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Prepared by:
Patricia Ann Avelino
Rena Faith Baradero
Judy Ann Barbarono
Rheda Norba
Julie Ann Tapican
MLS 3-A
ELECTROCHEMISTRY,
ELECTROPHORESIS, ISOELECTRIC
FOCUSING
Presented to: Asst. Prof. Maria Deanna B. Jolito, RMT, MSMT
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.
DIFFERENT TYPES OF ELECTROCHEMISTRY
POTENTIOMETRY
*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.
*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)
Reference Electrodes
*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 o n the concentration of
the analyte or any other ions in the solution
*Always treated as the left-hand electrode
Calomel electrode
*composed of
mercury/mercurous
chloride; It is dependable
but large, bulky, and
affected by temperature.
Silver-silver chloride electrode
*reference electrodes are
more compact and
handle temperature
fluctuations better
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.
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.
*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
Ion Selective Electrode
*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
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 standard reference electrode.
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
Coulometry & Amperometry
*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.
pO2 Gas Electrode
•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.
2 BASIC ELECTROCHEMICAL CELLS
*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.
*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.
COMPONENTS OF ELECTROCHEMISTRY
• 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.
• 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
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
use of cheaper more readily available starting materials
the possibility of reaching very high levels of product purity and selectivity
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.
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
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.
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
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.
DIFFERENT TYPES OF ELECTROPHORESIS
Moving Boundary or Frontal Electrophoresis*Involves separation of
molecules using homogenous solution
*No distinct zones are formed
*The Fraction resolved are those of albumin, , , globulins
Zonal Electrophoresis
*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
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.
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 gelPolyacrylamide gel
Agarose gels
•Purified agar
•After electrophoresis, it can be stained and read in a densitometer
•Long term storage
possible
Polyacrylamide Gel
•Gels with different pore sizes can be layered to provide good separation of molecules of different sizes
•Good resolution
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).
COMPONENTS OF ELECTROPHORESIS
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.
FACTORS INFLUENCING MIGRATION OF PARTICLES
Net electric charge of the particleSize and shape of the molecules Electric field strengthNature of the supporting medium Temperature of operation
ADVANTAGESVersatility in IdentificationAccuracy of Results Low cost to use
DISADVANTAGES:ToxicityElectrophoresis has limited sample analysisElectrophoresis measurements are not preciseOnly certain molecules can be visualized
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.
Interferences
Sample Contamination
Gel Problems
Improper Loading
Electrical Current Problems
Failed Visualization
Varied Measuring
PRINCIPLEIsoelectric 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.
Electrophoresis and IEF ideally has the same equipment used only that in IEF, the medium is pH gradient.
COMPONENTS OF ISOELECTRIC FOCUSING
Advantages
efficient economic (no sophisticated
equipment required) easy (clear, one-dimensional
separation principle) fast High capacity and resolution to 0.001
pH unit possible
Disadvantage
A disadvantage of IEF is that minor bands and aging bands are also seen and may cause confusion in interpretation.
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
Recommended