ANALYTICAL TECHNIQUES PART 2

ELECTROCHEMISTRY

Study of redox reactions

ELECTROCHEMISTRY

Involves the measurement of current

or voltage generated by the activity

of specific ion.

Galvanic Cell
Electrolytic Cell

Two basic electrochemical cells

Galvanic Cell

generates current

Electrolytic Cell

requires current

Current

The flow of electric charge across a certain element

Voltage

Electrical potential difference between two points

I

Symbol of Current

V

Symbol of voltage

A / amps / amperage

unit of current

V / Volts / Voltage

unit of voltage

1 ampere = 1 coulomb/second

SI unit for current

1 volt = 1 joule/coulomb

SI unit for voltage

Ammeter

Measuring intrument of current

Voltmeter

Measuring instrument of voltage

Current

Current or Voltage?
the effect

Voltage

Current or Voltage?
the cause

Current

voltage

______ cannot flow without _______

Voltage
current

________ can exist without _______.

POTENTIOMETRY

Measurement of the differences in voltage at a constant current

Nernst equation

POTENTIOMETRY is expressed in _________

pH

POTENTIOMETRY is used in the measurement of _______

direct measurement of electrical potential

Potentiometric methods of analysis involve the _____________________________ due to the activity of free ions.

Ion-Selective Electrodes (ISE)

composed of electrochemical transducer capable of responding to one given ion.

Ion-Selective Electrodes (ISE)

Very sensitive and specific to the ion it measures

Ion-Selective Electrodes (ISE)

Practical application: measurement of electrolytes namely Na+, K+, and Cl-

Na+, K+, and Cl-

electrolytes measured by potentiometry

Inert-metal electrodes

Metal electrodes

Membrane electrodes

Types of ISE

Inert-metal electrodes

(standard H electrode) for Na+ measurements

Metal electrodes

(Ag/AgCl electrode) for Cl- measurements

Membrane electrodes

(e.g. valinomycin gel electrode) for K+ measurements.

pH Electrode

ISE universally used in the laboratory

pH Electrode

Determines pH values quickly and accurately

Indicator electrode/ Glass electrode

Consists of a silver wire coated with AgCl, immersed into an internal solution of KCl, and placed into a tube containing a special glass membrane tip.

Indicator electrode/ Glass electrode

This membrane is only sensitive to

Hydrogen ions (H).

Composed of an internal wire made of either Silver – silver chloride system or Calomel electrode (mainly mercurious chloride)

Reference electrode

A filling solution of potassium chloride, and a permeable outer casing

Liquid junction

Protects the reference system from the medium to be measured without disconnecting the electrical potential between them.

Potassium chloride (KCl)

is a commonly used filling solution because K+ and Cl- have nearly the same mobilities.

Read-out device/meter (Voltmeter)

Numerically represent the pH measurements

acidic / acidic pH

If the number of H+ ions in the solution exceeds the number of H+ inside the pH meter probe; the solution will register an ________________.

alkaline/basic pH

If the number of H+ ions in the solution is less than the number of H+ inside the pH meter probe; the solution will register an __________________.

neutral pH

If the number of H+ ions in the solution is equal to the number of H+ inside the pH meter probe; the solution will register a _____________.

calibration

Do not measure the pH of unknown solutions without ____________.

7.0

Calibrate pH of unknown sol’n using a solution with a pH of ____.

3M KCI solution

Wash the electrode after use with clean water and store in ______________.

clean water

Apply ___________ to flush the electrode after the cleaning steps

pepsin

HCl

Protein containing samples

Immerse the electrode in _________ or _____ solution for a few hours.

thiourea

HCl
white

Sulfide containing samples

Immerse the electrode in _______ or _____ solution until the diaphragm of the electrode turns _________.

acetone
alcohol

Grease or organic compound samples

Apply ________ or _______ to clean the electrode for a few minutes

0.1 M NaOH
0.1M HCl

General Samples

Immerse the electrodes in _________ or ________ for a few minutes

Gas sensing electrodes

▪ Designed to detect specific gases

Amperometry
Potentiometry

Gas sensing electrodes

Examples are

▪ pO2 (Clark electrode)- ________

▪ pCO2 electrode (Severinghaus electrode)-

Enzyme electrodes

Determined by the reaction product of the immobilized enzyme

Urease

Glucose oxidase

Enzyme electrodes
Examples are:

• ________ – detects urea

• ______________ – detects glucose

COULOMETRY

Measurement of the amount of electricity (in coulombs) at a fixed potential.

