) to detect dopamine and serotonin. Zhang et al. [139] used MWCNT

based CFMEs were also used to detect ascorbic acid (AA). Kachoosangi et al. [140]

reported a sensitive electroanalytical method for determining paracetamol using

adsorptive stripping voltammetry at an MWCNT modified basal plane pyrolytic graphite

electrode (BPPGE). A simple and rapid electrochemical method has been developed for

the determination of ciprofloxacin based on a multi-wall carbon nanotubes film-modified

glassy carbon electrode (MWCNT/GCE) [141]. Several other types of nanocomposites of

CNT with different metal oxides like MnO2, NiO, TiO2, Pt or Au have also been prepared

and widely reported for electrocatalysis of different types of compounds [108]. Various

types of pharmaceutical have also been studied electrochemically using CNTs modified

electrode with good reproducibility and stability [142-144].

1.4.2 Graphene

Graphene (GR) is a two dimensional single atomic planar sheet of sp2

bonded

carbon atoms that are densely packaged into a honeycomb lattice structure, and is

essentially a very large polyaromatic hydrocarbon. GR has attracted strong scientific and

technological interest in recent years with its unique electronic, optical, mechanical,

thermal and electrochemical properties that are far superior to its counterparts [145]. It

holds great promise for many applications within the general field of electrochemistry

along with many applications, such as electronics, energy storage (supercapacitors,


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batteries, fuel cells, solar cells) and bioscience/biotechnologies. An essential

characteristic of an electrode material is its surface area, which is important in

applications such as energy storage, biocatalytic devices and sensors. It has an exposed

surface area of 2630 m2 g

-1which much greater than graphite (10 m

2 g

-1)5 and nearly two

times larger than that of CNTs (1315 m2 g

-1). Additionally, the electrical conductivity of

GR has been calculated to be 64 mS cm-1,

which is approximately 60 times more than that

of SWCNTs. Furthermore, the conductivity of GR remains stable over a vast range of

temperatures [146, 147]. Other advantages of GR that make it attractive for analytical

applications include its high mechanical strength, high elasticity and the absence of

metallic impurities that can affect the accuracy of a sensor. GR has a wide and diverse

impact within the fabrication and preparation of sensors and in electrocatalysis for

detecting a wide variety of compounds. Several sensors based on GR have also been

prepared and widely reported. Shang et al. [148] were the first researchers to use GR

based nanomaterials for electrochemical sensing for simultaneous determination of

determination of dopamine, ascorbic acid, and uric acid. Li et al. [149] used GR based

nanomaterials for the sensitive detection of dopamine in the presence of ascorbic acid.

Wang et al. [150] studied the electricatalytic analysis of some toxic ions like Pb2+

and

Cd2+

using graphene based composites. Schedin et al. [151] investigated the gas sensing

properties of graphene for gaseous molecules. pH sensing properties of graphene based

sensors were studied by Ang et al. [152] using hydroxyl and hydroxonium ions over the

pH range 2-12. Further, Zhu et al. [153] showed the promising electrochemical sensing

application of graphene for detection of biomolecules like catecholamines. GR has also


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been reported as support for Pt/Ru NPs for the electro-oxidation of methanol [154]. Wu et

al. [155] prepared chitosan dispersed graphene nanoflakes and immobilized on a GCE to

construct a graphene modified electrode and successfully applied for electrocatalysis of

cytochrome c. Kang et al. [156, 157] studied the electrochemistry of glucose oxidase

using GR/chitosan nanocomposite and also behaviour of paracetamol on graphene based

sensor using voltammetry. Due to its unique properties, GR has been considered as a

noble electrode material that has extensively been applied in recent years to detect a wide

range of compounds by electrochemical techniques.

