Potentiometric Analysis

• Based on potential
measurement of
electrochemical cells without
any appreciable current
• The use of electrodes to
measure voltages from
chemical reactions
potentiometer
• A device for measuring the potential of an
electrochemical cell without drawing a current
or altering the cell’s composition.
Potentiometric procedure:
Potentiometric measurements are made using a
potentiometer to determine the difference in
potential between a working (an indicator)
electrode and a counter (a reference) electrode.
- Cathode is the working/indicator electrode.
(right half-cell)
- Anode is the counter/reference electrode. (left
half-cell)
Ecell = Ec ─ Ea
Where : Ec is the reduction potential at the cathode.
: Ea is the reduction potential at the anode.
The role of the counter electrode is reduced to that
supplying a reference potential thus the counter
electrode is called the reference electrode.
 Reference Electrode: second ½ cell at a constant
potential
 Indicator Electrode: electrode that responds to
analyte and donates/accepts electrons
 Cell voltage is difference between the indicator and
reference electrode
 Components of a
Potentiometric Cell
1. Reference electrode
2. Salt bridge
3. Analyte
4. Indicator electrode
RE SB A IE
– Eref + Ej + Eind
 Reference electrode
• Half-cell with known potential (Eref)
• Left hand electrode (by convention)
• Easily assembled
• Rugged
• Insensitive to analyte concentration
▫ Reversible and obeys Nernst equation
▫ Constant potential
▫ Returns to original potential
Indicator electrode
• Generates a potential
(Eind) that depends on
analyte concentration
• Selective
• Rapid and reproducible
response
 Salt bridge
•Prevents mixing up of analyte
components

•Generates potential (Ej) =

negligible
Reference Electrodes
1. Standard Hydrogen Electrode
2. Calomel Reference Electrode
3. Silver/Silver Chloride
Reference Electrode
 SHE
• Hydrogen Gas
Electrode

• Pt (H2 (1 atm), H+ (1M)

Saturated Calomel Electrode
Hg2Cl2(s)+2e-2Hg(l)+2Cl-(aq)
• Aka SCE
• Easy to prepare
• Easy to maintain
• 0.2444 V at 25C
• Dependent on temp
• Toxic
SCE
Ag/AgCl Ref. Electrode

AgAgCl (satd),KCl (satd)

AgCl(s) + e-Ag(s)+Cl-(aq)
E = 0.199 V
 Liquid Junction Potential
• Liquid junction - interface between
two solutions containing different
electrolytes or different
concentrations of the same
electrolyte
• A junction potential occurs at every
liquid junction.
▫ Caused by unequal mobilities of the +
and - ions.
 Indicator Electrodes
I. Metallic IE
A. Electrodes of the First Kind
B. Electrodes of the Second Kind
C. Inert Metallic Electrodes (for Redox Systems)

II. Membrane IE
A. Glass pH IE
B. Glass IE for other cations
C. Liquid Membrane IE
D. Crystalline-Membrane IE

III. Gas Sensing Probes

METALLIC INDICATOR
ELECTRODES
 Electrodes of the First Kind
• Pure metal electrode in direct equilibrium with its
cation
• Metal is in contact with a solution containing its
cation.

M+n(aq) + ne-  M(s)

Disadvantages of First Kind Electrodes
• Not very selective
▫ Ag+ interferes with Cu+2

• May be pH dependent
▫ Zn and Cd dissolve in acidic solutions

• Easily oxidized (deaeration required)

• Non-reproducible response
Electrodes of the Second Kind
• Respond to anions by forming precipitates
or stable complex

• Examples:
1. Ag electrode for Cl- determination

2. Hg electrode for EDTA determination

Inert Metallic (Redox) Electrodes
• Inert conductors that respond to redox
systems
• Electron source or sink
• An inert metal in contact with a solution
containing the soluble oxidized and
reduced forms of the redox half-reaction.
• May not be reversible

• Examples:
▫ Pt, Au, Pd, C
 MEMBRANE
ELECTRODES
• Aka p-ion electrodes
• Consist of a thin membrane separating 2 solutions of
different ion concentrations
• Most common: pH Glass electrode
Glass pH Electrode
Properties of Glass pH electrode
• Potential not affected by the presence
of oxidizing or reducing agents
• Operates over a wide pH range
• Fast response
• Functions well in physiological
systems
• Very selective
• Long lifespan
 Theory of the glass membrane potential
• For the electrode to become operative, it must be soaked in water.
• During this process, the outer surface of the membrane becomes
hydrated.
• When it is so, the sodium ions are exchanged for protons in the
solution:
• The protons are free to move and exchange with other ions.

