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Size Exclusion Chromatography (SEC) : Principle

Size exclusion chromatography, also known as gel permeation chromatography or gel filtration chromatography, separates molecules based on their size. Small molecules penetrate the pores in the stationary phase and are eluted later, while large molecules that cannot enter the pores are eluted first. Common stationary phases used include cross-linked dextrans, agarose, and polyacrylamides in column lengths of 20-60 cm. Proteins, polymers, lipids, and other macromolecules can be separated using SEC.

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401 views12 pages

Size Exclusion Chromatography (SEC) : Principle

Size exclusion chromatography, also known as gel permeation chromatography or gel filtration chromatography, separates molecules based on their size. Small molecules penetrate the pores in the stationary phase and are eluted later, while large molecules that cannot enter the pores are eluted first. Common stationary phases used include cross-linked dextrans, agarose, and polyacrylamides in column lengths of 20-60 cm. Proteins, polymers, lipids, and other macromolecules can be separated using SEC.

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Ikkal
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Size Exclusion Chromatography (SEC)

• Molecular exclusion Chromatography


• Gel filtration chromatography
• Gel permeation chromatography.

• Principle:
• Molecules are separated according to their size.
• Small molecules penetrate the small pores in the
stationary phase, while large molecules do not.
Large molecules are eluted first.
History:
1960’s Porath and Flodin describe the separation of water soluble
macro molecules on cross-linked polydextran gels.

• Molecules are separated according to their size. Big molecules cannot


penetrate the small pores in the stationary phase. They are eluted by a volume
of solvent equal to the volume of mobile phase

Vr – V0
Kav =
Vt – V0

Vt V0: volume of the mobile phase (void volume)


Vr: retention volume for a solute
V0
Vt : total volume of the column, r2 x length

V0 found by e.g. Blue Dextran 2000


Molecular Exclusion Chromatography

Molecules are separated according to their size

SEC separation of two macromolecular sizes:


1.Sample mixture before entering the column Typical SEC calibration curve:
packing logarithm of M.Wt. versus
2.Sample mixture upon the head of the column retention volume
3.Size separation begins
4.Complete resolution
CHROMATOGRAPHY

•Mobile phase is delivered to a


column with the appropriate
stationary phase, using a
pump at a reproducible and
constant flow rate.

•0.01-1.0 mg of polymer
sample is injected.

•Sample is detected by at least


one detector.

•Due to the logarithmic


relation of mass and elution
time, a change in flow-rate of
0.1% can cause an error in
molar mass of up to 10%.
Stationary Phase
• Polymers of glucose:
– Cellulose
– Dextran (If cross-link is glycerin: Sephadex)
• Polymers of agarose
• Polyacrylamides

 Crossed-linked gels  Crossed-linked gels


Small pore size Big pore size
Exclude molecules with MM  700 Exclude molecules with MM  108

• The finer the particle, the greater the resolution, the slower
the flow rate of the column

• Particles with different pore sizes can be mixed to give a


wider molecular size separation range

• In general, two types of molecules can be separated provided


Columns
• Unlike the other modes of Usual size of SEC columns:
liquid chromatography, the
separation comes from the 7-8 mm diameter
stationary phase only. The
mobile phase should have no (analytical)
effect as long as the sample
is well dissolvable. 20-25 mm diameter
(preparative)
• Separation is carried out on
the pores which typically Length 20-60 cm
equals 40% of the total
column volume.

• As a result long columns, or Packing:


several columns are
required. •porous silica
•cross-linked organic gels
Columns
Selection of SEC columns:
• Small particle size (5µm) provides more theoretical plates

• Small particle size more sensitive to contamination

• Small particle size can result on shear degradation of large polymers

• Combination of packings with different separation range can be achieved by:


columns of different porosity or mixed bed columns

• Chemical nature of column packing crucial

Handling SEC columns:


• A column set should be always run in the same mobile phase (calibration, life of the
column)

• Should not be operated in backward position

• No air bubbles in columns during injection and assembly

• Always use a pre-column when in doubt about the quality of the sample
Applications

• Typical SEC applications:


– analysis of synthetic polymers and oligomers
– coal derived substances
– lipids
– proteins
– cellulose derivatives
– crude oil alkanes
Separation of proteins

Adenylate kinase 32000


Cytochrome C 12400
Enolase kinase 67000
Glutamate Dehydrogenase 290000
Lactate Dehydrogenase 140000
Choosing the pore width

Organic soluble polymers and styrene/divinylbenzene packings

The table lists the molecular weight range of separation for individual
pore size columns of styrene/divinylbenzene packings, based on
polystyrene chain length exclusion limits (in Angstroms)
Instrument parameters
• flow rate (0.1-10 ml/min, default 1 ml/min)

• injection volume (1-1000 l, default 10 l)

• temperature (25-220 ºC, default 40 ºC)

• column length (50-1200 mm, default 300 mm)

• column ID (2-20 mm, default 7.5 mm)

• connection (dead) volume (0-1000 µl)

• detector type (Refractive Index, Viscosity, Density, Lightscatter

• detector volume (1-1000 µl)

• detector path length (1-25 mm)

• detector time constant (0.01-5 s)


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