GEL CHROMATOGRAPHY

Dr. Imrana Khushk

Institute of Biotechnology and Genetic Engineering
University of Sindh, Jamshoro

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• Gel chromatography

• Gel permeation chromatography

• Size exclusion chromatography

• Gel filtration chromatography

• Molecular-sieve chromatography

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 Size Exclusion Chromatography

• Separates molecules according to their size, shape & molecular weight.

• It is usually applied to large molecules or macromolecular complexes

• Separates proteins, peptides, and oligonucleotides on the basis of size.

• Molecules move through a bed of porous beads, diffusing into the beads to
greater or lesser degrees.

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 Basic Principle
• Works by trapping smaller molecules in the pores of the adsorbent materials:

adsorption ("stationary phases").

• The larger molecules simply pass by the pores

because those molecules are too large to enter the
pores.

• Larger molecules therefore flow through the

column more quickly than smaller molecules

• In short : The smaller the molecule, the longer

the retention time.
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Pictorial representation of the working principle

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 STATIONARY PHASE:
• Semi-permeable, porous beads with a well-defined range of pore sizes.

• Smaller pore sizes: Rapid desalting of proteins or for protein purification.

• Intermediate pore sizes: Separate relatively small proteins.

• Very large pore sizes: Purification of biological complexes.

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 Stationary phase

 Dextran (Sephadex) gel: An α 1-6-polymer of glucose natural gel.

 Agarose gel: A 1,3 linked β-D-galactose and 1,4 linked 3,6-anhydro-α, L-

galactose natural gel.

 Acrylamide gel: A polymerized acrylamide, a synthetic gel.

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Fractionation range (Size Exclusion limit)
The average or maximum effective pore size defines what is called the fractionation
range or exclusion limit of the resin.

Molecules larger than the pore size pass straight through (are excluded). This is
called the exclusion limit.

Conversely, molecules below a certain size completely penetrate the pores and tend
to elute almost in the same position. This is called the permeation limit.

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Void volume

Void volume is the mobile phase hold-up volume. For example, if the stationary phase
occupies 40% of the total column volume, the void volume would be 60% of the total
column volume.

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•Elution: The process of passing the mobile phase through the
column, which carries the sample molecules along. Elution can be
isocratic (constant composition of the mobile phase) or gradient
(changing composition of the mobile phase).
•Eluate: The solution that comes out of the column after passing
through the stationary phase, containing the separated
components of the mixture.
•Elution Volume (Ve): The volume of mobile phase required to
elute a particular molecule or component from the column.
•Void Volume (Vo): The volume within the column that is outside
the pores of the gel beads, accessible only to molecules too large
to enter the pores. It represents the volume of the mobile phase in
the column excluding the volume occupied by the gel beads.
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•Retention Volume (Vr): The total volume from the point of injection to the
point at which a component is eluted, including the void volume.
•Partition Coefficient (Kav): A value that represents the degree to which a
molecule penetrates the gel matrix, calculated as (Ve - Vo) / (Vt - Vo), where
Vt is the total bed volume.
•Exclusion Limit: The molecular weight above which all molecules will elute
at the same volume (the void volume), because they are too large to enter
any of the pores.
•Fractionation Range: The range of molecular weights that a particular gel
filtration medium can effectively separate.
•Gel Filtration Standards: A set of molecules with known molecular weights
used to calibrate the gel filtration column, facilitating the determination of the
molecular weights of unknown substances by comparison.

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 Mobile Phase
Material Solvent

Synthetic elastomers Toluene

(polybutadiene , polyisoprene )
PS, PVC, Styrene-Butadiene Tetrahydrofuran
Rubber , Epoxy resins
Polyolefins Tri- chloro -benzene

Polyurethane Di- methylformamide

Proteins, polysaccharides Water / Buffers

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 Separation procedure
1- Preparation of column
 Swelling of the gel
 Packing the column
 Washing
2- Loading the sample onto the column using a syringe.
3- Eluting the sample and detection of components.

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 Columns
Commercially Available Columns

• Analytical column: 7.5–8mm diameters.

• Preparative columns: 22–25mm.

• Usual column lengths: 25, 30, 50, and 60 cm.

• Recently, narrow bore columns: 2–3mm

diameter have been introduced, which save time.

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 Factors affecting SEC
• Column Length: An increase in column length will enhance the resolution.
• Column Diameter: Increasing the column diameter increases the capacity of the column.
• Proper Column Packing : Column packing should be optimum It is important to
maximize resolution as:

• 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.
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 Detector
1. Concentration sensitive detectors
• Bulk Property Detectors- Refractive Index (RI) Detector
• Solute Property Detectors- Ultraviolet (UV) Absorption Detector
• Evaporative Detectors- Evaporative Light Scattering Detector
(ELSD)

2. Molar mass sensitive detectors

• Light Scattering Detectors
• Low Angle Light Scattering (LALS) Detectors
• Multiangle Light Scattering (MALS) detectors
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3. Viscosity Detectors: Differential Viscometers

Other :-

• Flame Ionization Detector (FID),

• A Mass Spectrometer or A Fourier Transform Infrared

(FTIR) Spectrometer

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Description Of Separation In SEC

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 Applications of SEC
 Proteins fractionation
 Purification
 Molecular weight determination.
 Separation of sugar, proteins, peptides, rubbers and others on the basis of
their size.
 Fractionation of molecules and complexes within a predetermined size range
 Size analysis and determination
 Removal of large proteins and complexes
 Buffer exchange
 Desalting
 Removal of small molecules such as nucleotides, primers, dyes, and
contaminants
 Assessment of sample purity
 Separation of bound from unbound radioisotopes
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 Pros and cons of SEC
Advantages Disadvantages
• Simplicity, reliability • Large size of columns need
& versatility large volumes of eluent often
• Ease of scale up creating excessive running
costs.
• Low chances of • When separating proteins by
sample loss SEC proteolysis increasing
• Less time of analysis problem.
• Low resolution compared to
other chromatographic
techniques
• Low sample handling capacity
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THANK YOU

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