1.4.3 Metal Nanoparticles

Metal nanoparticles (MNs) are one of most widely used materials in

electroanalytical investigations and have good potentials for constructing electrochemical

sensing platforms with high sensitivity and selectivity to detect target molecules based on

different analytical strategies. In order to further improve the sensitivity of

electrochemical detection, it is very necessary to find better electrode materials for

electroanalytical applications [108]. Over the last few decades, there has been an

increased interest in MNs for their use as sensors because of their unique structural and

surface chemistry. Microstructure of MNs plays an important role in revealing their

enhanced functions and application potential as analytical sensors. MNs based

electroanalytical techniques show enormous potentials for constructing enhanced

platforms for chemical sensing and biosensing [158, 159]. This is because that MNs can

effectively catalyze the redox processes of some molecules of analytical interest due to

their high conductivity, large surface area and good surface chemistry property, thus


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permit an improvement of the analytical performance of voltammetric techniques in

comparison to conventional electrodes. Functionalizing the surface of these entities

modifies their surface properties and offers an increment in their sensing capability and

selectivity towards catalytic processes occurring at their surface. A combination of

different nanoscaled MNs have been also gaining much interest for constructing high

performance electrochemical sensors. This is because composite nanomaterials could

provide larger active surface areas for the adsorption of target molecules and effectively

accelerate the electron transfer between electrode and detection molecules, which could

lead to a more rapid and sensitive current response [160-162]. In recent years, different

types of metal based hybrid functional nanomaterials have been reported for enhanced

electrochemical detection of different molecules. These typical hybrid nanomaterials

include CNT/silica coaxial nanocable supported Au/Pt hybrid NPs [163], polyaniline

nanofiber/high density Pt NP hybrids [164], GR/Pt or Au NP hybrids [160, 165] and high-

density Au/Pt hybrid NPs supported on TiO2 nanospheres [166, 167]. In addition, several

other advanced hybrid functional nanomaterials for electroanalytical applications have

been also reported. Huang et al. [161] demonstrated Pd NPs loaded carbon nanofibers as

electrode materials for sensing H2O2 and NADH at low potentials. Shan et al. [168]

presented a novel glucose biosensor based on immobilization of glucose oxidase in thin

films of chitosan nanocomposites of GR and Au NPs. GR in combination with various

metals is also known for heterogenous catalysis and led to the exploration of various GR-

metal systems, their structure and bonding behaviour. Silver nanoparticles decorated

SiO2/graphene oxide has been reported for H2O2 and glucose sensors [169]. SiO2 coated


22

graphene oxide imprinted polymer composite for electrochemical sensing of dopamine

has been also reported [170]. Cerium dioxide NP has also been reported for

electroanalysis of many compounds. Ispas et al. [171] studied the unique catalytic and

electrochemical properties of CeO2 as an electrode material to develop CeO2 based sensor

to determine hydrogen peroxide. A nanoceria modified Pt/Au composite electrode for the

electrochemical oxidation of methanol and ethanol in acidic media has been fabricated by

Anderson et al. [172] Also Pt/CeO2 composite electrode was constructed by Saha et al.

[173] and used to study glucose oxidase. A ceria/titania composite electrochemical

enzyme biosensor has been fabricated to study phenol and dopamine [174]. CeO2/indium

tin oxide electrode was applied for the study of cholesterol electrochemically [175].

Cu/CeO2 electrode was constructed by gammara et al. and used as catalyst for carbon

monoxide oxidation [176]. Thus, for analytical purposes, MNs have proved to be novel

electrode materials for constructing high performance electrochemical sensors with high

sensitivity and selectivity.

1.5 Surfactants in Electroanalysis

Being surface active, surfactants naturally have a very large impact on

electroanalysis. A prerequisite for surfactants to be surface active is the property to adsorb

at the interface between bulk phases electrode and solution [184]. Properties of

surfactants like adsorption at interface and aggregation into supramolecular structures are

advantageously used in electrochemistry [178-181]. Surfactants are able to modify and

control the properties of electrode surfaces. The use of surfactant structures to alter or

enhance reaction rates has a significant role in electroanalysis of compounds. A large


23

fraction of the research in controlling electrochemical reactions with surfactants as well as

aggregate characterization by electrochemical methods has been carried out since last few

years.

