Potentiometry: Principles and POTENTIOMETRIC CELL:

Applications
A typical cell for potentiometric analysis can be
represented as:
What is Potentiometry?

An analytical method for measuring electrical

potential between electrodes without drawing The potential of the cell we have just considered
current. Used to determine ion concentrations. is given by the equation:

Cell Potential (also called electromotive force

or emf): Voltage produced by an
electrochemical cell when two electrodes are
connected in a circuit.

Reference Electrode for

Component of Potentiometric cell:
Potentiometry:

- Reference electrode: A half-cell having a

Has a potential that is:
known electrode cell potential. Remains
constant at constant temperature and is - Accurately known
independent of the composition of the analyte
- Constant
solution.
- Completely insensitive to the composition of
- Indicator electrode: Indicator electrode +
the analyte solution
analyte electrode. Potential that has variation in
the concentration of analyte. Electrode is:
- Salt bridge: Usually KCl. Prevents component - Rugged
of analyte from mixing with reference electrode.
- Easy to assemble
Note: - Maintain a constant potential while passing
• Reference electrode is always left minimal currents
hand.
• KCl – ideal solution in salt bridge;
diffusion rates of K⁺ and Cl⁻ ions are
equal, unlike in the hydrogen
electrode, which is inconvenient and
poses a fire hazard.
Types of Reference Electrodes Liquid-junction Potentials:
for Potentiometry: Two electrolyte solutions of different
composition are in contact with one another,
1. Calomel electrode:
there is a potential difference across the
- Saturated Calomel electrode interface.

- Most widely used due to larger temperature

coefficient Indicator electrode
- Electrode potential of 0.2444 V at 25°C

- With agar – available translucent flakes, Used to measure the potential of the analyte
heteropolysaccharide made from East Indian solution comparing with that of reference
seaweed electrode. Its potential is directly proportional
Diagram of a typical commercial saturated to ion concentration.
calomel electrode: Types of Indicator Electrodes:
1. Metallic:

A. Electrode of the first kind:

- Pure metal that is in direct equilibrium with

its cation in the solution.

- Not widely used, as it is not selective and

respond on cation and easily with reduced
cations

B. Electrode of the second kind:

- Mercury serves as an indicator electrode of

the second kind for the EDTA anion Y⁴⁻.

- Potential of a silver electrode is proportional

to pCl (negative log of the chloride ion
- Memory Tip: Mercury + Calomel + KCl = SCE concentration)

C. Inert redox reaction:

2. Silver/silver chloride electrode: - Used to monitor redox systems

- 0.199 V at 25°C - Platinum, Gold, Palladium, Carbon

- The wire is dipped in a salt solution, usually - Platinum electrode: Convenient indicator
potassium chloride (KCl), saturated with silver electrode for titration involving standard cerium
chloride (AgCl). IV solutions
Note: calcium chloride, and a silver electrode that is
coated with silver chloride to form an internal
- Inert redox reactions: Reactions where reference electrode
electrons move but the electrodes don’t
change. C. Crystalline membrane electrode:

- Inert electrodes like platinum, gold, - Conduct electricity through solid medium
palladium, and carbon help in these - Membrane with cationic sites might be
reactions without getting involved. expected to respond selectively toward anions.
- Platinum electrodes are especially useful
for titrations, like those using cerium IV.
3. Ion Selective Field Effect Transistors (ISFET):

- Field effect transistor / metal oxide field effect

2. Membrane: transistor (MOSFET) is a tiny solid-state
semiconductor device.
- Aka. p-ion electrodes
- Widely used in computers and other electronic
- Data obtained from them are usually circuits
presented as p-functions, such as pH, pCa, or
pNO₃ - ISFETs advantages:

- More convenient way of determining a pH that - Ruggedness

involves measurement of potential - Small size
- pH measurement – glass electrode - Inertness toward harsh environments
A. Glass membrane electrode: - Rapid response
- The indicator electrode consists of a thin, pH- - Low electrical impedance
sensitive glass membrane sealed onto one end
of a heavy-walled glass or plastic tube.

