ELECTROCHEMISTRY

DR. MOSAAD NEGEM

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 Crystal Structure
Materials science focuses on materials that are already known to be useful or have the potential to be
developed for applications, either by compositional control to optimize properties or by fabrication
into desired forms, shapes or products. Materials science therefore includes whatever aspects of
chemistry, physics and engineering that are necessary to achieve the desired aims. Materials
chemistry is much more than just a subset of materials science, however, since it is freed from the
constraint of a focus on specific applications; materials chemists love to synthesize new materials and
measure their properties, some of which may turn out to be useful and contribute to the development
of new industries, but they do this within an overarching interest in new chemistry, new structures
and improved understanding of structure composition property relationships.
We see how to describe the regular arrangement of atoms in crystals and the symmetry of their
arrangement. Then we consider the basic principles of X-ray diffraction and see how the diffraction
pattern can be interpreted in terms of the distribution of electron density in a unit cell. X-ray
diffraction leads to information about the structures of metallic, ionic, and molecular solids. The solid
state includes most of the materials that make modern technology possible. The properties of solids
stem, of course, from the arrangement and properties of the constituent atoms, and one of the
challenges of this course is to see how a wide range of bulk properties, including rigidity, electrical
conductivity, and optical and magnetic properties, stem from the properties of atoms. One crucial
aspect of this link is the pattern in which the atoms (and molecules) are stacked together.
Crystallography
Solid state chemistry is concerned mainly with crystalline inorganic materials, their synthesis,
structures, properties and applications. A good place to begin is with crystal structures and crystal
chemistry. All necessary crystal structure information is contained in data on unit cells, their
dimensions and the positions or atomic coordinates of atoms inside the unit cell. Crystal chemistry
combines this basic structural information with information about the elements, their principal
oxidation states, ionic radii, coordination requirements and preferences for ionic/covalent/metallic
bonding. A working knowledge of the Periodic Table and the properties of elements is, of course,
invaluable to be able appreciate crystal chemistry, but conversely, knowledge of crystal structures
and especially crystal chemistry provides a very useful way to gain increased understanding of the
elements and their compounds.

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1.1 Lattices and unit cells: A unit cell is an imaginary parallelepiped that contains one unit of a
translationally repeating pattern. Unit cells are classified into seven crystal systems according to their
rotational symmetries. The Bravais lattices are the 14 distinct space lattices in three dimensions.
A crystal is built up from regularly repeating ‘structural motifs’, which may be atoms, molecules, or
groups of atoms, molecules, or ions. A space lattice is the pattern formed by points representing the
locations of these motifs (Fig. 1.1). The space lattice is, in effect, abstract scaffolding for the crystal
structure. More formally, a space lattice is a three-dimensional, infinite array of points, each of which
is surrounded in an identical way by its neighbours, and which defines the basic structure of the
crystal. In some cases there may be a structural motif centred on each lattice point, but that is not
necessary. The crystal structure itself is obtained by associating with each lattice point an identical
structural motif.

Fig. 1.1 Each lattice point Fig. 1.2 A unit cell is a Fig. 1.3 A unit cell can be chosen
specifies the location of a parallel-sided (but not in a variety of ways, as shown
structural motif (for example, a necessarily rectangular) here. It is conventional to choose
molecule or a group of figure from which the entire the cell that represents the full
molecules). The crystal lattice is crystal structure can be symmetry of the lattice. In this
the array of lattice points; the constructed by using only rectangular lattice, the rectangular
crystal structure is the collection translations (not reflections, unit cell would normally be
of structural motifs arranged rotations, or inversions). adopted.
according to the lattice.
The unit cell is an imaginary parallelepiped (parallel-sided figure) that contains one unit of the
translationally repeating pattern (Fig. 1.2). A unit cell can be thought of as the fundamental region
from which the entire crystal may be constructed by purely translational displacements (like bricks in
a wall). A unit cell is commonly formed by joining neighbouring lattice points by straight lines (Fig.
1.3). Such unit cells are called primitive. It is sometimes more convenient to draw larger non-
primitive unit cells that also have lattice points at their centres or on pairs of opposite faces.
An infinite number of different unit cells can describe the same lattice, but the one with sides that
have the shortest lengths and that are most nearly perpendicular to one another is normally chosen.
The lengths of the sides of a unit cell are denoted a, b, and c, and the angles between them are denoted
α, β, and γ (Fig. 1.4).
Unit cells are classified into seven crystal systems by noting the rotational symmetry elements they
possess. A cubic unit cell, for example, has four threefold axes in a tetrahedral array (Fig. 1.5). A
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monoclinic unit cell has one twofold axis; the unique axis is by convention the b axis (Fig. 1.6). A
triclinic unit cell has no rotational symmetry, and typically all three sides and angles are different
(Fig. 1.7). Table 1.1 lists the essential symmetries, the elements that must be present for the unit cell
to belong to a particular crystal system.
There are only 14 distinct space lattices in three dimensions. These Bravais lattices are illustrated in
Fig. 1.8. It is conventional to portray these lattices by primitive unit cells in some cases and by non-
primitive unit cells in others. A primitive unit cell (with lattice points only at the corners) is denoted
P. A body-centred unit cell (I) also has a lattice point at its centre. A face-centred unit cell (F) has
lattice points at its corners and also at the centres of its six faces.
Fig. 1.4 The notation for the sides and angles of a unit cell. Note that
the angle α lies in the plane (b,c) and perpendicular to the axis a.

Fig. 1.5 A unit cell belonging to the cubic Fig. 1.6 A unit belonging to the Fig. 1.7 A triclinic unit
system has four threefold axes, denoted monoclinic system has a twofold cell has no axes of
C3, arranged tetrahedrally. The insert axis (denoted C2 and shown in rotational symmetry.
shows the three fold symmetry. more detail in the insert).
A side-centred unit cell (A, B, or C) has lattice points at its corners and at the centres of two opposite
faces. For simple structures, it is often convenient to choose an atom belonging to the structural motif,
or the centre of a molecule, as the location of a lattice point or the vertex of a unit cell, but that is not
a necessary requirement.
Table 1.1 The seven crystal systems

Unit cell - The smallest repeat unit that shows the symmetry of the structure, eg 2D NaCl, 1, 2, 3
show symmetry of lattice (4 fold rotation) and correspond to the face of 3D unit cell. 4 and 5 do not
show symmetry. 6 has a 4 fold rotation axis and is smaller than 1, 2, 3. 6 is 2D unit cell.

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Fig. 1.8 The fourteen Bravais lattices. The points are lattice points and are not necessarily occupied
by atoms. P denotes a primitive unit cell (R is used for a trigonal lattice), I a bodycentred unit cell, F
a face-centred unit cell, and C (or A or B) a cell with lattice points on two opposite faces.
1.1a Symmetry
I- Rotational symmetry; symmetry elements and operations
Symmetry is most easily defined using examples. Consider the silicate tetrahedron shown in Fig.
4.9(a). If it is rotated about an axis passing along the vertical Si–O bond, then every 120◦ the
tetrahedron finds itself in an identical position. Effectively, the three basal oxygens change position
with each other every 120◦. During a complete 360◦ rotation, the tetrahedron passes through three
such identical positions. The fact that different (i.e. >1) identical orientations are possible means that
the SiO4 tetrahedron possesses symmetry. The axis about which the tetrahedron may be rotated is
called a rotation axis; it is an example of a symmetry element.
The process of rotation is an example of a symmetry operation. The symmetry elements that are
important in crystallography are listed in Table 4.2. There are two nomenclatures for labelling them,
the Hermann–Mauguin system used in crystallography and the Schonflies system used in
spectroscopy. Ideally, there would be only one system which everybody uses, but this is unlikely
to come about since (a) both systems are very well established, (b) crystallographers require elements
of space symmetry that spectroscopists do not, and vice versa, (c) spectroscopists use a more extensive
range of point symmetry elements than crystallographers. The symmetry element described above for
the silicate tetrahedron is a rotation axis, with symbol n. Rotation about this axis by 360/n degrees
gives an identical orientation and the operation is repeated n times before the original configuration
is regained. In this case, n = 3 and the axis is a threefold rotation axis. The SiO4 tetrahedron possesses
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four threefold rotation axes, one in the direction of each Si–O bond. When viewed from another angle,
SiO4 tetrahedra possess twofold rotation axes [Fig.4.9 (b)] the central Si and bisect the O–Si–O
bonds. Rotation by 180◦ leads to indistinguishable orientations of the tetrahedra. The SiO4
tetrahedron possesses three of these twofold axes.
Crystals may display rotational symmetries 2, 3, 4 and 6. Others, such as n = 5, 7, are never observed
in 3D crystal structures based on a regular periodic repetition of the unit cell and its contents. This is
shown in Fig. 4.9(c), where a fruitless attempt has been made to pack pentagons to form a complete
layer; thus, individual pentagons have fivefold symmetry but the array of pentagons does not. For
hexagons with sixfold rotation axes (d), a complete layer is easily produced; both the individual
hexagons and the overall array exhibit sixfold symmetry. This is not to say that molecules which have
pentagonal symmetry, n = 5, cannot exist in the crystalline state. They can, of course, but their fivefold
symmetry cannot be exhibited by the crystal as a whole.

Figure 4.9 (a) Threefold and (b) twofold rotation axes; (c) the impossibility of forming a complete
layer of pentagons; (d) a complete layer of hexagons.
1.2.3 Mirror symmetry
A mirror plane, m, exists when two halves of, for instance, a molecule can be interconverted by
carrying out the imaginary process of reflection across the mirror plane. The silicate tetrahedron
possesses six mirror planes, one of which, running vertically and perpendicular to the plane of the
paper. The silicon and two oxygens, 1 and 2, lie on the mirror plane and are unaffected by reflection.
The other two oxygens, 3 and 4, are interchanged on reflection. A second mirror plane lies in the
plane of the paper; for this, Si and oxygens 3, 4 lie on the mirror but oxygen 2, in front of the mirror,
is the image of oxygen 1, behind the mirror.

1.2.4 Centre of symmetry and inversion axes

A centre of symmetry exists when any part of a structure can be reflected through this centre of
symmetry, which is a point, and an identical arrangement found on the other side. An AlO6 octahedron
has a centre of symmetry located on the Al atom. If a line is drawn from any oxygen, e.g. 1, through
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the centre and extended an equal distance on the other side, it terminates at another oxygen, 2. A
tetrahedron, e.g. SiO4, does not have a centre of symmetry (a) The inversion axis, n, is a combined
symmetry operation involving rotation (according to n) and inversion through the centre. A 4
(fourfold inversion) axis is shown in (c). The first stage involves rotation by 360/4 = 90◦ and takes,
for example, oxygen 2 to position 2'. This is followed by inversion through the centre, at Si, and leads
to position 3. Oxygens 2 and 3 are therefore related by a 4 axis. Possible inversion axes in crystals
are limited to 1 , 2 , 3 , 4 and 6 for the same reason that only certain pure rotation axes are allowed.
The onefold inversion axis is not a separate symmetry element, but is simply equivalent to the centre
of symmetry; also, the twofold inversion axis is the same as a mirror plane perpendicular to that axis.

