size exclusion chromatography, its principle,instrumentations, advantages, disadvantages, applications

1. SIZE EXCLUSION
CHROMATOGRAPHY
Bindu Kshtriya
Khanchibindu95@gmail.com

2. INTRODUCTION
• Size exclusion chromatography is a mechanical sorting
of molecules based on the size of molecules in
solution.
• Small molecules are able to permeate more pores and
are retained longer than larger molecules.

3. TYPES OF SIZE EXCLUSION
CHROMATOGRAPHY
Two basic types of SEC are:-
❑ GEL PERMEATION CHROMATOGRAPHY (GPC)
• Uses a hydrophobic column packing material and a
non-aqueous mobile phase (organic solvent) to
measure the molecular weight distribution of synthetic
polymers.
❑ GEL FILTERATION CHROMATOGRAPHY(GFC)
• Uses a hydrophilic packing material and an aqueous
mobile phase to separate, fractionate, or measure the
molecular weight distribution of molecules soluble in
water, such as polysaccharides and proteins.

4. OBJECTIVE
• SEC is a widely used technique for the purification and analysis of
synthetic and biological polymers, such as proteins,
polysaccharides and nucleic acids.
• Mild, non destructive method for determining molecular weight.
• It has become a mature and well accepted technique for
characterizing both synthetic polymers and biopolymers.
• Standard technique for the analysis of monoclonal antibodies and
their aggregates.

5. GEL
• Gel is a familiar name. It refers to a fairly
soft, elastic material containing water. In the
scientific context, the term acquires a wider
meaning. A gel consists of a three
dimensional network. The structural
material, often consisting of cross linked
polymers, gives some mechanical stability.
The space within the gel not occupied by
structural material is filled with liquid. Liquid
occupies the main part of gel.

6. GEL PREPARATION
❑ Two methods of Gel Preparation:
■ Weighed amount of dry powder is mixed with excess of solvent
and allowed it to swell. The mixture is left as such till the
equilibrium condition is maintained.
■ In this method, gel slurry is warmed at about 100 degree Celsius
in boiling water bath. As a result gel swells in few days. The
slurry is then cooled and packed in the column.
• The gel can stored in the wet state and there is no
need to dry them, however for storage purpose,
sephadex and acrylamide gels can be dried without
any damage and can be brought back the wet state
very easily.

7. KEY POINTS
❑ The most convenient method to allow the gel to swell
in a particular solvent is
▪ If swelling is completed by heating ,then the resultant
slurry is allowed to cool before packing.
▪ If slurry is prepared in cold, it is necessary to
deareated to vacuum.

8. MECHANISM OF ACTION
Size exclusion column bed has three functional components:
❑ THE PORE VOLUME
• The pore volume refers to the pore-lumen space within
the particles.
❑ THE VOID VOLUME
• It refers to the excluded volume i.e., the space between
the particles.
❑ THE MATRIX VOLUME
• It refers to the solid component of the particles that fills
the column bed.

10. ▪ Molecules larger than the pore size can not enter the pores
and elute together as the first peak in the chromatogram.
▪ Molecules that can enter the pores will have an average
residence time in the particles that depends on the molecules
size and shape.
▪ Different molecules therefore have different total transit times
through the column.
▪ Molecules that are smaller the pore size, and have the
longest residence time on the column and elute together as
the last peak in the chromatogram.

12. INSTRUMENTATION
• INJECTION VALVE
• PUMP
• COLUMN
• DETECTOR
• RECORDER

13. WORKING
Pumps - for maintaining
constant rates of flow
Column-for separating the
samples
Detector – for quantifying
the result
Degasser

14. COMMERCIALLY AVAILABLE COLUMNS
• The typical column diameters are 7.5–8 mm for
analytical columns and 22–25 mm for (semi) preparative
columns; usual column lengths are 25, 30, 50, and 60
cm.
• The packing are based on either porous silica or semi
rigid (highly cross linked) organic gels, in most cases
copolymers of styrene and divinyl benzene.
• 125Å pore size for analysis of small proteins and
peptides
• 250Å pore size for most protein samples
• 450Å pore size for very large proteins and nucleic acids

15. Product pH stability Particle size
Superdex Peptide
Long term: 1–14
Short term: 1–14
13–15 μm
Superdex 75
Long term: 3–12
Short term: 1–14
13–15 μm
Superdex 200
Long term: 3–12
Short term: 1–14
13–15 μm
Superdex 30 prep grade
Long term: 3–12
Short term: 1–14
22–44 μm
Superdex 75 prep grade
Long term: 3–12
Short term: 1–14
22–44 μm
Superdex 200 prep grade
Long term: 3–12
Short term: 1–14
22–44 μm
COMMERCIALLY AVAILABLE COLUMNS AND
PROPERTIES:
Superdex 200 or Superdex 200 prep grade - especially suitable for the
separation of monoclonal antibodies from dimers and from contaminants o
lower molecular weight

16. SIZE EXCLUSION COLUMN
• It consist of a hollow tube tightly packed with extremely
small porous polymer beads designed to have pores of
different size.
• Lager the particles, faster is the elusion.