- 2 reference electrodes: Gas-Sensing Probes:

- External - Calomel - Principle: transfer of gas in the internal solution

- Internal – Ag/AgCl - A galvanic cell whose potential is related to the

concentration of gas solution
- pH sensing element – thin glass
membrane at the tip - Aka. Gas sensing electrodes (misnomer)

B. Liquid membrane: - Determine dissolved gases in water and other

solvents
- Direct potentiometric measurement of
numerous polyvalent cations as well as certain - Microporous membrane
ions - Fabricated from a hydrophobic polymer
- It consists of a conducting membrane that
selectively binds calcium ions, an internal
solution containing a fixed concentration of
Example: Point-of-Care testing (Blood Gases,
and Blood Electrolytes with Portable
Instrumentation)

Instrument For Measuring Cell Potential:

- pIon meter / ion meter (e.g., pH meter)

- Used for measurement of ions

- High resistance direct reading digital voltmeter

with internal resistance of more than 10 ohms

Direct Potentiometry:

- Rapid and convenient method for determining

the activity of a variety of cations and anions

- Comparison of potential of indicator electrode

and potential immersed in standard solution

Errors Affecting pH Measurement:

- Alkaline error – negative error

- Acidic error – positive error

- Dehydration

- Errors in low ionic strength solution

- Variation in junction potential

- Error in pH of standard buffer

Following Titrations By Potentiometry:

- Potentiometric titration involves measurement

of the potential of a suitable indicator electrode
as a function of titrant volume.

- Measurement is based on the titrant volume

CHROMATOGRAPHY

Introduction to Analytical Separations

• Separations are extremely important in

synthesis, in industrial chemistry, in the
biomedical sciences, and in chemical
analyses.
• Separations isolate the analyte from
Several separation methods that are in common use
potentially interfering constituents. include (1) chemical or electrolytic precipitation, (2)
• An interferent is a chemical species that distillation, (3) solvent extraction, (4) ion exchange, (5)
causes a systematic error in an analysis by chromatography, (6) electrophoresis, and (7) field-flow
enhancing or attenuating the analytical fractionation.
signal or the background.
• The goals of an analytical separation are
usually to eliminate or reduce interferences
Chromatographic Separations
so that quantitative analytical information
can be obtained from complex mixtures.
• With techniques such as chromatography,
• Chromatography is a widely used method for
quantitative information is obtained nearly
the separation, identification, and
simultaneously with the separation.
determination of the chemical components
in complex mixtures.
• It is a technique in which the components of
a mixture are separated based on differences
in the rates at which they are carried through
a fixed or stationary phase by a gaseous or
liquid mobile phase.

Separation principles. In (a), a mixture of four • The stationary phase in chromatography is a

components is completely separated so that each phase that is fixed in place either in a column
component occupies a different spatial region. In (b),
or on a planar surface.
a partial separation is shown. In the partial
separation, species A is isolated from the remaining
• The mobile phase in chromatography is a
mixture of B, C, and D. The reverse of the separation phase that moves over or through the
process shown is mixing at constant volume. stationary phase carrying with it the analyte
mixture. The mobile phase may be a gas, a
liquid, or a supercritical fluid.
 • Elution is a process in which solutes are
washed through a stationary phase by the
movement of a mobile phase.
• The mobile phase that exits the column is
termed the eluate.
• An eluent is a solvent used to carry the
components of a mixture through a
stationary phase.

• Chromatographic methods are of two basic

types:
o In column chromatography, the
stationary phase is held in a narrow
tube, and the mobile phase is forced
through the tube under pressure or
by gravity.
o In planar chromatography, the
stationary phase is supported on a
flat plate or in the pores of a paper,
and the mobile phase moves
through the stationary phase by • If a detector that responds to solute
capillary action or under the concentration is placed at the end of
influence of gravity. the column during elution and its signal
• Chromatographic methods fall into three is plotted as a function of time (or of
categories based on the nature of the mobile volume of added mobile phase), a series
phase: liquid, gas, and supercritical fluid. of peaks is obtained. Such a plot, called
a chromatogram.