Table 4.2 Symmetry elements

The identification of lattice planes

The spacing of the planes of lattice points in a crystal is an important quantitative aspect of its
structure. However, there are many different sets of planes (Fig. 1.9'), and we need to be able to label
them. Two-dimensional lattices are easier to visualize

Fig. 1.9' Some of the planes that can be drawn through the points of a rectangular space lattice and
their corresponding Miller indices (hkl): (a) (110), (b) (230), (c) (110), and (d) (010).
than three-dimensional lattices, so we shall introduce the concepts involved by referring to two
dimensions initially and then extend the conclusions by analogy to three dimensions. Consider a two-
dimensional rectangular lattice formed from a unit cell of sides a, b (as in Fig. 1.9'). Each plane in the
illustration (except the plane passing through the origin) can be distinguished by the distances at
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which it intersects the a and b axes. One way to label each set of parallel planes would therefore be
to quote the smallest intersection distances. For example, we could denote the four sets in the
illustration as (1a, 1b), (1/2 a,1/3 b), (−1a, 1b), and (∞a, 1b). However, if we agree to quote distances
along the axes as multiples of the lengths of the unit cell, then we can label the planes more simply
as (1, 1), (1/2 ,1/3 ), (−1, 1), and (∞, 1). If the lattice in Fig. 4.9' is the top view of a three-dimensional
orthorhombic lattice in which the unit cell has a length c in the z-direction, all four sets of planes
intersect the z-axis at infinity. Therefore, the full labels are (1, 1, ∞), ( 1/2, 1/3, ∞), (−1, 1, ∞), and (∞,
1, ∞).
The presence of fractions and infinity in the labels is inconvenient. They can be eliminated by taking
the reciprocals of the labels. As we shall see, taking reciprocals turns out to have further advantages.
The Miller indices, (hkl), are the reciprocals of intersection distances (with fractions cleared by
multiplying through by an appropriate factor, if taking the reciprocal results in a fraction). For
example, the (1, 1, ∞) planes in Fig. 4.9'a are the (110) planes in the Miller notation. Similarly, the (
1/2,1/3 , ∞) planes are denoted (230). Negative indices are written with a bar over the number, and
Fig. 4.9'c shows the ( 1 10) planes. The Miller indices for the four sets of planes in Fig. 1.9' are therefore
(110), (230), ( 1 10), and (010). Figure 1.10 shows a three dimensional representation of a selection of
planes, including one in a lattice with non-orthogonal axes.
The notation (hkl) refers to an individual plane. To specify a set of parallel planes we use the notation
{hkl}. Thus, we speak of the (110) plane in a lattice, and the set of all {110} planes that lie parallel
to the (110) plane. A helpful feature to remember is that, the smaller the absolute value of h in {hkl},
the more nearly parallel the set of planes is to the a axis (the {h00} planes are an exception). The
same is true of k and the b axis and l and the c axis. When h = 0, the planes intersect the a axis at
infinity, so the {0kl} planes are parallel to the a axis. Similarly, the {h0l} planes are parallel to b and
the {hk0} planes are parallel to c.
The Miller indices are very useful for expressing the separation of planes. The separation of the {hk0}
planes in the square lattice shown in Fig. 1.11 is given by
1 h2 + k 2 a
2
= d hk 0 = 1
4.1
d hk 0 a2 or (h 2 + k 2 ) 2

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Fig. 1.10 Some representative planes in three dimensions and their Miller indices.Note that a 0
indicates that a plane is parallel to the corresponding axis, and that the indexing may also be used for
unit cells with non-orthogonal axes.
By extension to three dimensions, the separation of the {hkl} planes of a cubic lattice is given by
1 h2 + k 2 + l 2 a
2
= or d hkl =
d hkl a2 (h 2 + k 2 + l 2 )
1
2

The corresponding expression for a general orthorhombic lattice is the generalization of this
expression:
1 h2 k2 l2
2
= 2
+ 2
+
d hkl a a a2

Fig. 1.12 The separation of the {220} planes is half that

Fig. 1.11 The dimensions of a unit cell
of the {110} planes. In general, the separation of the
and their relation to the plane passing
planes {nh,nk,nl} is n times smaller than the separation
through the lattice points.
of the {hkl} planes.

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Table 1.3 The seven crystal systems

Problem
1.1(a) Equivalent lattice points within the unit cell of a Bravais lattice have identical surroundings. What points within a
face-centred cubic unit cell are equivalent to the point (1/2, 0, 0)?
1.1(b) Equivalent lattice points within the unit cell of a Bravais lattice have identical surroundings. What points within a
body-centred cubic unit cell are equivalent to the point (1/2, 0, 1/2)?
1.2(a) Find the Miller indices of the planes that intersect the crystallographic axes at the distances (2a, 3b, 2c) and (2a,
2b, ∞c).
1.2(b) Find the Miller indices of the planes that intersect the crystallographic axes at the distances (1a, 3b, −c) and (2a,
3b, 4c).
1.3(a) Calculate the separations of the planes {111}, {211}, and {100} in a crystal in which the cubic unit cell has side
432 pm.
1.3(b) Calculate the separations of the planes {121}, {221}, and {244} in a crystal in which the cubic unit cell has side
523 pm.
1.4(a) The compound Rb3TlF6 has a tetragonal unit cell with dimensions a =651 pm and c =934 pm. Calculate the volume
of the unit cell.
1.4(b) Calculate the volume of the hexagonal unit cell of sodium nitrate, for which the dimensions are a =1692.9 pm and
c =506.96 pm.
1.5(a) The orthorhombic unit cell of NiSO4 has the dimensions a =634 pm, b =784 pm, and c =516 pm, and the density
of the solid is estimated as 3.9 g cm−3. Determine the number of formula units per unit cell and calculate a more precise
value of the density.
1.5(b) An orthorhombic unit cell of a compound of molar mass 135.01 g mol −1 has the dimensions a =589 pm, b =822
pm, and c =798 pm. The density of the solid is estimated as 2.9 g cm−3. Determine the number of formula units per unit
cell and calculate a more precise value of the density.
1.6(a) The unit cells of SbCl3 are orthorhombic with dimensions a =812 pm, b =947 pm, and c=637 pm. Calculate the
spacing, d, of the (411) planes.
1.6(b) An orthorhombic unit cell has dimensions a = 679 pm, b = 879 pm, and c = 860 pm. Calculate the spacing, d, of
the (322) planes.

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 POTENTIOMETRY
In potentiometry, information on the composition of a sample is obtained through the potential
appearing between two electrodes. Potentiometry is a classical analytical technique with roots before
the twentieth century. However, the rapid development of new selective electrodes and more sensitive
and stable electronic components since 1970 has tremendously expanded the range of analytical
applications of potentiometric measurements. Selective potentiometric electrodes are currently
widely used in many fields, including clinical diagnostics, industrial process control, environmental
monitoring, and physiology. For example, such devices are used in nearly all hospitals around the
globe for assessing several physiologically important blood electrolytes (K+, Na+, Ca2+, Mg2+, H+,
Cl−) relevant to various health problems. The speed at which this field has developed is a measure of
the degree to which potentiometric measurements meet the needs of the analytical chemist for rapid,
low-cost, and accurate analysis. In this part, the principles of direct potentiometric measurements,
based on ion-selective electrodes (ISEs), will be described. ISEs are chemical sensors with the longest
history. The field of ISE bridges fundamental membrane science with fundamental host–guest
chemistry.
1-Potentiometric method
The potentiometric method is required different equipment and operated using basic components
which are three; a reference electrode, the indicator electrode (metallic or ion selective electrode) and
the potential measuring device with accuracy to read change in potential about 0.2 V or less. The
potentiometry applies to determine the end points of the titrations; measurements of the pH of the
different solutions, measure the ion and compounds concentrations using ion-selective membrane
electrodes and detect thermodynamic equilibrium constants such as Ka, Kb, and Ksp.
General Principles

The diagrams of simple

potentiometric measurements

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B- Indicator Electrodes
In the potentiometry, the potential of the indicator electrode is proportional to the analyte activity.
Two classes of indicator electrodes are used in potentiometry: metallic electrodes and ion-selective
electrodes.
1- Metallic Electrode
Metallic electrode includes three types:

- Metallic Electrodes of the First Kind

An indicator electrode in which a metal is in contact with a solution containing its ion is called an
electrode of the first kind., If we place a copper electrode in a solution containing Cu2+, the electrode’s
potential due to the reaction (4)
Cu 2+ (aq) + 2e −  Cu(s) (4)
Nernst equation determines by the activity of Cu2+ as can be seen in eq. (5)
0.05916 1 0.05916 1
E = E Cu
o
2+
/ Cu
− log = +0.3419 − log .......(5)
2 a Cu 2 + 2 a Cu 2 +

If copper is the indicator electrode in a potentiometric electrochemical cell that also includes a
saturated calomel reference electrode:

SCE|| Cu2+(aq,aCu2+ =x) |Cu(s)

then we can use the cell potential to determine an unknown activity of Cu2+ in the indicator electrode’s
half-cell, using eq. (6)
0.05916 1
Ecell = Eind − E SCE + E j = +0.3419 − log − 0.268 + E j ....(6)
2 aCu 2 +

In general, if a metal, M, is in a solution of Mn+, the cell potential is

0.05916 1 0.05916
E cell = K − log =K+ log a M n
n aM n n

where K is a constant that includes the standard-state potential for the Mn+/M redox couple, the
potential of the reference electrode, and the junction potential.
The first kind is limited to the following metals: Ag, Bi, Cd, Cu, Hg, Pb, Sn, Tl, and Zn, due to
different reasons which include the slow kinetics of electron transfer at the metal–solution interface,
the formation of the metal oxides on the electrode’s surface, and interfering reactions-electrodes
Electrode of the first kind is extremely non-popular because the metallic indicator electrodes are
enormously non-selective and respond not only to their own ions but also to other more easily reduced
metal ions. Many metal electrodes can be used only in neutral or basic solutions because they dissolve

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in the presence of acids. Easily oxidized metal can be used only when analyte solutions are deaerated
to remove oxygen. Certain metals provide no reproducible potentials.
2- Electrodes of the Second Kind
Metal electrode responds to the activities of anions that form sparingly soluble precipitates or
stable complexes with such metal ions. We also can use this electrode to determine the activity of
anions if it is in equilibrium with Mn+. For example, the potential of an Ag electrode in a solution of
Ag+ can be obtained as in eq. (7)
1 1
E Ag + / Ag = E Ag
o
+ − 0.05916log = +0.7996 − 0.05916log
/ Ag
a Ag + a Ag + (7)

If we saturate the indicator electrode’s half-cell with AgI, the solubility reaction, eq. (8)
AgI ( s )  Ag + (aq) + I − (aq) (8)
determines the concentration of Ag+; thus
K sp, AgI
a Ag + =
aI − (9)

where Ksp, AgI is the solubility product for AgI. Substituting equation (9) into equation (7)
aI −
Ecell = +0.7996V − 0.05916log
K sp, AgI
(10)
we find that the potential of the silver electrode is a function of the activity of I–. If we incorporate
this electrode into a potentiometric electrochemical cell with a saturated calomel electrode:
SCE|| AgI(s), I-(aq,aI- =x) |Ag(s)

the cell potential is

Ecell = K − 0.05916log a I −
(11)
where K is a constant that includes the standard-state potential for the Ag+/Ag redox couple, the
solubility product for AgI, the reference electrode potential, and the junction potential.
If an electrode of the first kind responds to the activity of an ion that is in equilibrium with Mn+, we
call it an electrode of the second kind. Two common electrodes of the second kind are the calomel
and the silver/silver chloride reference electrodes.

