24 Ion/Molecular Exclusion chromatography

Dr. Varinder Kaur

Objectives: To study the basics of size exclusion chromatographyand know about following questions.

1.What is Size exclusion chromatography?

2. What is the mechanism of size exclusion chromatography?

3. Which type of stationary phases are used in SEC?

1. Description

Size exclusion chromatography has various applications like semi-preparative purifications and analysis. It separates the molecules on the basis of the difference in their size. In this technique, molecules do not bind the stationary phase instead the molecules percolate as per their size and shape. Moreover, use of buffer, adjustment of pH, and amounts of metal ions or cofactors is not necessary for the resolution in SEC. This technique avoids all the harsh conditions, therefore is usually used for biomolecules. The separation of biomolecules in aqueous systems by SEC is referred to as gel filtration chromatography (GFC), while the separation of organic polymers in non-aqueous systems is called gel permeation chromatography (GPC).

Principle

Size exclusion chromatography involves separation of components of a mixture based on their molecular size. The components separate due to their differential exclusion when they pass through stationary phase having different pore sizes in the cross linked polymeric gels or beads. The components elute from the stationary phase with different permeation rates. It involves moderate interaction with the components thus enabling high retention of biomolecular activity.

In SEC, the column filled with gel particles or porous stationary phase is equilibrated with mobile phase. The introduction of sample percolates the components with the mobile phase. The large molecules do not enter into the pores and are excluded through the vacant space not occupied by the particles. The smaller molecules are distributed in the mobile phase and the porous molecular sieves. This results in the fast movement of the larger particles as compared to the smaller particles.

The exclusion and inclusion of particles depends upon the size range chosen during the experiment. As per the parameters selected, all the components having size equal or larger than the critical mass behave identically and are excluded from the column. These components are eluted as ‘excluded volume’ of the column. The particles having size lower than the critical mass are captured by the pores and are eluted as ‘included volume’.

2. Mechanism of size exclusion chromatography

There are two possible mechanisms on the basis of which separation takes place. In SEC, the components are distributed within two liquid phases; 1) liquid present in the porous gel, 2) liquid present outside the gel. The distribution follows Steric exclusion mechanism, small components can enter in smaller and larger pores both, however, the larger components enter only in larger pores. Therefore, the different pore fractions are available to the molecules of different sizes. This means the distribution coefficients of components with different sizes differ in two liquid phases. The total volume (Vt) of the column packed with a gel that swell by the solvent is given by;

Vt = Vg + Vl + Vo

Where Vg is the volume occupied by the solid matrix of the gel

Vi is the volume of the solvent held in the pores or interstices

Vo is the free volume inside the gel particles

The diffusion equilibrium and the retention time of the component is given by

VR = V(int.) + KdV(int.)

Kd = Vi(acc)/V(total)

where Vi(acc) is the accessible pore volume

V(total) is the total pore volume

V(int.) is the interstitial volume

In secondary exclusion mechanism, the small molecules diffuse rapidly into the pores of the gel while larger molecules compete for vacant sites. The larger components move down and find relatively more unoccupied sites downwards. This result in the separation of smaller components collected at the upper end and larger components collected at the lower end.

3. Instrumentation of SEC

The instrument of SEC consists of same components as other chromatographic techniques. The block diagram of the instrumentation is given below.

3.1 Stationary phase

The stationary phase used in SEC is semi-permeable cross linked polymer with porous nature. It has well defined pores and the size of pores is controlled during cross linking. The stationary phases with smaller pore size are used for the purification or desalting of proteins. The medium size is used to separate relatively small proteins and large sized pores are used for biological samples. Usually, dextran (SephadexTM) polyacrylamide and dextranpolyacrylamide (SephacrylTM) are used for SEC. These are available in different pore sizes and are chosen on the basis of the size of the macromolecules to be separated. The main features of a stationary phase include inertness, inexpensiveness, mechanical stability, uniform size and shape and non-reactive towards various components of the sample. Some of the good stationary phases commonly used in various applications are discussed below.

Dextran: This gel is homopolysaccharide of glucose residues and can be prepared by varying the cross-linking to control the pore size. It is available as dry beads under the trade name Sephadex, which swell in the presence of water. Mainly, it is used for the separation of small peptides and globular proteins with small to average molecular mass.

Polyacrylamide:

It is also a gel and can be prepared by cross-linking N,N-methylenebisacrylamide. Its separation tendency is same as dextran and is available in various sizes. It is available as bio-gel P.

Agarose:

It is a gel composed of linear polymer of D-galactose and 3,6-anhydro-1-glalactose. It shows dual nature i.e. dissolved in boiling water and is converted to gel in cold water. Its pore size is larger as compared to the gels discussed before. Therefore, it is used for the separation of globular proteins and long molecules of DNA.

3.2 Mobile phase

The mobile phase is selected on the basis of the nature of components to be separated. Most commonly THF, chloroform, toluene, dimethyl formamide and mixed solvents are used to separate various components. In case of proteins and polysaccharides, aqueous buffers are used as mobile phase.The solvent should be able to dissolve the components of a mixture and must be inert towards various components. The detectable properties of the solvent must be different from the sample components. It should not be corrosive. Before using a mobile phase for the analysis, high purity solvent is passed through a filter paper of size 0.5 micron to remove the dust, insoluble salts etc.

3.3 Preparation of sample

A sample is dissolved in an appropriate solvent to get its dilute solution usually less than 1mg/mL. Sometimes higher concentrations are required for the samples with diverse molecular weight of components. The samples are filtered before loading into the instrument to prevent the clogging.

3.4 Columns

The columns of various lengths and diameters are available commercially. However, small columns are recommended to save the time and solvent. The particle size is selected depending upon the analyte. Generally particles with 5mm size are selected but small sized particles are more sensitive towards contamination. For high molecular weight, large particle size is recommended because small size of particles may damage the large sized analytes. The porosity of the column is also responsible for the separation of the components. Before operation, a column is saturated with the mobile phase and is handled with care.

3.5 Pump

The pumps are used to maintain the constant flow rate during the analysis. A change in the flow rate may cause error in the results. The in-line filters prevent the entry of particles and save the valves and pump deals from damage. These are of two types; syringe pump and reciprocating pump.

3.6 Detector

The detectors used with SEC are either concentration sensitive detectors like refractive index detectors, UV-Vis detectors and evaporative light scattering detectors, or molar mass sensitive detectors like low angle light scattering detectors, multiangle light scattering detectors and viscosity detectors.

4. Applications of SEC

1.  Purification

2.  Desalting

3.  Protein-ligand binding studies

4.  Protein folding studies

5.  Concentration of sample

6.  Copolymerisation studies

7.  Relative molecular mass determination

8.  Separation of sugars, peptides, rubbers and others

Example: Give a block diagram giving steps involved in the separation of polymers by SEC

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