1. ELECTROCHEMISTRY, ELECTROPHORESIS,
AND ISOELECTRIC FOCUSING
Presented by:
Anthony John Duran
Angelica Nhoj Gemora
Denfield Jan Pama
Siegefred Pue
Karen Grace Salao

2. ELECTROCHEMISTRY

3. ELECTROCHEMISTRY
 Involves measurement of current or voltage
generated by activity of specific ions.
 Electrical energy Chemical energy
Copper and silver nitrate
Cu AgNO3
2Ag+(aq) + Cu(s) 2Ag(s) + Cu2+(aq)

6. 2 TYPES OF ELECTROCHEMICAL CELLS
 Electrolytic cells
 nonspontaneous
chemical reactions are
forced to occur by the
input of electrical
energy.
 Consist of a container
for the reaction material
with electrodes
immersed in the
reaction material and
connected to a source
of direct current.

7.  Galvanic or
voltaic cell
 Spontaneous
redox reaction
produce electrical
energy
 the two halves of
the redox reaction
is separated,
requiring electron
transfer to occur
through an
external circuit.

8.  Electrochemistry includes:
 Potentiometry
 Amperometry
 Coulometry

9. POTENTIOMETRY: GENERAL PRINCIPLES
 Concentration of ions in solution is calculated from
the measured potential difference between the two
electrodes.
 This type of system includes at least two
electrodes, identified as an indicator electrode and
a reference electrode which act as the cathode and
anode respectively.

10.  Each electrode is in contact with either the sample
(in the case of the “indicator electrode”) or a
reference solution ( in the case of the “reference
electrode”).
 This method is made under conditions in which
essentially zero current is flowing through this
system.

11.  The difference is related to the molar concentration
of the solution as expressed by the Nernst
equation,
E = E°- (0.059/z)log (Cred/Cox)
Where: E= cell potential measured at 25°C
E°= standard redox potential
z= number of electrons involved
Cred= molar concentration of the reduced
form
Cox= molar concentration of the oxidized

12. STANDARD REDUCTION POTENTIALS

13.  System Components
 Liquid Junction
 Reference electrode
 Indicator or measuring electrode
 Readout device (Potentiometer)

15.  Liquid junction – also known as a salt bridge are
required to complete the circuit between the
reference and without contaminating anything.
Functions:
It allows electrical contact between the two
solutions.
It prevents the mixing of the electrode solutions.
It maintains the electrical neutrality in each half cell
as ions flow into and out of the salt bridge.

16.  Reference Electrode- is an electrochemical half-cell
that is used as a fixed reference for the measurement
of cell potentials.
 A half-cell with an accurately known electrode potential,
Eref, that is independent of the concentration of the
analyte or any other ions in the solution
 Always treated as the left-hand electrode
Examples:
Normal hydrogen electrode
Saturated calomel electrode
Ag-AgCl electrode

17. REFERENCE ELECTRODES
 Calomel electrode- composed of
mercury/mercurous chloride; It is dependable but
large, bulky, and affected by temperature.
 Silver/silver chloride- reference electrodes are
more compact and handle temperature fluctuations
better -- overall better & faster
 Normal Hydrogen Electrode- consists of a
platinized platinum electrode in a 1.228N HCl
solution with hydrogen at atmospheric pressure
bubbled over the platinum surface.

19.  Indicator Electrode- also called the measuring
electrode (platinum wire and carbon rod).
 It is immersed in a solution of the analyte, develops
a potential, Eind that depends on the activity of the
analyte.
 Is selective in its response
 It is the other electrochemical half-cell that responds
to changes in the activity of a particular analyte
species in a solution.
Example:
 Ion-Selective Electrodes

20. ION SELECTIVE ELECTRODE
 Is an indicator electrode that can respond to
individual types of anions or cations, and is one tool
that can be utilized for such a task.
Examples:
 Glass membrane Electrodes
 Gas-sensing Electrodes

21. pH electrode
 Selective for the
detection of hydrogen
ions.
 The measuring or
indicator electrode
has a “glass
membrane”
 pH is then determined
from potential
between the pH
electrode and a

22. PCO2 ELECTRODE
 Measurement of PCO2
in routine blood gases
 A modified pH electrode
with a CO2 permeable
membrane covering the
glass membrane surface
 A bicarbonate buffer
separates the
membranes
 Change in pH is
proportional to the
concentration of
dissolved CO2 in the
blood

23. COULOMETRY
 Coulometry is an electrochemical titration
where the titrant is electrochemically
generated and the endpoint is detected by
amperometry.

25. AMPEROMETRY
 Amperometry- is the measurement of the
current flow produced by an oxidation-
reduction reaction.
 A measure of the cell current when the
potential difference between indicator and
reference electrodes is controlled.