3- Inert Metallic Electrodes for Redox Systems

An electrode also can serve as a source of electrons or as a sink for electrons in an unrelated redox
reaction, in which case we call it a redox electrode. The Pt cathode is a redox electrode because its

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potential is determined by the activity of Fe2+ and Fe3+ in the indicator half-cell. Note that a redox
electrode’s potential often responds to the activity of more than one ion, which can limit its usefulness
for direct potentiometry. Several inert conductors can be used to monitor redox systems such as
platinum, gold, palladium, and Non-metal (carbon)
Membrane Electrodes
If metals are the only useful materials for constructing indicator electrodes, then there would be
few useful applications of potentiometry. In 1901 Fritz Haber discovered that there is a change in
potential across a glass membrane when its two sides are in solutions of different acidity. Membrane
electrode is fundamentally different from metal electrodes both in the design and in the principle.
The existence of this membrane potential led to the development of a whole new class of indicator
electrodes called ion-selective electrodes (ISEs). In addition to the glass pH electrode, ion-selective
electrodes are available for a wide range of ions. It also is possible to construct a membrane electrode
for a neutral analyte by using a chemical reaction to generate an ion that can be monitored with an
ion-selective electrode. The development of new membrane electrodes continues to be an active area
of research.
1.7.2.1. Ion selective electrode (ISE)
The ion-selective electrode is an indicator electrode capable of selectively measuring the activity of
a particular ionic species (known as the primary or analyte ion). Such electrodes exhibit a fast
response and a wide linear range, are not affected by color or turbidity, are not destructive, and are
very inexpensive. Ion-selective electrodes can be assembled conveniently in a variety of shapes and
sizes. Specially designed cells allow flow or microliter analyses Ion-selective electrodes are mainly
membrane-based devices, consisting of permselective ion-conducting materials, which separate the
sample from the inside of the electrode. On the inside is a filling solution containing the ion of interest
at a constant activity. The membrane is usually nonporous, water insoluble, and mechanically stable.
The composition of the membrane is designed to yield a potential that is primarily due to the ion of
interest (via selective binding processes, e.g., ion exchange, which occur at the membrane– solution
interface).The trick is to find a membrane that will selectively bind the analyte ions, leaving co-ions
behind. Membrane materials, possessing different ion recognition properties, have thus been
developed to impart high selectivity
The ion selective electrode measures the activity of an ion in a solution by determination the electric
potential formed across a membrane when the electrode is submerged in the solution. there are
different ISEs for various ions including H+, NH3, Br-, Cd2+, Ca2+, CO2, Cl-, Cu2+, CN-, F-, BF4-, I-,

Page 14 of 44
Pb2+, NO3, CLO4-, K+, Ag+, Na+ and many others. The ISE can take many forms, but all of them have
the basic parts. A combination electrode contains both a reference electrode and the ion selective
electrode housed in the same casing. A voltammeter and display is connected to the electrodes by a
cable or contained within the electrode housing. The principles of ISE based on a voltage produced
due ion-exchange occurring between the sample and internal electrolyte. Two sides of the membrane
produce potential similar to Galvanic Cell. ISEs all work on the basic of Galvanic cell. The galvanic
cell consists of two half-cell that are made from two different metals which are imersed into separated
solutions of their salts. The ISEs are utilized to determine endpoint and perform direct measurements
Ecell = EISE − Eref .
Reference (sample) || [A]sample (aq, aA=x) || [A]internal (aq, aA=y) || reference(internal)
where the ion-selective membrane is shown by the vertical slash separating the two solutions
containing analyte—the sample solution and the ion-selective electrode’s internal solution. The
electrochemical cell includes two reference electrodes: one immersed in the ion-selective electrode’s
internal solution and one in the sample. The cell potential, therefore, is shown in the following
eqation:
E𝐶𝑒𝑙𝑙= 𝑬𝒓𝒆𝒇,𝒊𝒏𝒕− 𝑬𝒓𝒆𝒇𝒆𝒓,𝒔𝒂𝒎 + 𝑬𝒎𝒆𝒎𝒃 + 𝑬𝒋 (𝟏)
where Emem is the potential across the membrane. Because the junction potential and the potential of
the two reference electrodes are constant, any change in Ecell is a result of a change in the membrane’s
potential.
The analyte interaction with the membrane generates a membrane potential if there is a difference
in its activity on the membrane’s two sides.

Figure General
design of the Ion
Selective
Electrode

Current is carried through the membrane by the movement of either holes or electrons present in the
membrane’s matrix. The membrane potential is given by the following Nernst-like equation 13.
RT ( a A )int (2)
Emem = Easym − ln
nF ( a A ) samp

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where (aA)samp is the analyte concentration in the sample, (aA)int is the concentration of analyte in the
ion-selective electrode’s internal solution, and n is the analyte charge. Ideally, Emem is zero when
(aA)int = (aA)samp. The term Easym, which is an asymmetry potential, accounts for the fact that Emem is
usually not zero under these conditions.
Substituting equation 2 into equation 1, assuming a temperature of 25 oC, and rearranging gives
RT (3)
Ecell = K + ln(a A ) samp
nF

where K is a constant that includes the potentials of the two reference electrodes, the junction
potentials, the asymmetry potential, and the analyte activity in the internal solution. Equation 14 is a
general equation and applies to all types of ion-selective electrodes. It should be noted again that ISEs
sense the activity, rather than the concentration of ions in solution. The term “activity” is used to
denote the effective (active) concentration of the ion. The difference between concentration and
activity arises because of ionic interactions (with oppositely charged ions) that reduce the effective
concentration of the ion. The activity of an ion i in solution is related to its concentration ci by the
following equation:
a A = f AC A

where fA is the activity coefficient. The activity coefficient depends on the types of ions present and
on the total ionic strength of the solution. The activity coefficient thus approaches unity (i.e., aA
=CA) in very dilute solutions.
- Advantages of ion selective electrode
a- Ion selective electrodes are relatively inexpensive, simple to use and have the extremely wide
range of applications and wide concentration range compared to other analytical techniques.
b- Ion selective electrodes are very rapid and immediate use such as time sensitive test (blood test).
c- Ion selective electrodes are valuable for continuous monitoring of changes in concentration for
example in potentiometric titrations or monitoring the uptake of nutrients or the consumption of
reagents.
d- They are useful in biological /medical applications because they measure the activity of the ion
directly rather than the concentration.
e- ISEs are one of the few techniques which can measure both positive and negative ions.
F- They are unaffected by the sample colour or turbidity.
g- ISEs can be used in aqueous solutions over a wide temperature range from 0 to 80oC.
h- non- destructive so they have no consumption of the analyte
i-non contaminating the sample
Page 16 of 44
Selectivity of Membranes
The term ‘‘Ion-selective electrodes’’ reflects the capability of ISEs to discriminate between ions.
Ideally, an ISE responds to only one kind of species in a mixed sample. Of course, the real-world
electrodes show only limited selectivity. The potentiometric selectivity of an electrode is its ability to
respond only to the target analyte ion in the presence of other ions. In other words, if the activity of
the target ion is the same, the electrode potential and the measured EMF (ideally) are also the same
whatever is the composition of the sample. Importantly, the potential of an ideally selective electrode
is constant at a constant activity of the analyte, but not necessarily at a constant concentration. The
selectivity of an ISE to ions in the presence of anthor ions is quantified with the so-called selectivity
coefficient: the parameter KA,B in the Nikolsky equation.
Equation (3) has been written on the assumption that the electrode responds only to the ion of interest,
A. In practice, no electrode responds exclusively to the ion specified. The actual response of the
electrode in a binary mixture of the primary and interfering ions (A and B, respectively) is given by
the Nikolskii–Eisenman equation:
nA
0.05916
Ecell = K + log(a A + K A, B aBn B )
nA

(a A )
K A, B = nA
nB
( aB )
Where nA and nB are the charge of the sample and interferent. (aA) and (aB) are the activities of analyte
and interfering yielding identical cell potentials. a quantitative measure of the electrode ability to
discriminate against the interfering ion (i.e., a measure of the relative affinity of ions A and B toward
the ion-selective membrane). For example, if an electrode is 50 times more responsive to A than to B,
kA,B has a value of 0.02. A kA,B value of 1.0 corresponds to a similar response for both ions. When kA,B
>> 1, then the ISE responds better to the interfering ion B than to the target ion A. Usually, kA,B is
smaller than 1, which means that the ISE responds more selectively to the target ion. The lower the
value of kA,B, the more selective is the electrode. Selectivity coefficients lower than 10−5 have been
achieved for several electrodes. For an ideally selective electrode, the kA,B would equal zero (i.e., no
interference). Obviously, the error in the activity aA due to the interference of B would depend on
their relative levels. The term nA/nB corrects for a possible charge difference between the target and
interfering ions. Normally, the most serious interferences have the same charge as the primary ion so
that nA/nB = 1. In practice, the contribution of all interfering ions present in the sample matrix (Σ kA,B

Page 17 of 44
anA/nB) should be included in the Nikolskii–Eisenman equation. For example, for a sodium electrode
immersed in a mixture of sodium, potassium, and lithium, the response is given by:
n n
Na + Na +
0.05916 n n +
Ecell =K+ log(a Na+ + K Na+ , K + a K +K + + K Na+ , Li+ a LiLi+ )
nA