Faraday’s Law

COULOMETRY follows __________ law

COULOMETRY

Involves electrochemical titration and is used for the measurement of chloride in both sweat and serum.

Chloride ISE

COULOMETRY is now obsolete and is being replaced today by

AMPEROMETRY

Used in the determination of :

▪ Partial pressure of Oxygen (pO2)

▪ Glucose

▪ Chloride

▪ Peroxidase

Polarography

▪ Utilizes the principle of amperometry

Polarography

Measurement of differences in current at a constant voltage

trace metals
O2

Vitamin C

amino acid concentrations

Polarography is used in the measurement of ____________ , ____, ___________ and ______________________.

Ilkovic equation

Polarography follows what equation?

Anodic Stripping Voltametry

the analyte is first concentrated onto the

surface of an electrode at a constant

potential and then goes back into solution as

the voltage is changed.

iron

lead

Anodic Stripping Voltametry is used in the measurement of ______ and ______.

Electrophoresis

is a separation technique based on the principle that a charged particle in solution will migrate towards one of the electrodes when placed in an electrical field.

Proteomics

large scale study of proteins particularly the structures and functions.

Genomics

study of genome (complete set of DNA or complete set of hereditary information)

basic type of electrophoresis that….

▪ Uses agarose gel

▪ Used in Genomics

Vertical

Electrophoresis

basic type of electrophoresis that….

▪ Uses Sodium Dodecyl Sulfate Polyacrylamide Gel (SDS- PAGE)

▪ Used in Proteomics

Ions

charged particles

Cations

(+) Charged ions

Anions

(-) Charged ions

Zwitterions

neutrally–charged ions

Cathode

(-) electrode; where reduction occurs

Anode

(+) electrode; where oxidation occurs.

Iontophoresis

migration of small charged ions

Zone Electrophoresis

migration of charged macromolecules.

Electrophoretogram

result of zone electrophoresis and consists of a macromolecule.

Driving Force (electrical power)

Supplies current or voltage

Support Medium (gel)

Provide a matrix that allows molecules to separate

Paper

Obsolete type of support medium

Cellulose acetate

(replacement for paper) – separates particles by molecular size.

Agarose Gel

separates particles by electrical charge (usually used for Genomics)

Starch Gel

separates particles by charge and size.

Polyacrylamide Gel

separates particles by charge and size (usually used for proteomics).

Buffer (Fluid)

Provides an ionic solution that allows current to pass through the water.

• Barbital (Veronal) Buffer : pH 8.6

• Tris-Borate-EDTA: pH 8.7

Most commonly used buffers

Sample

Can be pre-treated serum or DNA

Sample

Placed in wells created by comb

Sample

Usually combined with a dye (ethidium bromide) for easy visualization of migration.

Detecting System

Converts bands into algorithm to know the exact concentration of substances

▪ Densitometer

▪ Documentation System

Detecting System can be:

negative

net electric charge of cathode to anode

positive

net electric charge of anode to cathode

Large molecules

molecules that travels slowly

Small molecules

molecules that travels fast

Electric Field Strength

Increasing the strength of the field (increase Current) also increases migration

▪ Amino Black

▪ Ponceau S

▪ Oil red O

▪ Sudan Black

▪ Fat Red 7B

▪ Coomassie Brilliant Blue

▪ Silver Nitrate

▪ Nitrotetrazolium Blue

Stains