This document discusses various principles of electrochemistry including concentration measurement using the Nernst equation, reference electrodes such as silver-silver chloride and calomel electrodes, indicator electrodes that are selective for specific ions, and ion-selective electrodes for measuring ions like hydrogen, sodium, and ammonium. It also covers principles of electrophoresis such as particle migration in an electric field, gel electrophoresis using agarose or polyacrylamide gels, and two-dimensional electrophoresis. Factors that affect electrochemical measurements and applications in clinical analysis are summarized.

1. Prepared by:
Patricia Ann Avelino
Rena Faith Baradero
Judy Ann Barbarono
Rheda Norba
Julie Ann Tapican
MLS 3-A
Presented to: Asst. Prof. Maria Deanna B. Jolito, RMT, MSMT

2. PRINCIPLE
Electrochemistry
Involves the measurement of current or voltage generated
by activity of specific ions.
ions move from higher potential energy to low potential
energy thus moving from anode to cathode.

3. *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.

4. *Each electrode is in contact with either the sample or a reference
solution.
*This method is made under conditions in which essentially zero
current is flowing through this system.
*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)

5. *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 on the concentration of the analyte
or any other ions in the solution
*Always treated as the left-hand electrode

6. *composed of
mercury/mercurous
chloride; It is dependable
but large, bulky, and
affected by temperature.

7. *reference electrodes are
more compact and handle
temperature fluctuations
better

8. *consists of a platinized
platinum electrode in a
1.228N HCl solution with
hydrogen at atmospheric
pressure bubbled over the
platinum surface.

9. *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.
*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

10. *Very sensitive and selective for the ion it is measuring
*Used for measuring serum and urine electrolytes
*Used in the measurement of Na, K, Cl, Ca, Mg and NH3 in serum
3 Basic ISE classes:
*Ion-Selective glass – for H+, Na+, NH4
+
*Solid-state electrodes – Ag-AgCl membrane for chloride
determination in sweat
*Liquid ion-exchange – for pH determination

11. *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 standard
reference electrode.

12. • 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

13. *Coulometry is an
electrochemical titration
where the titrant is
electrochemically generated
and the endpoint is
detected by amperometry.
*Amperometry- is the
measurement of the current
flow produced by an
oxidation- reduction
reaction.

14. • Gas-sensing electrode 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.

15. *Electrolytic cells
- non-spontaneous 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.

16. *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.

17. • Two electrodes: the Anode and the Cathode
anode is the electrode where oxidation occurs;
cathode is the electrode where reduction occurs;
 Volt meter
measures the electric current. In Galvanic cells, this shows how much
current is produced; in Electrolytic cells, this shows how much current is
charging the system.

18. • Electrolyte
• conducting medium
• has contact with electrodes
• usually in aqueous solution of ionic compounds
• Salt Bridge
• joins the two halves of the electrochemical cell
• filled with a salt solution or gel
• keeps the solution separate
• Completes the circuit
• Wire- conductor for electrons to be transferred

20. 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
use of cheaper more readily available starting materials
the possibility of reaching very high levels of product purity and
selectivity

21. 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.

22. 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

23. 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.

24. 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.
Factor affecting Potentiometry
-undesired ions

25. PRINCIPLE
Electrophoresis
Involves the migration of charged particles in an electric
field. A charged particle or an ion will migrate toward the influence
of an externally applied electric field.

26. *Involves separation of
molecules using homogenous
solution
*No distinct zones are formed
*The Fraction resolved are those
of albumin, , ,  globulins

27. *Involves the use of support medium
*Fractions resolved are albumin, 1, 2, ,  globulins
*The charged particles are placed on a stabilizing
medium which will contain the proteins after
migration

28. 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.

29. 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.
Types of Gel:
 Agarose gel
Polyacrylamide gel

30. • Purified agar
• After electrophoresis, it
can be stained and read in
a densitometer
• Long term storage
possible

31. • Gels with different pore
sizes can be layered to
provide good separation of
molecules of different sizes
• Good resolution

32. 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).

33.  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.

35. Net electric charge of the particle
Size and shape of the molecules
Electric field strength
Nature of the supporting medium
Temperature of operation

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

37.  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.

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

39. PRINCIPLE
Isoelectric focusing
Involves the migration of proteins in a pH gradient created
but addition of an acid to the anodic area and a base to the cathode
area. They stop migrating when they reach their isoelectric points.

40.  Electrophoresis and IEF ideally has the same equipment
used only that in IEF, the medium is pH gradient.

41.  efficient
 economic (no sophisticated equipment
required)
 easy (clear, one-dimensional separation
principle)
 fast
 High capacity and resolution to 0.001 pH
unit possible

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

43. 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

44. THANK YOU 