Accordingly, an ISE displays a selective response when the activity of the primary ion is much larger
than the summation term of the interferents, specifically, aA >> ∑ kA,B aBnA/nB. Under this condition,
the effect of interfering ions is negligible, and changes in the measured potential can be related with
confidence to variations in the activity of the target ion. The selectivity coefficients thus serve as
guidelines as to how far a given ISE should be applicable for a particular analytical problem. Non-
selective ISEs are rarely useful for real-life applications (with the exception of their combination with
the operation of ISE arrays. In reality, equations with more than two components are rarely used.
Deviations from the Nikolski–Eisenman equation have been reported for various situations
(particularly for mixtures of ions of different charge, in the case of non-Nernstian behavior of
interfering ions, and due to the concentration dependence of kA,B). It is important for the analytical
chemist to realize the selectivity coefficient of a particular electrode.Various methods have been
suggested for determining the selectivity coefficient, including the fixed-interference method,
separate solution method, and the fixed primary ion method. The most popular fixed interference
method involves two solutions, one containing a constant concentration of the interfering ion and the
second, containing a zero concentration. The obvious approach to converting potentiometric
measurements from activity to concentration is to make use of an empirical calibration curve, such as
the one shown in Figure 3. Electrodes potentials of standard solutions are thus measured and plotted
versus the concentration. Since the ionic strength of the sample is seldom known, it is often useful to
add a high concentration of an electrolyte to the standards and the sample to maintain approximately
the same ionic strength (i.e., the same activity coefficient). The ionic strength adjustor is usually a
buffer (since pH control is also desired for most ISEs). The empirical calibration plot thus yields
results in terms of concentration. Theoretically, such a plot should yield a straight line, with a slope
of approximately 59/nA mV (Nernstian slope). Detection by means of ion-selective electrodes may be
performed over an exceedingly broad concentration range, which, for certain electrodes, may embrace
five orders of magnitude. In practice, the usable range depends on other ions in the solution. Departure
from the linearity is commonly observed at low concentrations (about 10−6 M) due to the presence of
coexisting ions. The extent of such departure (and the minimum activity that can be accurately

Page 18 of 44
measured) depend on the selectivity coefficient as well as upon the level of the interfering ion (Figure
nA

4). The detection limit for the analyte ion is defined by a A, min = K A, B a An B

Figure 3 Typical calibration plot for a monovalent ion.

and corresponds to the activity of A at the intersection of the asymptotes in the E/logaA calibration
plot, that is, where the extrapolated linear and zero-slope segments meet (see Fig 5). It is only when
the plot becomes almost horizontal that the activity measurement becomes impossible.

aB
aB
aB

Figure 4 The potential response of an ion-selective electrode versus activity of ion A in the
presence of different levels of an interfering ion B.

Figure 5 Determination of the detection limit of ion-selective electrodes.

a. Glass Electrodes

Page 19 of 44
Glass electrodes detect the activity of univalent ions. The selectivity of the thin ion-sensitive glass
membrane for these ions is obtained by change the chemical composition of the membrane.
pH Electrodes
The most common potentiometric device is the pH electrode. This electrode has been widely utilized
for pH measurements for several decades. Besides direct pH measurements, the pH glass electrode is
commonly employed as the transducer in various gas and biocatalytic sensors, involving proton-
generating/consuming reactions. Its remarkable success is attributed to its outstanding analytical
performance, in particular its extremely high selectivity for hydrogen ions, its remarkably broad
response range, and its fast and stable response. The phenomenon of glass selectivity was reported by
Cremer in 1906. Glass pH electrodes of different configurations and dimensions have been in routine
use since the early 1940s following their commercial introduction by A. Beckman. A schematic of a
commonly used configuration is shown in Figure 6. This consists of a thin, pH-sensitive glass
membrane sealed to the bottom of an ordinary glass tube. The composition of the glass membrane is
carefully controlled. Usually, it consists of a three-dimensional silicate network, with negatively
charged oxygen atoms, available for coordinating cations of suitable size. The first commercial glass
electrodes were manufactured using Corning 015, a glass with a composition that is approximately
22% Na2O, 6% CaO and 72% SiO2. Some of the more popular glasses have three-component
compositions of 80% SiO2–10% Li2O–10% CaO. Inside the glass bulb are a dilute hydrochloric acid
solution and a silver wire coated with a layer of silver chloride. The electrode is immersed in the
solution whose pH is to be measured, and connected to an external reference electrode. (In the so-
called combination electrode, the external reference electrode is combined with the ion-selective
electrode into one body.) The rapid equilibrium established across the glass membrane, with respect
to the hydrogen ions in the inner and outer solutions, produces a potential:
When immersed in an aqueous solution for several hours, the outer approximately 10 nm of the
membrane’s surface becomes hydrated, resulting in the formation of negatively charged sites, —SiO–
. Sodium ions, Na+, serve as counter ions. Because H+ binds more strongly to —SiO– than does Na+,
they displace the sodium ions
H + + − SiO− Na +  − SiO− H + + Na +

giving rise to the membrane’s selectivity for H+. The transport of charge across the membrane is
carried by holes or electrons according to the type of silicate (p- or n-types semiconductors .
Cell Potentials for pH measurements

Page 20 of 44
 The potentials of the two reference electrodes (internal and external ) depend on the
electrochemical characteristics of their respective redox couples. The potential across the glass
membrane: depends on the physicochemical characteristics of the glass and its response to ionic
concentrations on both sides of the membrane. Four potential are produced including EAg,AgCl and
EAg,AgCl , are reference electrode potentials that are constant. A 3rd potential is the junction potential
Ej across the salt bridge that separates the calomel electrode from the analyte solution. The 4th and
most important potential is the boundary potential, Eb, which varies with the pH of the analyte
solution.
E𝐶𝑒𝑙𝑙= 𝑬𝒓𝒆𝒇,𝒊𝒏𝒕− 𝑬𝒓𝒆𝒇𝒆𝒓,𝒔𝒂𝒎 + 𝑬𝒃 + 𝑬𝒋
The potential of a glass electrode using Corning 015 obeys the equation
Ecell = K + 0.0591log aH +

A pH range of approximately over 0.5 to 9. At more basic pH levels the glass membrane is more
responsive to other metal ions, such as Na+ and K+. Replacing Na2O and CaO with Li2O and BaO
extends the useful pH range of glass membrane electrodes to pH levels greater than 12.
Glass membrane pH electrodes are often available in a combination form that includes both the
indicator electrode and the reference electrode. The use of a single electrode greatly simplifies the
measurement of pH. An example of a typical combination electrode is shown in glass electrode,
Figure 6 .
Because the typical thickness of an ion-selective electrode’s glass membrane is about 50 mm, they
must be handled carefully to avoid cracks or breakage. Glass electrodes usually are stored in a solution
of a suitable storage buffer recommended by the manufacturer, which ensures that the membrane’s
outer surface is fully hydrated. If your glass electrode does dry out, you must recondition it by soaking
for several hours in a solution containing the analyte. The composition of a glass membrane changes
over time, affecting the electrode’s performance. The average lifetime for a typical glass electrode is
several years. Whenever there is a charge imbalance across any material, there is an electrical
potential across the material: – the concentration of protons inside the membrane is constant and the
concentration outside is determined by the concentration, or activity, of the protons in the analyte
solution. This concentration difference produces the potential difference that we measure with a pH
meter.
A glass electrode system contains two reference electrodes: the external calomel electrode or
silver/silver chloride electrode and the internal silver/silver chloride electrode. The internal
electrode consists of a thin, pH-sensitive glass membrane sealed onto one end of a heavy-walled glass

Page 21 of 44
or plastic tube. A small volume of dilute hydrochloric acid with saturated KCl with silver chloride is
contained in the tube. A silver wire in this solution forms a silver/silver chloride reference electrode.
It is the thin glass membrane bulb at the tip of the electrode that responds to pH.

Figure 6 Schematic diagram showing a combination glass electrode for measuring pH.
Glass Membrane Potential
The boundary potential is determined by potentials, E1 and E2, which appear at the two surfaces of
the glass membrane from the reactions:
− SiO− H + (s)int  H + (aq)int + −SiO− ( s)

− SiO− H + (s)ext  H + (aq)ext + −SiO− (s)

where int. refers to the interface between the interior of the glass and the internal solution and ext.
refers to the interface between the analyte solution and the exterior of the glass. These two reactions
cause the two glass surfaces to be negatively charged with respect to the solutions with which they
are in contact.
The resulting difference in potential between the two surfaces of the glass is the boundary
potential, which is related to the activities of hydrogen ion in each of the solutions by the Nernst-
like equation:
Eb = E Int − E Ext = −0.0591log
(a )
H + int
(a )
H + ext

For a glass pH electrode, the hydrogen ion activity of the internal solution is held constant:
E b = C + 0.0591 log( a H + ) ext

C = −0.0591 log( a H + ) int

Page 22 of 44
Describing Selectivity
The effect of an alkali metal ion on the potential across a membrane can be accounted for by inserting
an additional term:
Eb = C + 0.0591log(aH + + K H , M aM + )

where kH,B is the selectivity coefficient for the electrode. Selectivity coefficients range from zero (no
interference) to values greater than unity. Before using the pH electrode, it should be calibrated using
two (or more) buffers of known pH. Many standard buffers are commercially available, with an
accuracy of •±0.01 pH unit. Calibration must be performed at the same temperature at which the
measurement will be made; care must be taken to match the temperature of samples and standards.
The exact procedure depends on the model of pH meter used. Modern pH meters, such as the one
shown in Figure 7, are microcomputer-controlled, and allow double-point calibration, slope
calculation, temperature adjustment, and accuracy to •0.001 pH unit, all with few basic steps. The
electrode must be stored in an aqueous solution when not in use, so that the hydrated gel layer of the
glass does not dry out. A highly stable response can thus be obtained over long time periods.
Measurements of pH can also be performed using other types of potentiometric sensors. Non-glass
electrodes offer various advantages for certain pH measurements (particularly intravascular and
intraluminal clinical applications, food assays, and operation in fluoride media), including ease of
preparation, low electrical resistance, and safety in handling. The most common examples are the
quinhydrone electrode in which the response is due to a proton transfer redox reaction (of the
quinone–hydroquinone couple) and the antimony electrode (based on the redox reaction between
antimony and antimony oxide involving protons). Other metal–-metal oxide couples, such as
palladium–-palladium oxide, have been applied for pH measurements. Membrane electrodes based
on various neutral hydrogen ion carriers (e.g., tridodecylamine) can also be employed. The resulting
electrodes exhibit excellent selectivity, reproducibility, and accuracy, but their dynamic range is
inferior compared with glass electrodes.
Errors in pH measurements
1. pH measurements cannot be more accurate than standards (±0.01)
2. Junction potential: exists if composition of the analyte is different from that of the pH standards.
To minimize this, use pH standards with the same composition. Junction potential dependent on ionic
strength of solution – Ej may be a significant error if test solution has different ionic strength than
buffers
3. Junction potential drift: exists when there is formation of AgCl (precipitation) or Ag