Charge is slowly carried

by migration of Na+
across glass membrane

Potential is determined
by external [H+]
 Alkaline error
• Exhibited at pH > 9
• Electrodes respond to
H+ and alkali cations
• C,D,E and F:
measured value is <
true value
▫ Electrode also
responds to other
cations
• Higher pH at lower
[Na+]
Acid error
• Exhibited at pH
< 0.5

• pH readings are
higher (curves
A and B)
▫ Saturation effect
with respect to
H+
 Selectivity Coefficient
• No electrode responds exclusively to one kind of ion.
▫ The glass pH electrode is among the most selective, but it
also responds to high concentration of Na +.
• When an electrode used to measure ion A, also
responds to ion X, the selectivity coefficient gives
the relative response of the electrode to the two
different species.

response to X
k A, X 
response to A
▫ The smaller the selectivity coefficient, the less interference
by X.
 Selectivity Coefficient
• Measure of the response of an ISE to other ions

Eb = L’ + 0.0592 log (a1 + kHBb1)

• kHB = 0 means no interference

• kHB  1 means there is interference

• kHB < 1 means negligible interference

LIQUID MEMBRANE
ELECTRODES
 Liquid Membrane Electrodes
• Potential develops across the interface between
the analyte solution and a liquid ion exchanger
(that bonds with analyte)
• Similar to a pH electrode except that the
membrane is an organic polymer saturated with a
liquid ion exchanger
• Used for polyvalent ions as well as some anions

• Example:
• Calcium dialkyl phosphate insoluble in water, but
binds Ca2+ strongly
 0.1 M CaCl2

Responsive to Ca2+
 Characteristics of Ca +2 ISE
• Relatively high sensitivity
• Low LOD
• Working pH range: 5.5 – 11
• Relevant in studying physiological processes
 A +
K -selective electrode
• Sensitive membrane
consists of
valinomycin, an
antibiotic
CRYSTALLINE-
MEMBRANE
ELECTRODES
Crystalline-Membrane Electrodes
• Solid state electrodes
• Usually ionic compound
• Crushed powder, melted and formed
• Sometimes doped to increase
conductivity
• Operation similar to glass membrane
Crystalline-Membrane Electrodes
• AgX membrane: Determination of X-
• Ag2S membrane: Determination of S-2
• LaF3 membrane: Determination of F-
 F - Selective Electrode
• A LaF3 is doped with EuF2.
• Eu2+ has less charge than the La3+, so an
anion vacancy occurs for every Eu2+.
• A neighboring F- can jump into the vacancy,
thereby moving the vacancy to another site.
• Repetition of this process moves F- through
the lattice.
Fluoride Electrode
GAS SENSING
PROBES
 Gas Sensing Probes
• A galvanic cell whose potential is related to
the concentration of a gas in solution
• Consist of RE, ISE and electrolyte solution
• A thin gas-permeable membrane (PTFE)
serves as a barrier between internal and
analyte solutions
• Allows small gas molecules to pass and
dissolve into internal solution
• O2, NH3/NH4+, and CO2/HCO3-/CO32-
Gas
Sensing
Probe
 DIRECT POTENTIOMETRY
• A rapid and convenient method of
determining the activity of cations/anions
 Potentiometric Measurement
• Ionic composition of standards must be
the same as that of analyte to avoid
discrepancies

• Swamp sample and standard with inert

electrolyte to keep ionic strength
constant

• TISAB (Total Ionic Strength Adjustment

Buffer) = controls ionic strength and pH
of samples and standards in ISE
measurements
 Potentiometric Measurement
1. Calibration Method

2. Standard Addition
Method
 Special Applications:
Potentiometric pH Measurement
using Glass electrode
• One drop of solution
• Tooth cavity
• Sweat on skin
• pH inside a living cell
• Flowing liquid stream
• Acidity of stomach
 Potentiometric Titration
• Involves measurement of the potential
of a suitable indicator electrode as a
function of titrant volume
• Provides MORE RELIABLE data than
the usual titration method
• Useful with colored/turbid solutions
• May be automated
• More time consuming
Potentiometric Titration Curves
 Contents
• Ion selective electrodes (ISE’s) and CO sensor (examples
2
of potentiometric sensors)
• Oxygen sensor (based on the fuel cell principle)
• Enzyme based glucose sensor (amperometric) and urea
(potentiometric)
• Immunosensor (amperometric)
• From ISFET to ISN’t FET (potentiometric)
 Ion selective electrodes (ISE’s)