26. PRINCIPLE
 In the presence of some conductive buffer. If an electrolytic
potential is applied to the solution through a working
electrode, then the measured current depends (in part) on
the concentration of the analyte. Measurement of this
current can be used to determine the concentration of the
analyte directly.
 However, the difficulty is that the measured current depends
on several other variables, and it is not always possible to
control all of them adequately. This limits the precision of
direct amperometry.

27.  If the potential applied to the working electrode is
sufficient to reduce the analyte , then the
concentration of analyte close to the working
electrode will decrease.
 If the potential applied to the working electrode is
great enough (an overpotential), then the
concentration of analyte next to the working
electrode will depend entirely on the rate of
diffusion.

28. This can be seen in the following equation:
Q = It = znF
Where:
z = the number of electrons involved in the
reaction
n = the number of moles of analyte in the sample
Where: = Faraday’s constant (96485 C/mol of
F
electrons)
Q= the electrical charge
I= the current
t= the time

29.  pO2 Gas Electrodes
 Gas-sensing electrodes that use amperometric
or current-sensing electrolytic cell as indicator.
 They consist of a gas permeable membrane
(polypropylene) which allows only dissolved
oxygen to pass through.

31. ADVANTAGES
 less hazardous process
 elimination or minimization of polluting
byproducts requiring disposal
 process simplification so that an otherwise
multistep chemical route is simplified to one or
two steps

32.  use of cheaper more readily available starting
materials
 the possibility of reaching very high levels of
product purity and selectivity

33. DISADVANTAGES
 requires the use of a solvent to solubilize
the reactants and products
 Water is the ideal solvent but too often organic
solvents or co-solvents are required
 supporting electrolytes to carry the current are very
often needed
 Electricity is required in all electrochemical processing
which may or may not be a critical factor, depending on
where the process is located.

34. APPLICATION
 Use of the potential measurements to give direct
information on the activity, or concentration of an
analyte in a sample
 pH measurements
 Use of potential measurements to follow the course
of titration, as occurs in a potentiometric titration.
 Measurement of chloride in body fluids such as
sweat, urine and CSF.
 Determination of ascorbic acid or vitamin C

35. INTERFERENCES
 Errors in ISE measurement can result in any ion
determination if data are not collected for standards and
samples at approximately the same temperature, since the
Nernst equation that governs the calibration of potential
versus concentration is temperature dependent.
 Response of an ISE to a non-analyte or an interferent ion in
the sample.

36.  Components in certain sample matrices also can
change the sensitivity of an electrode by adsorbing
to its surface, thereby blocking access of the
analyte.
 Sensitivity of the glass pH electrode may be
reduced for some electrodes at pH values above 10
(i.e. sodium error) because of the interference of
monovalent cations in high concentrations,
especially Na+.
 In solutions of pH less than 1, low water activities
also may give rise to measurement error.

37. ELECTROPHORESIS

38. ELECTROPHORESIS
 Method of separation and purification
 Involves migration of charged particles in an electric
field
 It is suitable for the separation and the quantitation of
proteins in body fluids.
 Is a tool that is used by clinical laboratory
scientists/medical technologists to separate molecule
prior to molecule identification.

39. GENERAL PRINCIPLES
 The electrical field is applied to a solution through
oppositely charged electrodes placed in the
solution.
 An ion then travels through the solution toward the
electrode of opposite charge: positively charged
particles move to the negatively charged electrode,
and negatively charged particles migrate to the
positively charged electrode.

40.  The separation of analytes by
electrophoresis has two key requirements:
 There must be a difference in how analytes
interact with the separation system.
 The bands or peaks for the analytes must be
sufficiently narrow to allow them to be resolved.
 The sample is separated into bands where
each band has molecules containing similar
mobility.

41. FACTORS INFLUENCING MIGRATION OF
PARTICLES
 Net electric charge of the particle
 Size and shape of the molecules
 Electric field strength
 Nature of the supporting medium
 Temperature of operation

43. COMPONENTS
 power source with a voltmeter and voltage regulator
 electrophoresis tank that holds the electrophoresis
buffer
 an anode and a cathode connected with the power
source
 a glass plate that holds the gel and is submerged into
the electrophoresis buffer
 a comb which is used to make the sample wells in the
agar before it solidifies.

44. TYPES OF ELECTROPHORESIS
 Moving boundary or frontal
electrophoresis
 It involves separation of molecules using
homogenous solution.
 No distinct zones are
formed. The fractions
resolved are those of albumin,
α, β, and γ globulins.