Page 23 of 44
(reduction) at the porous plug. To minimize this, recalibrate the electrode every 2 h.
Also, Caused by slow changes in [KCl] and [AgCl]→ re-calibrate.
4. Equilibration time: It takes time for an electrode to equilibrate with the analyte solution, esp. in a
poorly buffered solution (pH varies greatly).
5. Dehydration of glass membrane: If the membrane has dried out, recondition it in water for several
hours before use. Hydration of Glass Surface – glass electrodes must be kept hydrated for good
measurement. It must be rehydrated for 24 hrs if it dries out. Because it will cause noisy readings,Or
A dry electrode will not respond to H+ correctly
6. Temperature: A pH meter should be calibrated at the temperature at which pH measurements will
be made. Temperature of electrodes, calibration buffers and sample solutions must be the same
primarily because of T in Nernst Eq.
7. Na or alkaline error: when H+ is very low and Na+ is high, the pH electrode responds to Na+ as if it
were H+. So the apparent H+ is higher, or apparent pH is lower. Alkaline error or sodium error occurs
when pH is very high (e.g., 12) because Na+ concentration is high (from NaOH used to raise pH) and
H+ is very low. Electrode responds slightly to Na+ and gives a lower reading than actual pH. This is
related to the concept of selectivity coefficients where the electrode responds to many ions but is most
selective for H+. Problem occurs because Na+ is 10 orders of magnitude higher than H+ in the solution.
8. Acid error: In strong acid, perhaps the glass surface is saturated with H+, so the apparent H+ is
lower, the apparent pH is higher, see Figure 8. Acid Error, electrode reads slightly higher than the
actual pH in very acidic solutions.
9. pH measurements are only as good as the buffers used to calibrate.
10. Response Time related to activity for all potentiometric electrodes is fast at high activity
(concentration) however it is slow at low concentrations.

Figure 8 The alkaline and acid errors of several glass pH electrodes: A, Corning 015/H2SO4; B,
Corning 015/HCl; C, Corning 015/1M Na+; D, Beckman-GP/1M Na+; E, L&N BlackDot/1M Na+; F,
Beckman E/1M Na+;G, Ross electrode.
Glass Electrodes for Other metal ions
Page 24 of 44
From the early days of glass pH electrodes, alkaline solutions were noted to display some interference
on the pH response. Deliberate changes in the chemical composition of the glass membrane (along
with replacements of the internal filling solution) have thus led to electrodes responsive to monovalent
cations other than hydrogen, including sodium, ammonium, and potassium. This usually involves the
addition of B2O3 or Al2O3 to sodium silicate glasses, to produce anionic sites of appropriate charge
and geometry on the outer layer of the glass surface.The observation that the Corning 015 glass
membrane responds to ions other than H+ led to the development of glass membranes with a greater
selectivity for other metal ions. Membrane with a composition of 11% Na2O, 18% Al2O3 or B2O3,
and 71% SiO2 is used as an ion-selective electrode for Na+.
For example, the sodium- and ammonium-selective glasses have the compositions 11%Na2O –
18%Al2O3 –71%SiO2 and 27%Na2O – 4%Al2O3 –69%SiO2, respectively. Unlike sodium silicate
glasses (used for pH measurements), these sodium aluminosilicate glasses possess what may be
termed AlOSiO− sites with a weaker electrostatic field strength and a marked preference for cations
other than protons. The overall mechanism of the electrode response is complex but involves a
combination of surface ion exchange and ion diffusion steps. To further minimize interference from
hydrogen ions, it is desirable to use solutions with pH values higher than 5. Improved mechanical and
electrical properties can be achieved using more complex glasses containing various additives. Other
glass ion-selective electrodes have been developed for the analysis of Li+, K+, Rb+, Cs+, NH4+, Ag+,
and Tl+. Table 1 shows the different chemical composition of ISE for Na+, Li+ and K+ .
b- Solid-State Ion-Selective Electrodes
Considerable work has been devoted to the development of solid membranes that are selective
primarily to anions. A solid-state ion-selective electrode uses a membrane consisting of either a
polycrystalline inorganic salt or a single crystal pellets, or mixed crystals of an inorganic salt.
b1-Ion-selective electrodes for sulphide:
We can fashion a crystalline solid-state ion-selective electrode by sealing of single crystal of 1–2 mm
thick pellet of Ag2S, or single crystal mixture of Ag2S and a second silver salt or another metal
sulphide—into the end of a non-conducting plastic cylinder, filling the cylinder with an internal
solution containing the analyte, and placing a reference electrode into the internal solution. Figure 9
shows a typical design.
The membrane potential for a Ag2S pellet develops as the result of a difference in the extent of the
solubility reaction
Ag 2 S ( S )  2 Ag + (aq) + S 2− (aq)

Page 25 of 44
on the membrane’s two sides, with charge carried across the membrane by Ag+ ions. When we use
the electrode to monitor the activity of Ag+, the cell potential is
Ecell = K + 0.0591log a Ag +

The membrane also responds to the activity of S2-, with a cell potential of
0.0591
Ecell = K − log aS 2−
2
If we combine an insoluble silver salt, such as AgCl, with the Ag2S, then the membrane potential also
responds to the concentration of Cl–, with a cell potential of
Ecell = K − 0.0591log aCl −
By mixing Ag2S with CdS, CuS, or PbS, we can make an ion-selective electrode that responds to
the activity of Cd2+, Cu2+, or Pb2+. In this case the cell potential is
0.0591
Ecell = K + log aM 2+
2

Figure 9 Schematic diagram of a solid-

state electrode. The internal solution
contains a solution of analyte of fixed
activity.

where a M2+ is the activity of the metal ion.

The selectivity of these ion-selective electrodes is determined by the relative solubility of the
compounds. A Cl– ISE using a Ag2S/AgCl membrane is more selective for Br– (KCl–/Br– = 102) and
for I– (KCl–/I– = 106) because AgBr and AgI are less soluble than AgCl. If the activity of Br– is
sufficiently high, AgCl at the membrane/solution interface is replaced by AgBr and the electrode’s
response to Cl– decreases substantially. Most of the polycrystalline ion-selective electrodes listed in
Table 2 can be used over an extended range of pH levels. The equilibrium between S2– and HS– limits
the analysis for S2– to a pH range of 13–14.
b2-Ion-selective electrodes for F-
An example of a very successful solid-state sensor is the fluoride-ion selective electrode. Such a
single-crystal device is by far the most successful anion-selective electrode. The membrane of a F–
ion-selective electrode is fashioned from a single crystal of LaF3, which is usually doped with a small
amount of EuF2 to enhance the membrane’s conductivity. Because EuF2 provides only two F–ions—
Page 26 of 44
compared to the three F– ions in LaF3, each EuF2 produces a vacancy (“holes”) in the crystal’s lattice.
Fluoride ions pass through the membrane by moving into adjacent vacancies. As shown in Figure 10,
by jumping from one vacancy defect to another), thus establishing the desired potential difference.
The LaF3 membrane is sealed into the end of a non-conducting plastic cylinder, which contains a
standard solution of F–, typically 0.1 M NaF, and a Ag/AgCl reference electrode. Such a solid-state
membrane derives its selectivity from restriction of the movement of all ions, except the fluoride of
interest. The latter moves by migration through the crystal lattice. The membrane potential for a F–
ISE results from a difference in the solubility of LaF3 on opposite sides of the membrane, with the
potential given by
Ecell = K − 0.0591 log a F −

One advantage of the F– ion-selective electrode is its freedom from interference. The only significant
exception is OH– (KF–/OH– = 0.1), which imposes a maximum pH limit for a successful analysis. Below
a pH of 4 the predominate form of fluoride in solution is HF, which does not contribute to the
membrane potential. For this reason, an analysis for fluoride must be carried out at a pH greater than
4. Hence, the electrode is limited to use over the pH range of 0–8.5. The electrode exhibits at least a
1000 : 1 preference for fluoride over chloride or bromide ions. Unlike a glass membrane ion-selective
electrodes, a solid-state ISE does not need to be conditioned before it is used, and it may be stored
dry. The surface of the electrode is subject to poisoning, as described above for a Cl– ISE in contact
with an excessive concentration of Br–. If an electrode is poisoned, it can be returned to its original
condition by sanding and polishing the crystalline membrane.

Figure 10 Migration of the fluoride ion through the LaF3 lattice (doped with EuF2). The vacancies
created within the crystal cause jumping of neighboring F− into the vacancy.
c. Liquid Membrane Electrodes
Liquid-membrane-type ISEs, based on water-immiscible liquid substances impregnated in a
polymeric membrane, are widely used for direct potentiometric measurements. Such ISEs are
particularly important because they permit direct measurements of several polyvalent cations as well
as certain anions. The polymeric membrane separates the test solution from the inner compartment,
containing a standard solution of the target ion (into which a silver–silver chloride wire is dipped).
The filling solution usually contains a chloride salt of the primary ion, as desired for establishing the

Page 27 of 44
potential of the internal silver–silver chloride wire electrode. The membrane-active (recognition)
component can be an ion exchanger or a neutral macrocyclic compound. The selective extraction of
the target ion at the sample–membrane interface creates the electrochemical phase boundary potential.
The membranes are commonly prepared by dissolving the recognition element, a plasticizer (e.g., o-
nitrophenyl ether, which provides the properties of liquid phase), and the PVC in a solvent such as
tetrahydrofuran. (The recognition element is usually present in 1–3% amount.) Slow (overnight)
evaporation of the solvent leaves a flexible membrane of 50–200 nm thickness, which can be cut
(with a cork borer) and mounted on the end of plastic tube. The ion-discriminating ability (and hence
the selectivity coefficient) depends not only on the nature of the recognition element but also on the
exact membrane composition, including the membrane solvent and the nature and content of the
plasticizer. The extraction properties of the membrane can be further improved by adding ion pairing
agents to the plasticizer.
c1-Ion Exchanger Liquid membrane Electrodes
Another class of ion-selective electrodes uses a hydrophobic membrane containing a liquid organic
complexing agent that reacts selectively with the analyte. Three types of organic complexing agents
have been used: cation exchangers, anion exchangers, and neutral ionophores. A membrane
potential exists if the analyte’s activity is different on the two sides of the membrane. Current
is carried through the membrane by the analyte.
One of the most successful liquid membrane electrodes is selective toward calcium. Such an
electrode relies on the ability of phosphate ions to form stable complexes with the calcium ion. It uses
a liquid cation exchanger, consisting of an aliphatic diester of phosphoric acid [(RO)2PO−2 with R
groups in the C8–C16 range], that possesses high affinity for calcium ions. which uses a porous
plastic membrane saturated with the cation exchanger di-(n-decyl) phosphate. As shown in Figure
12, the membrane is placed at the end of a non-conducting cylindrical tube, and is in contact with two
reservoirs. The outer reservoir contains di-(n-decyl) phosphate in di-n-octylphenylphosphonate,
which soaks into the porous membrane. The inner reservoir contains a standard aqueous solution of
Ca2+ and a Ag/ AgCl reference electrode. Calcium ion-selective electrodes are also available in which
the di-(n-decyl) phosphate is immobilized in a polyvinyl chloride (PVC) membrane, eliminating the
need for the outer reservoir containing di-(n-decyl) phosphate.