Surfactants find several applications in electroanalytical chemistry. Solubilization

of organic compounds in surfactant aggregates and electrode surface modification are

well known phenomenon in modern electrochemistry [182]. Introduction of surfactants in

this area of work adds a new and useful dimension to study redox mechanisms at

chemically modified electrodes. Also use of surfactants minimizes the use of high cost

hazardous solvents in electrochemical studies of compounds. Various compounds of

analytical interest have been determined electrochemically in solubilized systems of

different surfactants at chemically modified electrodes using electroanalytical techniques

[183]. Surfactants aggregate as bilayer, cylinders or surface micelle adsorbed on the

surface of electrode above the critical micelle concentration (CMC). Dang et al. [184]

have demonstrated the use of cationic surfactant CTAB on the surface of an acetylene

black electrode could significantly decrease the overpotential of dioxygen reduction, and

increase the reduction peak current of oxygen. Choi et al. [185] reported SDS for

enhanced electrocatalytic activity towards methanol oxidation. CTAB/Clay modified

glassy carbon electrode confined with ferrocene dicarboxylic acid was found to determine

ascorbic acid [186]. The voltammetric peak enhancement of pharmaceuticals in the

presence of surfactants is the result of fast electron transport between electrode surface

and the analyte have been reported by different studies. Angeles et al. [187] showed the

effect of SDS micelles in the selective determination of dopamine in the presence of


24

ascorbic acid in the presence of uric acid, and showed good anti-fouling properties

towards surface active materials. Also Dale et al. [188] have reported much on fabrication

method of graphene using surfactants. Furthermore, Vittal et al. [189] have successfully

reviewed the beneficial role of surfactants in electrochemistry and in modification of

electrodes.

1.6 Electrochemical Characterization of Sensors

1.6.1 Scanning Electron microscopy

To study the morphological characteristics of fabricated chemically modified

electrodes, scanning electron microscopy (SEM) is usually carried out that produces

images of fabricated sensor by scanning it with a focused beam of electrons. The

electrons interact with atoms in the sample, producing various signals that can be detected

and that contain information about the sample's surface topography and composition

[190]. The electron beam is generally scanned in a raster scan pattern and position of

beam is combined with the detected signal to produce an image. Accelerated electrons in

SEM carry significant amounts of kinetic energy that is dissipated as a variety of signals

produced by electron-sample interactions when the incident electrons are decelerated in

the solid sample. These signals include secondary electrons (that produce SEM images),

backscattered electrons (BSE), diffracted backscattered electrons (EBSD that are used to

determine crystal structures and orientations of minerals), photons (characteristic X-rays

that are used for elemental analysis and continuum X-rays), visible light and heat.

Secondary electrons and backscattered electrons are commonly used for imaging samples.

Secondary electrons are most valuable for showing morphology and topography on


25

samples and backscattered electrons are most valuable for illustrating contrasts in

composition in multiphase samples. The SEM is routinely used to generate high

resolution images and to show spatial variations in chemical compositions.

1.6.2 Eletrochemical Impedance Spectroscopy

Electrochemical impedance spectroscopy (EIS) is a technique of measuring the

electrical impedance of a substance as a function of the frequency of an applied electrical

current. Impedance is a measure of the resistance to the flow of an alternating current.

EIS have been widely applied to the characterization of electrode processes and complex

interfaces [191]. These measurements are carried out at different ac frequencies. In EIS,

the sample under investigation is excited by a small amplitude ac sinusoidal signal of

potential or current in a wide range of frequencies and the response of the current or

voltage is measured. Frequency sweeping in a wide range from high-to low-frequency

enables the reaction steps with different rate constants, such as mass transport, charge

transfer, and chemical reaction. For typical impedance measurements, a small excitation

signal (e.g., <20 ~ 30 mV) is used, so that the electrochemical cell is considered as a

pseudo linear system. In this condition, a sinusoidal potential input to the electrochemical

cell leads to a sinusoidal current output at the same frequency.