• It is useful for both qualitative and

quantitative analysis.
Column Chromatographic
Separations

Concentration profiles of solute bands A

and B at two different times in their
migration down the column.
 Common Forms of Column o The target protein binds to the
Chromatography ligand in the mobile phase and is
recovered from the column. This
protein–ligand interaction can also
• Size-exclusion chromatography or gel- be disrupted with a change in pH or
filtration chromatography ionic strength.
o separates molecules on the basis of
size
o useful way in separating proteins of
varied molecular weights.
o The stationary phase consists of
cross-linked gel particles which is
usually in bead form.
o The bead consists of one or two
polymers, usually a combination of
dextran or agarose and
polyacrylamide.

Column
• Ion-exchange chromatography

o Shares similarity with affinity

chromatography but uses a ligand
with a positive or negative charge.

o A negatively charged resin is

a cation exchanger, and a positively
charged one is an anion exchanger.

o Proteins that have a net charge

opposite to that of the exchanger
stick to the
column, exchanging places with the
bound counterions. Proteins that
have no net charge or have the
same charge as the exchanger elute.

• Affinity chromatography
o uses the specific binding properties
of many proteins; produces very
pure proteins
o Stationary phase is also made up of
polymeric material that is covalently
linked to some compound, called
a ligand.
• Gas Chromatography chromatography (GLC) and gas-solid
o In gas chromatography, the chromatography (GSC).
components of a vaporized sample o In gas-liquid chromatography, the
are separated by being distributed mobile phase is a gas, and the
between a mobile gaseous phase stationary phase is a liquid that is
and a liquid or a solid stationary retained on the surface of an inert
phase held in a column. solid by adsorption or chemical
o In performing a gas chromatographic bonding.
separation, the sample is vaporized o In gas-solid chromatography, the
and injected onto the head of a mobile phase is a gas, and the
chromatographic column. stationary phase is a solid that
o Elution is brought about by the flow retains the analytes by physical
of an inert gaseous mobile phase. adsorption.
o In contrast to most other types of o Gas-solid chromatography permits
chromatography, the mobile phase the separation and determination of
does not interact with molecules of low-molecular-mass gases, such as
the analyte. The only function of the air components, hydrogen sulfide,
mobile phase is to transport the carbon monoxide, and nitrogen
analyte through the column. oxides.
o Two types of gas chromatography
are encountered: gas-liquid
SPECTROSCOPY Properties of Electromagnetic
• Studies the interactions of radiation and Radiation
matter
• Spectroscopic analytical methods are based
on measuring the amount of radiation • Electromagnetic radiation is a form of
produced or absorbed by molecular or energy that is transmitted through space
atomic species of interest. at enormous velocities.
• Can be classified according to the region of o Light is a form of
the electromagnetic spectrum used or electromagnetic radiation in the
produced in the measurement. UV/visible region. It can also
o γ-ray travel readily in vacuum.
o X-ray o It can be described as a wave
o Ultraviolet (X-ray) with properties of wavelength,
o Visible frequency, velocity, and
o Infrared (IR) amplitude.
o Microwave
o Radio-frequency (RF)
• Spectrochemical methods have provided
perhaps the most widely used tools for the
elucidation of molecular structure as well as
the quantitative and qualitative
determination of both inorganic and WAVE CHARACTERISTICS OF LIGHT
organic compounds. • The amplitude of an electromagnetic
wave is a vector quantity that provides a
measure of the electric or magnetic
field strength at a maximum in the
wave.
• The period (p) of an electromagnetic
wave is the time in seconds for
successive maxima or minima to pass a
point in space.
• The frequency of an electromagnetic
wave is the number of oscillations that
occur in one second and is equal to 1/p.
• The unit of frequency is the hertz (Hz),
which corresponds to one cycle per
second, that is, 1 Hz = 1 s-1.
THE SPEED OF LIGHT
• In a vacuum, light travels at its
maximum velocity.
• This velocity, which is given the
special symbol c = 2.99792 x 108
m s-1.
 PARTICLE NATURE OF LIGHT: PHOTONS
• In many radiation/matter interactions, it The Electromagnetic Spectrum
is useful to emphasize the particle
nature of light as a stream of photons or
quanta. • Note that the visible region, to which our
• A photon is a particle of electromagnetic eyes respond, is only a tiny fraction of
radiation having zero mass and an the entire spectrum (ROY G BIV).
energy of hv. • Spectrochemical methods that use not
• The energy of a single photon to its only visible but also ultraviolet and
wavelength, frequency, and infrared radiation are often called optical
wavenumber by methods.