Page 28 of 44
 Figure 12 Schematic diagram showing a
liquid-based ion-selective electrode for
Ca2+. The structure of the cation exchanger,
di-(n-decyl) phosphate, is shown in red.

The membrane potential for the Ca2+ ISE develops as the result of a difference in the extent of
the complexation reaction
𝑪𝒂𝟐+ (𝒂𝒒) + 𝟐(𝑪𝟏𝟎 𝑯𝟐𝟏 𝑶)𝟐 𝑷𝑶−
𝟐 (𝒎𝒆𝒎) ↔ 𝑪𝒂[(𝑪𝟏𝟎 𝑯𝟐𝟏 𝑶)𝟐 𝑷𝑶𝟐 ]𝟐 (𝒎𝒆𝒎)

on the two sides of the membrane, where (mem) indicates a species that is present in the membrane.
The cell potential for the Ca2+ ion-selective electrode is
𝟎. 𝟎𝟓𝟗𝟏
𝑬𝒄𝒆𝒍𝒍 = 𝑲 + 𝒍𝒐𝒈 𝒂𝑪𝒂𝟐+
𝟐
The selectivity of this electrode for Ca2+ is very good, with only Zn2+ showing greater selectivity.
Table 3 lists the properties of several liquid-based ion-selective electrodes. An electrode using a liquid
reservoir can be stored in a dilute solution of analyte and needs no additional conditioning before use.
The lifetime of an electrode with a PVC membrane, however, is proportional to its exposure to
aqueous solutions. For this reason these electrodes are best stored by covering the membrane with a
cap along with a small amount of wetted gauze to maintain a humid environment. Before using the
electrode it is conditioned in a solution of analyte for 30–60 minutes. Calcium activities as low as
0.5x10-6 M can be measured, with selectivity coefficients of KCa,Mg and KCa,K of 0.02 and 0.001,
respectively. Such potential response is independent of the pH over the pH range 5.5 to 11.0. Above
pH 11, Ca(OH)2is formed, while below 5.5, protons interfere. Because of its attractive response
characteristics, the calcium ISE has proved to be a valuable tool for the determination of calcium ion
activity in various biological fluids.
Anion exchangers, such as lipophilic quaternary ammonium salts (e.g., see Figure 13) or
phosphonium salts, have been employed for the preparation of anion-selective sensors. The resulting
ISEs usually lack an anion recognition function, and hence display anion selectivity corresponding to
the anion partition into the supporting hydrophobic membrane. This gives rises to the following
selectivity order, which is known as the Hofmeister series:

Page 29 of 44
large lipophilic anions >ClO4− >IO4−>SCN−>I−>NO3 −>Br- >Cl-> HCO3- >H2PO4- (i.e., with
maximum response to lipophilic anions). Accordingly, several commercial sensors (e.g.,NO3-
“selective” electrodes), based on ion-exchange type membranes, suffer an interference from lipophilic
anions (e.g., ClO4-). Sensors responsive to anionic macromolecules have also been developed. A very
successful example is the use of the quaternary ammonium salt tridodecylmethylammonium chloride
(TDMAC) for detecting the clinically important drug heparin. Apparently, the polyionic heparin is
favorably extracted into the membrane through ion-pairing interaction with the positively charged
nitrogen atoms (Figure 14). Such an extraction process results in a steady-state change in the phase
boundary potential at the membrane–sample interface. Analogous potentiometric measurements of
other macromolecular polyanionic(e.g., polyphosphates, DNA) or polycationic (e.g., protamine,
polyarginine) species, based on the use of various lipophilic ion exchangers, have been prepared. Ion
exchange electrodes sensitive to large organic cations have also been described. For example, PVC
membranes containing diononylnaphthalenesulfonic acid (DNNS) have been used for the detection
of drugs of abuse (e.g., opiate alkaloids). Such organic-responsive electrodes.

Figure 13 The quaternary

alkyl ammonium chloride

Figure 14 the
measurements of heparin
using the TDM/PVC
membrane
Liquid membrane Neutral Carrier Electrodes
In addition to charged liquid ion exchangers, liquid membrane electrodes often rely on the use of
complex forming neutral-charged carriers. Since the early 1980s much effort has been devoted to the
isolation or synthesis of compounds containing cavities of molecule-sized dimensions. Such use of
chemical recognition principles has made an enormous impact on widespread acceptance of ISEs. For
example, most blood electrolyte determinations are currently being performed with ionophore-based
sensors, either with centralized clinical analyzers or with decentralized disposable units. Neutral
carriers can be natural macrocyclic molecules or synthetic crown compounds (e.g, cyclic polyethers)
capable of enveloping various target ions in their pocket. Electron-donor atoms, present in the polar
host cavity, further facilitate and influence the interaction with the target ion. For example, while
oxygen-containing crown ethers form stable complexes with alkali or alkali earth metals, sulphur-

Page 30 of 44
containing ones are best suited for binding heavy metals. The extent of this interaction is determined
by the “best fit” mechanism, with larger ions that cannot fit in the molecular cavity, and smaller ones
that are weakly coordinated. Often, a subunit group is added to the crown compound to impart higher
selectivity (through a steric/blockage effect) and improved lipophilicity. Overall, these ionophores
serve as reversible and reusable binding reagents that selectively extract the target analyte into the
membrane.
Such binding event creates the phase boundary potential at the membrane–sample interface. To ensure
reversible binding, it is essential to keep the free energy of activation of the analyte–ionophore
reaction sufficiently small. A host of carriers, with a wide variety of ion selectivities, have been
proposed for this task. Most of them have been used for the recognition of alkali and alkaline metal
cations (e.g., clinically relevant electrolytes). A classical example is the cyclic depsipeptide
valinomycin (Figure 15), used as the basis for the widely used ISE for potassium ion. This doughnut-
shaped molecule has an electron-rich pocket in the center into which potassium ions are selectively
extracted. For example, the electrode exhibits a selectivity for K+ over Na+ of approximately
30,000.The basis for the selectivity seems to be the fit between the size of the potassium ion (radius
1.33 Å) and the volume of the internal cavity of the macrocyclic molecule. The hydrophobic side
chains of valinomycin stretch into the lipophilic part of the membrane. In addition to its excellent
selectivity, such an electrode is well behaved and has a wide working pH range. Strongly acidic media
can be employed because the electrode is 18,000 times more responsive to K+ than to H+. A Nernstian
response to potassium ion activities, with a slope of 59 mV/pK+, is commonly observed from 10−6 to
0.1M. Such attractive performance characteristics have made the valinomycin ISE extremely popular
for clinical analysis for blood potassium. Many other cyclic and noncyclic organic carriers with
remarkable ion selectivities have been used successfully as active hosts of various liquid membrane
electrodes. These include the 14-crown-4-ether for lithium; 16-crown-5 derivatives for sodium; bis
(benzo-18-crown-6 ether) for cesium; the ionophore ETH 1001[(R,R)-N,N′-bis(11-
ethoxycarbonyl)undecyl-N,N′-4,5-tetramethyl-3,6-dioxa octanediamide] for calcium; the natural
macrocyclics nonaction and monensin for ammonia and sodium ; respectively; the ionophore
ETH1117 for magnesium, calixarene derivatives for sodium and lead; and macrocyclic thioethers for
mercury and silver. Some common ionophores used for sensing different cations are displayed in
Figure 16. Anion-selective liquid membrane electrodes have also been developed, based on the
coordination of the anionic guest to host materials, such as metallophorphyrin or hydrophobic vitamin
B12 derivatives, alkyl tin compounds or macrocyclic polyamines.

Page 31 of 44
 Figure 15 Valinomycin.

Figure 16 Structure of neutral carriers used in liquid membrane ion-selective electrodes.

d- Gas-sensing electrodes
Real-time monitoring of gases, such as carbon dioxide, oxygen, and ammonia, is of great importance
in many practical environmental, clinical, or industrial situations. Gas-sensing electrodes are highly
selective devices for measuring dissolved gases. They are reliable and simple, exhibit excellent
selectivity. Gas sensors usually incorporate a conventional ion-selective electrode surrounded by a
thin film of an intermediate electrolyte solution and enclosed by a gas-permeable membrane. An
internal reference electrode is usually included, so that the sensor represents a complete
electrochemical cell. The gas (of interest) in the sample solution diffuses through the membrane and
comes to equilibrium with the internal electrolyte solution. In the internal compartment, between the
membrane and the ion-selective electrode, the gas undergoes a chemical reaction, consuming or
forming an ion to be detected by the ion-selective electrode.
d1- Carbon Dioxide Sensors
Carbon dioxide devices were originally developed to measure the partial pressure of carbon dioxide
in blood. This electrode is used today for various automated systems for blood gas analysis.

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The basic design of a gas-sensing electrode is shown in Figure 17 consisting of a thin membrane that
separates the sample from an inner solution containing an ion-selective electrode. The membrane is
permeable to the gaseous analyte, but impermeable to non-volatile components in the sample’s
matrix. The gaseous analyte passes through the membrane where it reacts with the inner solution,
producing a species whose concentration is monitored by the ion-selective electrode. For example, in
a CO2 electrode, CO2 diffuses across the membrane where it reacts in the inner solution to produce
H3O+.
𝑪𝑶𝟐 (𝒂𝒒) + 𝟐𝑯𝟐 𝑶(𝒍) ↔ 𝑯𝑪𝑶− +
𝟑 (𝒂𝒒) + 𝑯𝟑 𝑶 (𝒂𝒒) 𝑰
The change in the activity of H3O+ in the inner solution is monitored with a pH electrode, for which
the cell potential is given by equation:
𝑬𝒄𝒆𝒍𝒍 = 𝑲 + 𝟎. 𝟎𝟓𝟗𝟏 𝒍𝒐𝒈 𝒂𝑯+
To find the relationship between the activity of H3O+ in the inner solution and the activity CO2 in
the inner solution we rearrange the equilibrium constant expression for reaction I; thus
𝒂𝑪𝑶𝟐
𝒂 𝑯 𝟑 𝑶+ = 𝑲 𝒂
𝒂𝑯𝑪𝑶−𝟑
where Ka is the equilibrium constant. If the activity of HCO3– in the internal solution is sufficiently
large, then its activity is not affected by the small amount of CO2 that passes through the membrane.
Substituting above equation into the previous equation gives
𝑬𝒄𝒆𝒍𝒍 = 𝑲′ + 𝟎. 𝟎𝟓𝟗𝟏 𝒍𝒐𝒈 𝒂𝑪𝑶𝟐
where K′ is a constant that includes the constant for the pH electrode, the equilibrium constant for
reaction of the carbon dioxide and water and the activity of HCO3– in the inner solution.