When the real part of the impedance is plotted on the axis of the abscissa and the

imaginary part is plotted on the axis of the ordinate, we get a nyquist plot. In the nyquist

plot, a vector of length |Z| is the impedance and the angle between this vector and the real

axis is a phase shift, Ø. The randles circuit (Fig. 1.2) [191] is the simplest and most


26

common electrical representation of an electrochemical cell. It includes a resistor with a

resistance of Rct, an interfacial charge-transfer resistance connected in parallel with a

capacitor with a capacitance of C and this RC electrical unit is connected in series with

another resistor with a resistance of Rs the solution resistance. Nyquist plot for a randles

cell is a semicircle with two intercepts on the real axis in the high and low frequency

regions. The former is the Rs while the latter is the sum of the Rs and Rct. The diameter of

the semicircle is therefore equal to the charge transfer resistance.

Figure 1.2: A typical randles circuit to fit impedance data

1.7 Electroanalytical Method Validation

Method validation is the process used to confirm that the analytical procedure

employed for a specific test is suitable for its intended use. It is an analytical procedure is

the process by which the performance characteristics of the procedure meet the

requirements for the intended analytical applications [31, 32]. Results from method

validation can be used to judge the quality, reliability and consistency of analytical results

that is an integral part of good analytical practices. The objective of any analytical

measurement is to obtain consistent, reliable and accurate data. Validated analytical


27

methods play a major role in achieving this goal. There are various analytical procedures

that needs to be validated are as follows,

1.7.1 Analytical Procedure

The analytical procedure refers to the way of performing the analysis. It should

describe in detail the steps necessary to perform each analytical test. This may include but

is not limited to the sample, the reference standard and the reagents preparations, use of

the apparatus, generation of the calibration curve and use of the formulae for the

calculation.

1.7.2 Specificity

Specificity is the ability to assess the analyte in the presence of components which

may be expected to be present. Typically these might include impurities, excipients,

degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be

compensated by other supporting analytical procedure. Analytical techniques that can

measure the analyte response in the presence of all potential sample components should

be used for specificity validation.

1.7.3Precision

Precision of an analytical procedure is the closeness of the agreement between a

series of measurements obtained from multiple sampling of the same homogeneous

sample under the prescribed conditions. Precision is considered at three levels

repeatability, intermediate precision and reproducibility. Repeatability expresses the

precision under the same operating conditions over a short interval of time. It is also

termed intra-assay precision. Repeatability to be tested from at least six replications are


28

measured at 100 percent of the test target concentration or from at least nine replications

covering the complete specified range. Intermediate precision is determined by

comparing the results of a method run within a single laboratory over a number of days.

Reproducibility expresses the precision between laboratories usually applied to

standardization of methodology. The objective of reproducibility is to verify that the

method provides the same results in different laboratories.

1.7.4 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an accepted

reference value and the value found. This is sometimes termed trueness. It can also be

described as the extent to which test results generated by the method and the true value

agree. Accuracy can be assessed by analyzing a sample with known concentrations and

comparing the measured value with the true value as supplied with the material.

1.7.5 Recovery

After extraction of the analyte from the matrix and injection into the analytical

instrument, its recovery can be determined by comparing the response of the extract with

the response of the reference material dissolved in a pure solvent. The concentration

should cover the range of concern and should include concentrations close to the

quantitation limit, one in the middle of the range and one at the high end of the calibration

curve. Validation methodology recommends accuracy to be assessed using a minimum of

nine determinations over a minimum of three concentration levels covering the specified

range (for example, three concentrations with three replicates each). Accuracy should be


29

reported as percent recovery by the assay of known added amount of analyte in the

sample or as the difference between the mean and the accepted true value, together with

the confidence intervals.

1.7.6 Linearity

Linearity of an analytical procedure is its ability to obtain test results that are

directly proportional to the concentration amount of analyte in the sample. Linearity is

determined by a series of five to six injections of the standards whose concentrations span

80–120 percent of the expected concentration range. The response should be directly

proportional to the concentrations of the analytes.

