ELECTROPHORESIS AND

CHROMATOGRAPHY
BY: TANIA WAHAB
SOBIA GILLANI
HUMA IMTIAZ
 CONTENTS
• INTRODUCTION
• PRINCIPLE
• COMPONENTS
• OPERATION
• QUALITY CONTROL
• APPLICATIONS
 ELECTROPHORESIS
• Term means: migration with electricity.
• Involves the separation of components of a sample by differential rate of
migration in the presence of electric field.
• Theory was first proposed by Prof Ferdinand F reuss by doing experiment
on migration of colloidal clay particals
 PRINCIPLE
• Molecules moves with the speed dependent on their charge ,shape and
size and get separated in the presence of an electric field.
 COMPONENTS
• Gel casting assembly.
• Buffer container or electrophoresis tank
• Power supply
• Glass plate to hold the gel
• Comb to load sample in the gel before solidification
Cont…
 OPERATION
• Gel is prepared by adding powedered agarose to liquid boiling the mixture
• Comb is already placed which create rows of well for sample loading
• This agarose is then poured into casting tray and allow to solidify at room temp
• Gel is solidified and comb is removed and load standards and sample in wells
• Apply desired voltage to initiate electrophoresis
• Seperated products can be seen by placing the gel on UV trans illuminator and
calculated by comparing with standards
 QUALITY CONTROL
• Calibration of equipment
• Good quality and properly working of standards
• The standards should be run to check the validity
• Chemical must be purchased from suppliers who guarantee the purity
• Do not use expire reagent
• Buffers for QC of reagent must be tested according to SOPs
 APPLICATIONS
• Separation of protiens, DNA ,RNA and other macromolecules
• Purifications and analysis of vaccine and antibiotics
 CHROMATOGRAPHY
• Separation technique
• Used for the efficient separation of a number of similar components
present in a mixture.
 PRINCIPLE
• Physical method of separation that distribute components to separate
between two phases moves in a definite direction.
• Substances are separated based on their differential distribution between
two phases i,e mobile phase and stationary phase
 COMPONENTS
• Stationary phase
• Mobile phase
• Filter paper
• Staining solution
• Glass jar
• Clip
 OPERATION
• Whatman sheet for chromatography is selected
• 10 micro liter of sample (eg amino acid) is applied to one end of paper
• Spot is dried
• Paper is placed hanging in the tank using a clip . Lowe end of paper where test
solution was applied is diped into the solvent present in tank where remaining
part is hanging vertically
• Solvent travel up the paper by capillary action and takes with it the unknown
substance
 Cont..
• Different substances have different mobility rate, they will be separated at
different places over a vertical line.
• Paper is dried and stained.
• Test solutions are identified by comparing with standards.
• Rf values can be calculated by following formula:
Rf=distance travelled by the solute/distance travelled by the solvent.
Conti…
 QUALITY CONTROL
• Standard should of good quality.
• Every test run should include the standard.
• Standardization and accuracy is critical in advance chromatography.
• All results should be documented.
 APPLICATIONS
• For qualitative and quantitative analysis of large number of compounds
e.g amino acids, proteins, carbohydrates…etc
• For analysis of drugs, hormones and vitamins
• Advantageous to determine the molecular weight of proteins.