Figure 17 Schematic diagram of

a gas-sensing membrane
electrode.

Similarly, by using different membranes, it is possible to obtain potentiometric sensors for gases
such as sulfur dioxide or nitrogen dioxide. Membrane coverage of other ion-selective electrodes
(e.g., chloride) can be used for the sensing of other gases (e.g., chlorine). The composition of the
inner solution changes with use, and both the inner solution and the membrane must be replaced

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periodically. Gas-sensing electrodes are stored in a solution similar to the internal solution to
minimize their exposure to atmospheric gases.
e-ELECTROCHEMICAL BIOSENSORS
Electrochemical biosensors combine the analytical power of electrochemical techniques with the
specificity of biological recognition processes. The aim is to biologically produce an electrical signal
that relates to the concentration of an analyte. For this purpose, a biospecific reagent is either
immobilized at a suitable electrode, which converts the biological recognition event into a quantitative
amperometric or potentiometric response. Such biocomponent– electrode combinations offer new
powerful analytical tools that are applicable to many challenging problems. Two general categories
of electrochemical biosensors may be distinguished, depending on the nature of the biological
recognition process: biocatalytic devices (utilizing enzymes, cells, or tissues as immobilized
biocomponents) and affinity sensors (based on antibodies, membrane receptors, or nucleic acids).
e1-Enzyme-Based Electrodes
Enzymes are proteins that catalyze chemical reactions in living systems. Such catalysts are not only
efficient but also extremely selective. Hence, enzymes combine the recognition and amplification
steps, as needed, for many sensing applications. Enzyme electrodes are based on the coupling of a
layer of an enzyme with an appropriate electrode. Such electrodes combine the specificity of the
enzyme for its substrate with the analytical power of electrochemical devices. As a result of such
coupling, enzyme electrodes have been shown to be extremely useful for monitoring a wide variety
of substrates of analytical importance in clinical, environmental, and food samples.
Practical and Theoretical Considerations
The operation of an enzyme electrode is illustrated where immobilized enzyme layer is chosen to
catalyze a reaction, which generates or consumes a detectable species:
S + C ⎯⎯ ⎯→ P + C  (6.1)
Enzyme

where S and C are the substrate and coreactant (cofactor), and P and C′ are the corresponding
products. The choice of the sensing electrode depends primarily on the enzymatic system employed.
For example, amperometric probes are highly suitable when oxidase or dehydrogenase enzymes
(generating electrooxidizable hydrogen peroxide or NADH species) are employed, pH–glass
electrodes for enzymatic pathways which result in a change in pH, while gas (carbon dioxide)
potentiometric devices will be the choice when decarboxylase enzymes are used.
e1.1- Glucose Sensors

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The determination of glucose in blood plays a crucial role in the diagnosis and therapy of diabetes.
Electrochemical biosensors for glucose have played a key role in the move toward simplified wide
scale glucose testing, and have dominated the $5 billion/year diabetes monitoring market. The glucose
amperometric sensor, developed and represents the first reported use of an enzyme electrode. The
electrode is commonly based on the entrapment of glucose oxidase (GOx) between polyurathene and
permselective membranes on a platinum working electrode (Figure 19).The liberation of hydrogen
peroxide in the enzymatic reaction
Glu cos e + O2 ⎯Glu
⎯cos⎯e−⎯ ⎯⎯→ Gluconic − acid + H 2O2 ....... 6.4
oxidase

can be monitored amperometrically at the platinum surface:

H 2 O2 ⎯⎯ ⎯⎯⎯ ⎯→ O2 + 2 H + + 2e.......... 6.5
Electrode( anode)

The multilayer membrane coverage (of Figure 19) improves the relative surface availability of oxygen
and excludes potential interferences (common at the potentials used for detecting the peroxide
product). Electrocatalytic transducers based on Prussian Blue layers or metallized carbons, which
preferentially accelerate the oxidation of hydrogen peroxide, are also useful for minimizing potential
interferences. The enzymatic reaction can also be followed by monitoring the consumption of the
oxygen cofactor. Glucose oxidase (and other oxidoreductase enzymes) do not directly transfer
electrons to conventional electrodes because their redox centers are surrounded by a thick protein
layer. Such insulating shell introduces a spatial separation of the electron donor–acceptor pair, and
hence an intrinsic barrier to direct electron transfer, in accordance to the distance dependence of the
electron transfer rate. The interfacial electron transfer rate is thus dependent on the distance between
the enzyme redox centre and the electrode surface, that is, on the depth of the redox group inside the
protein shell, and the orientation of the protein on the surface. As a result of using artificial
(diffusional) electron-carrying mediators, measurements become insensitive to oxygen fluctuations
and can be carried

Page 35 of 44
 Pt anode

Ag cathode

Figure 19 Schematic of a “first generation” glucose biosensor

Figure 20 “Second generation” enzyme electrodes : sequence of events that occur in a mediated
system (ox = oxidation; red = reduction).
out at lower potentials that do not provoke interfering reactions from coexisting electroactive species
(Figure 20). Many organic and organometallic redox compounds have been considered for this role
of enzyme mediator. Some common examples are displayed in Figure 21. In particular, ferricyanide
and ferrocene derivatives (e.g., Figure 21a) have been very successful for shuttling electrons from
glucose oxidase to the electrode by the following scheme:
Glu cose + GOx(ox) → Gluconic − acid + GOx( red ) ....... 6.7

GOx( red ) + 2M ( ox ) → GOx( ox ) + 2M ( red ) + 2 H + ....... 6.8

2M ( red ) → 2M ( ox ) + 2e....... 6.9

where M(ox) and M(red) are the oxidized and reduced forms of the mediator. This chemistry has led to
the development of hand-held battery-operated meters for personal glucose monitoring in a single
drop of blood. Other classes of promising mediators for glucose oxidase are quinone derivatives,
ruthenium complexes, phenothiazine compounds, and organic conducting salts [particularly
tetrathiafulvalene–tetracyanoquinodimethane

Page 36 of 44
Figure 22 (a) Composition of an electron-relaying redox polymer and (b) use of the polymer for
electrical “wiring” of an enzyme to the electrode surface.
. An elegant nondiffusional route for establishing electrical communication between GOx and the
electrode is to “wire” the enzyme to the surface with a long polymer having a dense array of electron
relays [e.g., osmium(bipyridyl) bound to poly(vinyl pyridine), Figure 22a]. Such a polymeric chain
is flexible enough to fold along the enzyme structure (Figure 22b). The resulting three-dimensional
redox-polymer/enzyme network offers high current outputs and stabilizes the mediator to the surface.
It has been successfully used in a commercial painless forearm blood glucose monitoring system.
Nanoscale materials, such as gold nanoparticles or carbon nanotubes, have also shown to be extremely
useful for “plugging” an electrode into GOx. An even more elegant possibility is the chemical
modification of the enzyme with the redox-active mediator. Glucose electrodes of extremely efficient
electrical communication with the electrode can be generated by the enzyme reconstitution process.
For this purpose, the flavin active center of GOx is removed to allow positioning of the electron-
mediating ferrocene unit prior to reconstitution of the enzyme (Figure 23). Ultimately, these and
similar developments would lead to minimally invasive subcutaneously implanted (needle-type) and
noninvasive devices for continuous real-time monitoring of glucose. Such probes would offer a tight
control of diabetes, in connection with an alarm detecting hypo- or hyperglucemia or for a future
closed-loop insulin release system .

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Figure 23 Electrical contacting of a flavoenzyme by its reconstitution with a relay FAD semi-synthetic
cofactor.
e1.2- Ethanol Electrodes
The reliable sensing of ethanol is of great significance in various disciplines. The enzymatic reaction
of ethanol with the cofactor nicotinamide adenine dinucleotide (NAD+), in the presence of alcohol
dehydrogenase (ADH)
C2 H5OH + NAD + ⎯⎯⎯→C2 H5O + NADH
ADH

serves as a basis of amperometric sensors for ethanol. Reagentless devices based on the
coimmobilization of ADH and NAD+ to various carbon or platinum anodes are employed for this task
(e.g., Figure 24). NAD+ is regenerated electrochemically by oxidation of the NADH, and the resulting
anodic current
is measured:
NADH ⎯⎯⎯→ NAD + + H
ADH +
+ 2e.......... ..... 6.11
To circumvent high overvoltage and fouling problems encountered with reaction (6.11) at
conventional electrodes, much work has been devoted to the development of modified electrodes with
catalytic properties for NADH. Immobilized redox mediators, such as the phenoxazine Meldola Blue
or phenothiazine compounds, have been particularly useful for this task. Such mediation should be
useful for many other dehydrogenase- based biosensors. High sensitivity and speed are indicated from
the flow injection response. Alcohol biosensing can be accomplished also in the presence of alcohol
oxidase, based on measurements of the liberated peroxide product.

Figure 24 Reagentless ethanol

bioelectrode.

e1.3-Urea Electrodes
The electrode is an ammonium ion-selective electrode surrounded by a gel impregnated with the
enzyme urease. The generated ammonium ions are detected after 30–60 s to reach a steady-state
potential. Alternately, the changes in the proton concentration can be probed with glass pH or other
pH-sensitive electrodes. As expected for potentiometric probes, the potential is a linear function of
the log [urea] in the sample solution. New opportunities (particularly assays of new environments or
monitoring of hydrophobic analytes) accrued from the finding that enzymes can maintain their
biocatalytic activity in organic solvents.
Page 38 of 44
 One example of an enzyme electrode is the urea electrode, which is based on the catalytic
hydrolysis of urea by urease
⎯→ 2 NH 4+ + CO32−
CO( NH 2 ) 2 + 2H 2 O ⎯urease
⎯

Figure 25 shows one version of the urea electrode, which modifies a gas-sensing NH3 electrode by
adding a dialysis membrane that traps a pH 7.0 buffered solution of urease between the dialysis
membrane and the gas permeable membrane. When immersed in the sample, urea diffuses through
the dialysis membrane where it reacts with the enzyme urease to form the ammonium ion, NH4+,
which is in equilibrium with NH3.
NH4+ (aq) + H2 O(l) ↔ H3 O+ (aq) + NH3 (aq)
The NH3, in turn, diffuses through the gas permeable membrane where a pH electrode measures the
resulting change in pH. The response of the electrode to the concentration of urea is given by
𝐸𝑐𝑒𝑙𝑙 = 𝐾 − 0.0591 𝑙𝑜𝑔 𝑎𝑢𝑟𝑒𝑎
Another version of the urea electrode (Figure 26) immobilizes the enzyme urease in a polymer
membrane formed directly on the tip of a glass pH electrode. In this case the response of the electrode
is 𝒑𝑯 = 𝑲𝒂𝒖𝒓𝒆𝒂
Few potentiometric biosensors are commercially available. As shown in Figure 26, however, it is
possible to convert an ion-selective electrode or a gas-sensing electrode into a biosensor.

