Govt. Nagarjuna P.g.
college of science, Raipur
Department of Biochemistry
“ GEL FILTRATION CHROMATOGRAPHY
“
Guided by Presented by
Dr. M. K. Dwivedi (Hod) SEEMA SINHA
Miss. Yamini sahu
MSC - 2nd sem.
➢ Also known as ,
❏ Gel permeation chromatography .
❏ Gel filtration chromatography.
❏ Size Exclusion chromatography ,
( Molecular exclusion chromatography )
❏ Molecular sieve chromatography .
SYNOPSIS
➢ Introduction
➢ Principle
➢ Instrumentation
➢ Digram
➢ Application
➢ Reference
INTRODUCTION
➢ Is a chromatographic technique in which seperation of component is Based on the
difference in molecular weight or size .
➢ It is one of the effective methods used to isolate and analyze the
Bio-macromolecules .
➢ The stationary phase consists of Beads containing pores.
➢ When gel is packed into a column and percolated with a solvent.
➢ It permits large molecules to pass faster without pentration of pores.
➢ Smaller molecules spend more time inside the beads and therefore is eluted later.
➢ When an organic solvent is used as a mobile phase called as gel permeation
chromatography.
➢ When an aqueous solution is used as a mobile phase called as gel filtration
chromatography.
PRINCIPLE
➢ The stationary phase in this method is an open network of polymers which are
cross linked to each other to from the pores of consistent size.
➢ When mobile phase containing mixture of solutes of various size is passed
through the column.
➢ The molecules which are too large to enter the pores are excluded completely
and gel eluted with mobile phase.
➢ The molecules which are smaller in size diffuse in and out of the pores, travelled
quite large path eluted later.
➢ So, the components of mixture gel eluted from column according to their relative
molecular size.
➢ Large molecules eluted First.
➢ Smaller molecules eluted at last.
Instrumentation and methodology of gel chromatography
➢ Colomn
➢ the columns used in gel chromatography brooder and longer than used for
adsorption and partition chromatography .
➢ The diometer of column used in gel chromatography.
➢ Range from 10-20 mm.
➢ The column contains glass wool or filter paper at its lower end
➢ Which allows flow of mobile phase but prevent the exit of stationary phase.
Gel ,
➢ Second important component of gel chromatography.
➢ Use, as a stationary phase.
➢ Properties -
➢ Chemically non reactive .
➢ Pores size equal.
➢ Gel particals stable.
➢ Low swelling ability .
Types of Gels ,
Gel which are commonly used include -
➢ Cross linked dextrons (Sephodex )
➢ Agarose ( sepharose, Bio-Gel A )
➢ Polyacrylamide (Bio-Gel P )
➢ Polyacrylmorphine ( Enzocryl Gel )
➢ Polystyrenes ( Bio Beads S )
Gel preparation
➢ Two common ways are -
➢ Mixing dry powder gel with an excess of mobile phase
➢ Allow if to swell.
➢ Leave until the equilibrium condition is achieved.
➢ This process takes few hours to few days.
➢ Mix the powder gel with an excess solvent (m.p.)
➢ Worm the slurny to about 100;c for 30min in a water bath.
Packing of column
➢ Clamp the glass column in a vertical position
➢ Fill the column with gel slumy carefully to avoid air bubbles.
➢ Let the suspension settle down.
➢ Keep the solvent just above the surface of the gel.
Apply of the sample ,
➢ Glass pipettes are used to pour the sample at the top of the gel surface.
➢ Followed by washing with mobile phase to ensure complete sample has
passed into gel .
Elution development
➢ After sample application add more solvent carefully to the height 2-5cm.
➢ After that connect the column with mobile phase reservoir so that hight
of solvent maintain at appropriate high .
➢ The sample components are separated by continuous flow of solvent
through the column.
Collection and analysis of eluent
➢ Collect the eluent in a series of the test tubes.
➢ Either at fixed volume or time intervals.
➢ Various detectors are used are -
➢ Differential refractometers, UV-visible spectrophotometers , flame
ionisation detector, electrical conductivity detectors etc.
Gel filtration chromatography - An overview
Internal volume (Vi)
APPLICATION
➢ Molecular weight determination .
➢ Separation of large molecular weight compounds like
proteins, carbohydrates , peptides , nucleic acids etc.
➢ Purification of enzymes , hormones , antibodies, etc .
➢ Desalting of biomolecules.
➢ Separation of sugar , Rubbers , polystyrenes etc.
Reference.
➢ Google slides
➢ YouTube
THANK YOU