Ag/AgCl Ag/AgCl

Inner solution

Frit
analyte

Inner solution
Membrane (e.g. potassium,
sodium, pH, etc.)
Inner reference electrode/ inner
solution/membrane/analyte (external
solution)/external
reference electrode
Ion selective electrodes (ISE’s)
• A traditional pH measurement with
a glass electrode is the best known
potentiometric ion selective
electrode (ISE) (e.g. a thin glass layer
with this composition 22% Na2O,
6% CaO, 72% SiO2)
• There is no change in the inner
solution and there is no actual
contact between inner and outer
solution for any potentiometric
probe or sensor
• Contact with the solution is always
through the external reference
electrode (Luggin capillary)
Ion selective electrodes (ISE’s)

• Often reference and glass electrode

are combined in one single structure
(How would you make such a thing
? See homework Q 1)
• The resistance (impedance) of this
sensor is very high (glass layer) so
that the input amplifier of the pH
meter must be very high (the input
impedance of the meter must be at
least 100 > than that of the sensor!)
• Very high impedance can make the
measurement noisy. The smaller the
sensor the bigger this problem
becomes.
 Ion selective electrodes (ISE’s)
• The so-called Donnan potential is
established on both sides of the glass
membrane-the potential on one side is
kept constant through the internal
reference solution while the other side is
determined by the analyte solution
• For other ions than protons (cations and
anions) other membranes are available
(see e.g. LaF3 for F- and a wide variety of
polymeric membranes)
 Ion selective electrodes (ISE’s)

• An ion selective polymeric membrane

is often made by mixing an ionophore
(e.g. valinomycin, a natural occuring
antibiotic) with PVC and a plasticizer
(to make the rigid plastic more
flexible)
• In these types of ISE’s one sometimes
does not use an internal reference
solution at all or one incorporates a
hydrogel to replace the aqueous
solution . This makes the electrode
easier to handle and store. Especially
with no internal reference electrode
drift tends to be larger!
• The polymeric ISE’s lend themselves
well to miniaturization and cost
reduction (it is much more difficult to
miniaturize a glass pH electrode)
 Ion selective electrodes (ISE’s)

• By making ISE’s planar Evaporated Ag film

(e.g. on a polyimide sheet)

many sensors can be made Insulator layer

in parallel (i.e. batch

fabnrication). From 3D
structures to 2D !
• Mass production can make
them very small (e.g. 2 by 3
mm), cheap (perhaps
Chloridized Ag i.e. AgCl
Hydrogel on reference
disposable), reproducible electrodes with internal
electrolytes
and even electronics might
be integrated (see below Ion selective membrane

under ISFETs)
 Carbon dioxide sensor

• Gases that react with water freeing or

absorbing a proton in the electrolyte
may be detected by a pH sensitive
detector element e.g. glass or IrOx 1. CO2 + H2 O  H2CO - 3
• Example gases: CO2, NH3, H2S, etc.
• A direct proportionality exists
 H+ + HCO- 3  2H + + CO2- 2
between the concentration of the
neutral gas and the measured pH e.g.
in the case of CO2 ( with NaHCO3 for
2. NH3 + H2O  NH + 4 + OH-
internal electrolyte) i.e.
3. H2S + H2 O  HS- + H3O +
K aCO 2
aH + 
a HCO - 3
Carbon dioxide sensor

Ecell = E ind - Eref (1)

As the indicator is only H+ sensitive, and the potential of the reference is a
constant (because of the constant chloride concentration in the electrolyte), we have

Ec ell  K1  0.059log aH 
(at 25o C) (2)
CO2 penetrates through the gas permeable membrane and will react with the
electrolyte in the agar hydrogel:
CO2 + H2 O = H+ + HCO3 - (3)

a H   K' PCO2 aH 2 O / aHCO 

3 (4)
As the activities for H2 O and HCO3 - are constant in the electrolyte, the
voltage of the sensor cell should be:

Ec ell  K2  0.059log PCO2

(5)
Carbon dioxide sensor (3D)
Carbon dioxide sensor (MEMS
version)
• A pH, CO2 and oxygen
electrochemical sensor array for in-
vivo blood measurements was made
using MEMS techniques
• The pH and CO2 sensors are
potentiometric and the oxygen sensor
is amperometric (see further in this
class)
• The pH sensor is an ISE based on a
pH sensitive polymer membrane.
• The CO2 sensor is based on an IrOx
pH sensor and a Ag/AgCl reference
electrode. .
 Electrochemical oxygen sensor (fuel
cell)
"Fuel cell" oxygen sensors consist of a diffusion barrier, a sensing
electrode (cathode) made of a noble metal such as gold or silver,
and a working electrode made of a metal such as lead or zinc
immersed in a basic electrolyt (such as a solution of potassium
hydroxide).