17. •Increasing the column length will enhance the resolution, and
increasing the column diameter increases the capacity of the
column.
•Proper column packing is important to maximize resolution: An
over packed column can collapse the pores in the beads,
resulting in a loss of resolution.
• An under packed column can reduce the relative surface area
of the stationary phase accessible to smaller species, resulting
in those species spending less time trapped in pores.
POINTS TO REMEMBER

18. DETECTORS
• TYPES
➢ Concentration sensitive detectors
U.V absorption ,refractive index (R.I ) detectors, (I.R)
absorption and density detectors
➢ Molecular weight sensitive detectors
Low angle light scattering detectors (LALLS) ,Multi
angle light scattering (MALLS) detectors..

19. CHROMATOGRAM
• Extent of retention depends on the size of the included
molecules relative to the pores.
• Small molecules will enter all pores.
• Intermediate molecule, due to velocity of mobile phase,
will not be able to diffuse into pores that may fit, thus
will bee retained less effectively.
• Initial peak contains totally excluded solute.
• Final peak contains totally included solutes.

20. DESCRIPTION OF SEPARATION IN SEC

21. ADVANTAGES
• It can be carried out at room temperature and the sample are
not decomposed because no exposure to high temperature.
• Rapid, routine analysis.
• Identify high mass components even in low concentration.
• Can analyze poly disperse samples.
• Branching studies can be done
• Absolute molecular weight can be obtained.

22. • Short and well defined separation times.
• Narrow bands, which leads to good sensitivity.
• Freedom from sample loss because solute do not
interact with stationary phase.
• Absence of column deactivation brought about by
interaction of solute with the packing.

23. DISADVANTAGE
• Filtrations must be performed before using the
instrument to prevent dust and other particulates from
ruining the columns and interfering with detectors.
▪ Bad response for very small molecular weights.
• Sensitive for flow rate variation, internal standard
should be used whenever possible.
• High investment cost.

24. • Only a limited number of bands can be accommodated
because the time scale of chromatogram is short
• Inapplicability to samples of similar size, such as
isomers.10% difference in molecular mass is required
for reasonable resolution

25. APPLICATIONS
• Purification
• Desalting
• Concentration of dilute solutions
• Molecular weight determination
• Protein binding study

26. PURIFICATION
• Purification of biological macromolecules like viruses,
protein, enzymes hormones ,nucleic acids, antibodies
and polypeptides.
• Separation of low molecular weight component from
mixture.
• examples separation of low molecular weight Dextran
from corn syrup oil.

27. DESALTING
• Large molecules of biological origin are separated from
inorganic or ionisable species is known as desalting.
• By the use of a column of sephadex G-25,solution of
high molecular weight compounds can be desalted.
• In this the high molecular weight substances move with
the void volume while the low molecular weight
components are distributed between the mobile and
stationary phase and hence move slowly.

28. APPLICATION OF DESALTING
• Desalting is faster and more efficient technique than
dialysis.
• It helps in removal of phenol from nucleic acid
preparations ,ammonium sulphate from protein
preparations as well as monosaccharides from
polysaccharides and amino acids from proteins.

29. • Solution of high molecular weight substances can
be concentrated by the addition of dry G-
sephadex (coarse).
• Water and low molecular weight substances
remain in solution.
• After ten minutes the gel is removed by
centrifugation, leaving the high molecular weight
material in a solution whose concentration has
increased but whose ph and ionic strength are
unaltered.
CONCENTRATION OF DILUTE SOLUTION

30. PROTEIN BINDING STUDIES
• Reversible binding of a ligand to a macromolecule such
as protein including receptor proteins.
• A sample of proteins/ ligands mixture is applied to
column of gel which has previously equilibrated with
solution of ligand of same concentration in mixture.
• Sample is eluted with buffer and concentration of
ligand and protein in the effulent are determined.
• Early fractions will contain unbound ligand,but the
subsequent appearance of the protein will result in an
increase in the total amount of ligand (bound plus
unbound)

31. MOLECULAR WEIGHT DETERMINATION
• Sephadex G-25 and G-50 have been used in the
removal of low molecular weight molecules from high
molecular weight natural product molecules.
• The effulent volume of globular protein are largely
determined by their molecular weight.
• The effluent volume is approximately a linear function
of the logarithm of molecular weight.

32. ABSOLUTE SIZE-EXCLUSION
CHROMATOGRAPHY
Absolute size-exclusion chromatography (ASEC)
is a technique that couples a dynamic light
scattering (DLS) instrument to a size exclusion
chromatography system for absolute size
measurements of proteins and macromolecules
as they elute from the chromatography system.

33. ADVANTAGE OF DYNAMIC LIGHT SCATTERING(DLS)
• A big advantage of DLS coupled with SEC is the
ability to obtain enhanced DLS resolution.
• Batch DLS is quick and simple and provides a
direct measure of the average size but the
baseline resolution of DLS is 3 to 1 in diameter

34. RECENT ADVANCES IN SEC
Size-exclusion chromatography (SEC) process is now available to purify
DNA-wrapped carbon nanotubes (DNA-CNT) and to sort them into
fractions of uniform length. A type of silica-based column resin was
identified that shows minimum adsorption of DNA-CNT.

35. THANK YOU
BINDU KSHTRIYA