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

THANK YOU