Chapter 1 General Introduction
1
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
Chapter 1 General Introduction
2
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].
Chapter 1 General Introduction
3
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
Chapter 1 General Introduction
4
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.
Chapter 1 General Introduction
5
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
Chapter 1 General Introduction
6
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.
Chapter 1 General Introduction
7
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
Chapter 1 General Introduction
8
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
Chapter 1 General Introduction
9
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
Chapter 1 General Introduction
10
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
Chapter 1 General Introduction
11
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
Chapter 1 General Introduction
12
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
Chapter 1 General Introduction
13
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
Chapter 1 General Introduction
14
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
Chapter 1 General Introduction
15
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
Chapter 1 General Introduction
16
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
Chapter 1 General Introduction
17
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]
Chapter 1 General Introduction
18
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,
Chapter 1 General Introduction
19
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
Chapter 1 General Introduction
20
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
Chapter 1 General Introduction
21
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
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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)
Chapter 1 General Introduction
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.
Chapter 1 General Introduction
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
Chapter 1 General Introduction
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.
Chapter 1 General Introduction
33
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