45.  Zonal electrophoresis
 Involves the use of a support medium.
 The fractions resolved are albumin, α1, α2, β and
γ globulins.
 The charged particles are placed on a stabilizing
medium which will contain the proteins after
migration.

46.  PAPER ELECTROPHORESIS- It is the form of
electrophoresis that is carried out on filter paper. This
technique is useful for separation of small charged
molecules such as amino acids and small proteins.
• FILTER PAPER- It is the stabilizing medium.
• APPARATUS- Power pack, electrophoretic cell that
contains electrodes, buffer reservoirs, support for
paper, transparent insulating cover.

48.  GEL ELECTROPHORESIS- It is a technique used
for the separation of Deoxyribonucleic acid,
Ribonucleic acid or protein molecules according to
their size and electrical charge using an electric
current applied to a gel matrix.
What is a gel?
 Gel is a cross linked polymer whose composition
and porosity is chosen based on the specific weight
and porosity of the target molecules.
Types of Gel:
 Agarose gel
 Polyacrylamide gel

49.  Agarose gels
 Purified agar
 After
electrophoresis,
it can be stained
and read in a
densitometer
 Long term
storage
possible

50.  Polyacrylamide
Gel
 Gels with different
pore sizes can be
layered to provide
good separation of
molecules of
different sizes
 Good resolution

51.  TWO-DIMENSIONAL ELECTROPHORESIS- the
standard electrophoretic separation in one direction
is followed by SDS-PAGE in the perpendicular
direction.
 This technique combines the technique IEF (first
dimension), which separates proteins in a mixture
according to charge (PI), with the size separation
technique of SDS-PAGE (second dimension).

53. VIDEO 

54. PROCEDURE
 Serum is applied to the
support media and the
protein dissolves in the
buffer, giving them an
electric charge
 A specific amount of
current is applied for a
specific amount of time
 As the current flows
through the media, the
electrically charged
molecules migrate along
the supporting media

55.  The negatively charged protein molecules migrate
towards the oppositely charged electrode.
 The sample is separated into bands where each band
has molecules containing similar mobility.
 Once the medium has been stained and the
background of the medium support has been cleared,
the electrophoretic pattern can be scanned through a
densitometer.

56. STAINING OF THE SUPPORTING MEDIUM
 Staining fixes the
protein to the membrane
by denaturing
 Makes the fractions
visible
 Decolorization is used to
remove background
color
 Each peak in each
column represents a
different band of
molecules that migrated
together

57. ADVANTAGES:
 Versatility in Identification
 Accuracy of Results
DISADVANTAGES:
 Toxicity
 Electrophoresis has limited sample analysis
 Electrophoresis measurements are not precise
 Only certain molecules can be visualized

58. APPLICATION
 Specific protein analysis
 Identification and quantitation of hemoglobin and its subclasses
Identification of monoclonal proteins in either serum or urine.
Separation and quantitation of major lipoprotein and lipid classes
Isoenzyme analysis
Western blot technique to identify a specific protein.
Southern blot techniques to identify specific nucleic acid sequence.

59. INTERFERENCES
 Sample Contamination
 Gel Problems
 Improper Loading
 Electrical Current Problems
 Failed Visualization
 Varied Measuring

60. ISOELECTRIC FOCUSING

61. ISOELECTRIC FOCUSING
 Involves the migration of proteins in a pH gradient.
 Addition of acid to the anodic area of the
electrolyte cell and a base to the cathode area.
 It is the pH where the net charge of the protein
molecule is zero.
 Isoelectric focusing
requires solid support
such as agarose gel and
polyacrylamide gel.

62. PRINCIPLES
 Protein in a mixture can be precipitated depending
on its isoeletric point.
 IEF requires stable pH gradient which can be
formed by using mixture of specially designed
amphoteric molecules known as ampholytes.
 When electric field is applied, a pH gradient is
established, that is negatively charged ampholytes
move towards anode and positively charged
towards cathode and align themselves according to
their pIs.

63. VIDEO 

65. ADVANTAGES
 IEF offers the following advantages:
 efficient
 economic (no sophisticated equipment required)
 easy (clear, one-dimensional separation
principle)
 fast
 High capacity and resolution to 0.001 pH unit
possible

66. DISADVANTAGE
 A disadvantage of IEF is that minor bands and
aging bands are also seen and may cause
confusion in interpretation.

67. APPLICATIONS
 Useful in measuring serum acid phosphatase isoenzyme.
 Detects oligoclonal immunoglobulin bands in CSF and isoenzyme
of creatine kinase and alkaline phosphatase in serum.
 Applied in the assay of Acid Phosphatase isoenzyme.
 General characterization of proteins by pI purity determination of
proteins.
 Discrimination of caseins
 Routine clinical analyses

68. THANK YOU 