Oxygen diffusing into the sensor is reduced to hydroxyl ions at the

cathode:

O2 + 2H2O + 4e- -------- 4 OH-

Hydroxyl ions in turn oxidize the lead (or zinc) anode:

2Pb + 4OH- ------------- 2PbO + 2H2O + 4e-

2Pb + O2 ----------------- 2PbO

Fuel cell oxygen sensors are current generators. The amount of

current generated is proportional to the amount of oxygen
consumed (Faraday's Law).
 Enzyme based sensor

• Enzymes are high-molecular weight Pt- anode (+)

biocatalysts (proteins) that increase the
rate of numerous reactions critical to life
Ag cathode (-)
itself
• Enzyme electrodes are devices in which the
analyte is either a substrate (also called Immobilized glucose oxidase
reactant) or a product of the enzyme (e.g. in cellulose-diacetate with heparin)
reaction, detected potentiometrically or
amperometrically
Polyurethane membrane
• Example : glucose sensor substrate
(glucose) diffuses through a membrane to
the enzyme layer where glucose is
converted
• Both oxygen (which is being consumed)
and H2O2 (which is being produced) can be
measured electrochemically (in an Glucose oxidase (in presence of oxygen)
amperometric technique), or the local pH
Glucose H 2O2 + gluconic acid
change can be monitored (in a
potentiometric measurement)
Enzyme based sensor

• Amperometric glucose sensor

based on peroxide oxidation, Anodic
+i
l
• Plateau of limiting current is i

proportional to the peroxide

concentration which in turn is
proportional to glucose - - -
typical 0.6 to 0.8 V vs Ag - +
cathode
+ 0.6 V
• Glucose oxidase is an oxidase
type enzyme, urease is a
hydrolytic type enzyme:
-i
Cathodic
Urease
CO (NH 2 )2  CO2 + 2 NH3
2H
O
Enzyme based sensor

• A potentiometric urea sensor

may consist of two pH sensors V
one with the enzyme coated on IrOx IrOx
its surface and one without (the
reference electrode) urease
• The electrode with the urease
will sense a local pH change
• The pH difference bewteen the
two electrodes is proportional to
the urea concentration
• As an example two IrOx
electrodes may be used
 Immunosensors

• Affinity pairs: An enzyme/ substrate combination is only one example of

an affinity pair, in nature there are many other examples of affinity pairs
based on molecular recognition (think about double stranded DNA)
• Affinity pairs exhibit tremendous binding selectivity for each other
through their intricate 3D molecular structures (lock and key)
• A much more selective affinity pair than enzyme / substrate pair is the
antigen/antibody pair (AgAb) -- KA (affinity constant) values of 106-1012
LM-1 vs 102-106 LM-1 (as a consequence enzyme sensors may be
reversible while imunosensors are irreversible but much more selective)
• In an immunosensor one measures the concentration of either an
antibody or an antigen by measuring an event triggered by the binding
of an antigen/antibody- usually a label is involved (e.g. an enzyme, an
isotope, a chromophore, etc.) , a direct detection of the binding event
(without label) is very difficult but is being attempted in various
research labs.
 Immunosensors

• One example of an immunosensor is an enzyme based immunosensor where the

label is an enzyme--see next slide
• Typically an antigen (the same antigen we are trying to determine in the
unknown solution) is labeled with an enzyme (say catalase) and added to the
unknow sample in which the sensor is placed
• The labeled antigen competes with native (unlabeled antigen) for reaction with
the antibody, which is immobilized on an electrode surface
• Unbound labeled antigen is washed off and substrate for the enzyme (H2O2 in the
case of catalase) is added
• The enzyme decomposes H2O2 and the oxygen is picked up by the underlying
oxygen sensor
• The oxygen current decreases with increasing concentration of the nonlabeled
native antigen in the sample solution
• The enzyme reaction will produce many detectable species per bound AbAg pair,
hence the name “enzyme amplification.”
Immunosensors

Enzyme labeled antigen

Immobilized antibody

Antigen Competition for sites on the antibody

Oxygen permeable membrane

Immobilized antibody

Oxygen sensor

Oxygen permeable membrane

Oxygen sensor
Immunosensors

Wash the unbound antigen away and add H 2O2

Oygen is formed

Oxygen permeable membrane

Oxygen sensor

The oygen signal is lower the higher the amount of native antigen
From ISFET to ISN’T FET
Homework
1. Design a combination glass electrode. Explain
how it works.
2. Design a planar immunosensor. How could you
incorporate a good reference?
3. Explain how a potentiometric CO 2 sensor works.
4. List a list of reasons why the ISFET did not
become a commercial success.