1.7.7 Range

The range of an analytical procedure is the interval between the upper and lower

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

the analytical procedure has a suitable level of precision, accuracy and linearity.

1.7.8 Limit of Detection

The detection limit (LOD) of an individual analytical procedure as the lowest

amount of analyte in a sample which can be detected but not necessarily quantitated as an

exact value. LOD is usually expressed as the concentration of the analyte in the sample,

for example, percentage, parts per million (ppm) or parts per billion (ppb). It is estimated

from the standard deviation (σ) of the response and the slope (S) of the calibration curve

that can be easily estimated by the linear regression equation. LOD can be found by the

following equation (viii),

LOD = 3σ/S (viii)


30

1.7.9 Limit of Quantification

The quantitation limit (LOQ) of an individual analytical procedure is the lowest

amount of analyte in a sample which can be quantitatively determined with suitable

precision and accuracy. Quantitation limit is a parameter of quantitative assays for low

levels of compounds in sample matrices, and is used particularly for the determination of

impurities and/or degradation products. The quantitation limit is generally determined by

the analysis of samples with known concentrations of analyte and by establishing the

minimum level at which the analyte can be quantified with acceptable accuracy and

precision. LOQ can be estimated by the following equation (ix),

LOQ = 10 σ/S (ix)

Where S is the slope of the calibration curve of the analyte and σ is the standard deviation

of the responses obtained from the linear regression equation of the calibration curve.

1.7.10 Robustness and Ruggedness

The robustness of an analytical procedure as a measure of its capacity to remain

unaffected but deliberate variations in method parameters. It provides an indication of the

reliability of the procedure during normal usage. Robustness tests examine the effect that

operational parameters have on the analysis results. Ruggedness is the degree of

reproducibility of results obtained under a variety of conditions, such as different

laboratories, analysts, instruments, environmental conditions, operators and materials.


31

1.7.11Stability

Stability is the measure of the bias in the assay results generated during a pre

selected time interval. For validating the developed method, it is necessary to evaluate the

stability of the procedure for it is taken into normal and routine practice.

1.8 Scope of work

The present work incorporates the electrocatalytic determination of some

pharmaceuticals like cabergoline {N-[3-(dimethylamino)propyl]-N-

[(ethylamino)carbonyl]-6-(2-propenyl)-8g-ergoline carboxamide} which is an

ergot derivative and used in the treatment of prolactinomas and in progressive phase

treatment of parkinson's disease; tizanidine {5-chloro-4-(2- imidazolin-2-ylamino)-2,1,3

benzothiadiazole hydrochloride} which is a muscle relaxant and very helpful in relieving

spasm; dabigratran etexilate {Ethyl 3 - {[(2-{[(4-{N'-hexyloxycarbonyl carbamimidoyl}

phenyl) amino] methyl} - 1 - methyl-1H-benzimidazol-5-yl) carbonyl] (pyridin-2-yl-

amino) propanoate} which is a direct thrombin inhibitor, an anticoagulant drug and is

used in antithrombotic treatments and febuxostat {2-[3-cyano-4-(2-methylpropoxy)

phenyl]-4-methylthiazole-5-carboxylic acid}which is a non purine inhibitor of xanthine

oxidase used in the treatment of hyperurecemia and chronic gout. These pharmaceuticals

have been studied in different solvent systems using chemically modified electrodes as

electrochemical sensors. Different nanomaterials like multi walled carbon nanotubes,

graphene, cerium dioxide nanoparticles, aluminium titanate nanopowder have been used

for the fabrication of electrochemical sensors to enhance the speed, sensitivity and

stability of the redox processes and electrochemical properties of drugs have been studied


32

using voltammetric techniques. These sensors are capable of being incorporated into

robust, portable, or miniaturized devices, enabling tailoring for electrochemical

applications. Moreover, the combination of various nanomaterials into composites in

order to explore their synergistic effects has become an interesting area of research. Also

the use of surfactants in electroanalysis minimizes the use of high cost, hazardous organic

solvents and promotes the approach of green chemistry in electrochemistry.


33

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