• where h is Planck’s constant

(6.63 x 10-34 J·s)
• c = 2.99792 x 108 m s-1
• v = frequency
• λ = wavelength

PARTICLE NATURE OF LIGHT: PHOTONS

• Wavenumber and frequency are directly

proportional to the photon energy. Spectroscopic Measurements

• Wavelength is inversely proportional to

energy. • Spectroscopists use the interactions of
• SI unit of energy is in Joules (J). radiation with matter to obtain
information about a sample.
• Before exposure to the stimulus, the
analyte is predominately in its lowest-
energy or ground state.
• The stimulus then causes some of the
analyte species to undergo a transition
to a higher-energy or excited state.
 • Emission spectroscopy usually refers to
methods in which the stimulus is heat or
electrical energy
• Chemiluminescence spectroscopy refers to
excitation of the analyte by a chemical
reaction.
• A familiar example of
chemiluminescence is found in the
light emitted by a firefly.
Chemiluminescence involving a
biological or enzyme reaction is
often termed bioluminescence.
Basic Spectroscopy
• The popular light stick is another
Figure 1. The basic scheme of photobiological events:
(photobiology.info)
familiar example of
the primary photoactive molecule M absorbs light and chemiluminescence.
is promoted to its excited state, M*. It then undergoes • Measurement of the radiant power emitted
a chemical transformation to one or more intermediate as the analyte returns to the ground state
species I, and finally yields a product P. The vertical can give information about its identity and
position of the bars in the diagram represents the concentration.
energy content of each species. • The results of such a measurement are often
expressed graphically by a spectrum, which
is a plot of the emitted radiation as a function
of frequency or wavelength.

• In absorption spectroscopy, we
measure the amount of light absorbed
• Information about the analyte is acquired
as a function of wavelength. Absorption
by measuring the electromagnetic
measurements can give both qualitative
radiation emitted as it returns to the
and quantitative information about the
ground state or by measuring the amount
sample.
of electromagnetic radiation absorbed as a
• In photoluminescence spectroscopy
result of excitation.
the emission of photons is measured
following absorption.
 • The most important forms of
photoluminescence for analytical
purposes are fluorescence and
phosphorescence spectroscopy.

Beer-Lambert Law or Beer’s Law

• Also called the absorption law

• Quantitatively states how the amount of

attenuation depends on the
concentration (c) of the absorbing
molecules and the path length (b) over
which absorption occurs.

• Longer path length and higher

concentration of analyte translate to a
higher attenuation of energy.
What Each Symbol Means:

• P = The amount of light that comes out

of the sample (after passing through).

• P0 = The amount of light you started

with (before it hit the sample).

• %T = The percentage of the original

light that made it through the sample.

• According to Beer’s law, absorbance is

directly proportional to the
• The transmittance (T) of the solution is concentration of the absorbing species,
the fraction of incident radiation c, and to the path length, b, of the
transmitted by the solution. It is often absorbing medium as expressed by:
expressed as a percentage and called the
percent transmittance (%T).

• The absorbance (A) of a solution is

related to the transmittance in a
logarithmic manner.

• As the absorbance of a solution • a is a proportionality constant called the

increases, the transmittance decreases. absorptivity.

• When we express the concentration in

the equation above in moles per liter
 and b in cm, the proportionality
constant is called the molar
absorptivity (Ɛ), where Ɛ has the units L
mol-1 cm-1

• Thus,

Sample Problem:

A 7.25 x 10-5 M solution of potassium

permanganate has a transmittance of 44.1%
when measured in a 2.10-cm cell at a
wavelength of 525 nm. Calculate (a) the
absorbance of this solution and (b) the molar
absorptivity of KMnO4.

(a) A = -logT = -log0.441

= -(-0.356) = 0.356

(b) ε = A/bc

= 0.356/(2.10 cm x 7.25 x 10-5 mol L-1)

= 2.34 x 103 L mol-1 cm-1