Figure 25 Schematic diagram

showing an enzyme-based po-
tentiometric biosensor for urea.

f- Tissue and Bacteria Electrodes

The limited stability of isolated enzymes, and the fact that some enzymes are expensive or even not
available in the pure state, has prompted the use of cellular materials (plant tissues, bacterial cells,
etc.) as a source of enzymatic activity. For example, the banana tissue (which is rich with polyphenol
oxidase) can be incorporated by mixing within the carbon paste matrix to yield a fast-responding and
sensitive dopamine sensor (Figure 27). These biocatalytic electrodes function in a manner similar to
that for conventional enzyme electrodes (i.e., enzymes present in the tissue or cell produce or consume
a detectable species). Other useful sensors rely on the coupling of microorganisms and
electrochemical transducers. Changes in the respiration activity of the microorganism, induced by the
Page 39 of 44
target analyte, result in decreased surface concentration of electroactive metabolites (e.g., oxygen),
which can be detected by the transducer.
Figure 27The mixed
tissue (banana)–
carbon paste sensor
for dopamine.

6.1.2 Affinity Biosensors

Affinity electrochemical biosensors exploit selective binding of certain biomolecules (e.g.,
antibodies, receptors, or oligonucleotides) toward specific target species for triggering useful
electrical signals. The biomolecular recognition process is governed primarily by the shape and size
of the receptor pocket and the ligand of interest (the analyte). The high specificity and affinity of
biochemical binding reactions (such as DNA hybridization and antibody–antigen compexation) lead
to highly selective and sensitive sensing devices.
f1- Immunosensors
Immunoassays are among the most specific of the analytical techniques, provide extremely low
detection limits, and can be used for a wide range of substances. As research moves into the era of
proteomic, such assays become extremely useful for identifying and quantitating proteins.
Immunosensors are based on immunological reactions involving the shape recognition of the antigen
(Ag) by the antibody (Ab) binding site to form the antibody/antigen (AbAg) complex:
Ab + Ag  AbAg .......... ... 6.13

The antibody is a globular protein produced by an organism to bind to foreign molecules, namely,
antigens, and mark them for elimination from the organism. The remarkable selectivity of antibodies
is based on the stereospecificity of the binding site for the antigen, and is reflected by large binding
constants (ranging from 105 to 109 L/mol). Antibody preparations may be monoclonal or polyclonal.
The former are produced by a single clone of antibody-producing cells, and thus have the same
affinity. Polyclonal antibodies, in contrast, are cheaper but possess varying affinities. Electrochemical
immunosensors, combining specific immunoreactions with an electrochemical transduction, have
gained considerable attention. Such sensors are based on labeling of the antibody (or antigen) with an
enzyme that acts on a substrate and generate an electroactive product that can be detected
amperometrically. Enzyme immunosensors can employ competitive or sandwich modes of operation
(Figure 28). In competitive-type sensors, the sample antigen (analyte) competes with an enzyme-
labeled antigen for antibody-binding sites on a membrane held on an amperometric or potentiometric
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sensing probe. After the reaction is complete, the sensor is washed to remove unreacted components.
The probe is then placed in a solution containing the substrate for the enzyme, and the product or
reactant of the biocatalytic reaction is measured. Because of the competitive nature of the assay, the
measurement signal is inversely proportional to the concentration of the analyte in the sample. Several
enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, and catalase, have
been particularly useful for this task.

Figure 28 Enzyme immunosensors based on the competitive or sandwich modes of operation.

Sandwich-type sensors are applicable for measuring large antigens that are capable of binding two
antibodies. Such sensors utilize an antibody that binds the analyte antigen, which then binds the
enzyme-labeled antibody. After removal of the nonspecifically adsorbed label, the probe is placed
into the substrate- containing solution, and the extent of the enzymatic reaction is monitored
electrochemically.
f2- DNA Hybridization Biosensors
Background and Principles
Nucleic acid recognition layers can be combined with electrochemical transducers to form new and
important types of affinity biosensors. The use of nucleic acid recognition layers represents an
exciting area in biosensor technology. Electrochemical DNA hybridization biosensors offer
considerable promise for obtaining sequence-specific information in a simpler, faster, and cheaper
manner, compared to traditional hybridization assays. Such strategies hold an enormous potential for
clinical diagnosis of genetic or infectious diseases, for the detection of food contaminating organisms,
for early warning against biowarfare agents, for environmental monitoring, or in criminal
investigations. The basis for these devices is the DNA base pairing. Accordingly, these sensors rely
on the immobilization of a relatively short [ (base pair)] single-stranded DNA sequence (the “probe”)
on the transducer surface, which, on hybridization to a specific complementary region of the target
DNA, gives rises to an electrical signal (Figure 29). A wide range of chemistries have been exploited
for monitoring electrochemically the DNA hybridization. These can be divided into two major

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principles, involving the use of labels generating an electrical signal or label-free protocols. The
hybridization event can thus be detected via the increased current signal of an electroactive indicator
(that preferentially binds to the DNA duplex), or due to captured enzyme or nanoparticle tags, or from
other hybridization-induced changes in electrochemical

Figure 29 Steps involved in the detection of a specific DNA sequence using an electrochemical DNA
hybridization biosensor.
parameters (e.g., capacitance or conductivity). Control of the probe immobilization (e.g., linking
chemistry, surface coverage) is essential for assuring high reactivity, orientation and/or accessibility,
and stability of the surface bound probe, as well as for avoiding non-specific binding/adsorption
events.
f2.1 Electrical Transduction of DNA Hybridization
Several studies have demonstrated the utility of electroactive indicators for monitoring the
hybridization event. Such redox-active compounds have a much larger affinity for the resulting
duplex (compared to their affinity to the probe alone). Their association with the surface duplex thus
results in an increased electrochemical response. Very successful has been the use of a threading
intercalator ferrocenyl naphthalene diimide (FND), which binds to the DNA duplex more tightly than
do the usual intercalators and displays a negligible affinity to the single-stranded probe.
The use of enzyme labels to generate electrical signals also offers great promise for ultrasensitive
electrochemical detection of DNA hybridization. This can be accomplished by combining the
hybridization step with an electrochemical measurement of the product of the enzymatic reaction.
The potential of enzyme labels for electrical detection of DNA hybridization was demonstrated using
horseradish peroxidase or alkaline phosphatase. Such enzymatic amplification facilitated
measurements down to the z mol (3000 copies) level. It is also possible to exploit different rates of
electron transfer through sand ds-DNA for probing hybridization (including mutation detection) via
the perturbation in charge migration through DNA. It was found that such charge transport is very
sensitive to the DNA structure and perturbations in the structure and exploited such DNA-mediated
charge transport chemistry for detecting single-base mutations and DNA damage. Increased attention

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has been given to new indicator-free electrochemical detection schemes that greatly simplify the
sensing protocol. Such direct, label- free, electrical detection of DNA hybridization can be
accomplished by monitoring changes in the conductivity of conducting polymer molecular interfaces,
such as by using DNA-substituted or doped polypyrrole films. It is also possible to exploit changes
in the intrinsic electroactivity of DNA accrued from the hybridization event. Among the four nucleic
acids bases, the guanine moiety is most easily oxidized and is most suitable for such label-free
hybridization detection. A greatly amplified guanine signal, and hence hybridization response, can be
obtained by using the electrocatalytic action of a Ru(bpy)32+ redox mediator. Such mediated guanine
oxidation is illustrated in Figure 30. Ultimately, these developments will lead to the introduction of
miniaturized (on-chip) sensor arrays, containing numerous microelectrodes (each coated with a
different oligonucleotide probe) for the simultaneous hybridization detection of multiple DNA
sequences. The new gene chips would integrate a microfluidic network, essential for performing all
the steps of the bioassay, and would thus address the growing demands for shrinking DNA
diagnostics, in accordance to the market needs in the twenty-first century.

Figure 30 Schematic representation of guanine oxidation mediated by a ruthenium complex.

f2.2- Receptor-Based Sensors
Another promising and new sensing avenue is the use of chemoreceptors as biological recognition
elements. Receptors are protein molecules embedded in the cellular membrane, and specifically bind
to target analytes. The receptor–analyte (host–guest) binding can trigger specific cellular events, such
as modulation of the membrane permeability, or activate certain enzymes, which translate the
chemical interaction to electrical signals. For example, ion channel sensors, utilizing receptors in a
bilayer lipid membrane, couple the specific binding process with intense signal amplification. The
latter is attributed to the opening/closing switching of the ion flux through the membrane (Figure 31).
A single selective binding event between the membrane receptor and the target analyte can thus result
in an increase of the transmembrane conduction that involves thousands of ions. Unlike most antibody
bindings (aimed at specific substances), receptors tend to bind to classes of substances (possessing
common chemical properties that dictate the binding affinity). Accordingly, receptor-based
biosensors are usually class-specific devices. Instead of isolating, stabilizing, and immobilizing
chemoreceptors onto electrodes, it is possible to use intact biological sensing structures for
Page 43 of 44
determining relevant chemical stimulants. This novel concept was illustrated with antennule
structures of the blue crab. Such structures are part of the crab food-

Figure 31 Schematic representation of the ion permeability modulation for cation responsive
voltammetric sensors based on negatively charged lipid membranes. Complexation of the guest cation
to the phospholipid receptors causes an increase of the permeation for the anionic marker ion.
locating system, and thus can be exploited for the determination of amino acids. Similarly, various
drugs can be monitored with respect to their stimulation of nerve fibers in the crayfish walking leg.
A flow cell based on such a neuronal sensor is shown in Figure 32. Such a sensor responds to stimulant
compounds at extremely low levels (down to 10−15M), with very short response times.
In addition to the use of bioreceptors, it is possible to design artificial molecules that mimic
bioreceptor functions. Such artificial receptors (hosts) can be tailored for a wide range of guest
stimulants. For example, cyclodextrin derivatives have been used to provide a shape discrimination
effect in connection with ion channel sensors. The receptors are incorporated within artificial lipid
membranes. Artificial sensor arrays (emulating biological sensory systems, e.g., the human nose) are
also being explored in various laboratories. High sensitivity and selectivity can be achieved also by
using the receptor recognition process as an in situ preconcentration step.

Figure 32 Neuronal sensing-top view of the flow cell with mounted antennule and various electrode
connections

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