Size-exclusion Chromatography prof. aza Department of Pharmacy, Andalas University STIFI Perintis, Padang STIFAR, Pekan Baru STIFI Bhakti Pertiwi, Palembang.

Size-exclusion Chromatography

prof. azaDepartment of Pharmacy,Andalas UniversitySTIFI Perintis, PadangSTIFAR, Pekan BaruSTIFI Bhakti Pertiwi, Palembang

Principle• Size exclusion Chromatography is basically different

from all other chromatographic methods in that a sample molecule size classification process rather than any interaction phenomena forms the basis of separation.

• The column then appears to be filled with massive, impenetrable beads and, as no attractive forces are apparent, i.e there is no interaction between beads and sample molecules.

• Interaction, such as a adsorption, ion exchange, and partitioning, should be absent in the ideal steric-exclusion system.

• The technique is known by many names (gel permeation, gel filtration, molecular sieving), and is applicable to a wide range of materials covering both high and low molecular weights. It has been most popularly used for fractionation of polymers, but is being used more and more for small molecule separations.

Principle, continued• r molecule > r pore, no pore volume

accessible First peak• r molecule < r pore, available pore

volume is a function of molecular radius, separation according to molecular size.

• r molecule <<< r pore, all pore volume available Final peak

Principle, continued• The elution volume or time is a function of

molecular size alone.• As the separation is completed after only a

small amount of mobile phase has passed though, the peak capacity is limited.

• Two types of molecules can be separated, provided that there is at least a 10 % difference in their molar masses.

• The van Deemter curve does not follow the normal pattern of passing though a minimum and having a step gradient when flow rates are low. The low rate of macromolecule diffusion mean that the B term is negligible and separation performance increases the more slowly the chromatography is carried out.

• There are no interaction equilibria, hence relatively large samples can be separated.

Pictorial Representation of Exclusion Chromatography, Large solutes cannot penetrate into the pores of the packing, hence they will move with solvent. Small molecules can diffuse into the stagnant pools and are retained.

The volume of an exclusion column

• Void volume, Vo that volume occupied by the flowing mobile phase between the particles.

• That volume occupied by the solid portion of the packing.

• The Pore Volume (Vp) – that volume occupied by stagnant mobile phase.

The distribution coefficient

K = Va/VpVA = K.Vp

VR = Vo + VA

VR = Vo + K.Vp

K = a distribution coefficient of a particular solute as the ratio of the pore volume accessible to the solute (VA) to the total pore volume (Vp).The range of K is 0 to 1, since if a solute is totally excluded K = 0, and if a solute totally permeates the pores K = 1

Typical Calibration and Corresponding Chromatogram for Exclusion Chromatography

Size-exclusion chomatography of styrene oligomers (Heitz and Fresenius, 1975. Condition column, 1000 cm x 2 mm i.d. (coiled); stationary phase, Merckogel 6000, mobile phase, dimethylformamide.

Calibration graph for a size-exclusion column.

Pore width and separating range of a typical commercial column set.

Separation of a mixture

Solvents for Exclusion Chromatography

Soft Gels (Gel filtration)• These materials are typified by cross-linked

dextrans, the most popular being Sephadex®. Soft gel pickings swell the solvent (generally water) is necessary to soak them in the solvent at least overnight so that no changes in volume will occur during use.

• Soft gels are particularly useful for separating water-soluble polymers, they have found wide use in the characterization of proteins and enzymes.

• They have not found wide us in HPLC since they are very fragile and cannot withstand more than about 150 psi.

Soft Gels for Exclusion Chromatography Columns

Semirigid gels• They are derived from polystyrene which has

been cross-linked with various percentages of divinylbenzene to give semirigid structure. They are commonly used in organic solvents, such as tetrahydrofuran, acetone etc, since aqueous solutions do not wet the surface of polystyrene beads.

• They are populer for separating complex polymer samples, such us rubbers and plastics.

• The main disadvantage of these large particle packing (dp 37-75 µm) is that mass transfer slow. To achieve adequate separation, slow flow rates must be used and this leads to long analysis times.

Semirigid Large-Particle Packings

Rigid Packing• These packing are invariably from glass or

silica. They are available in a wide range of pore sizes and in large and small particle diameters.

• Their strength removes limits on flow rates since higher pressures can be used.

• Both aqueous and organic solvent may be used and changeover is rapid.

• A word of caution- alkaline solutions with pH >7.5 should be avoided.

• The major disadvantages with these packing is frequently bothersome adsorption effects. The beads can be treated with Carbowax 400 or polyethylenglycol. In addition the beads may be treated with various silylating reagents to deactivate active site.

Rigid Packing for Exclusion Chromatography Columns

Exclusion Separation of Normal Paraffins on TSK Gel, mobile phase tetrahydrofuran.

Analysis of Fruit Juice Extracts by Combined Exclusion and Partition chromatographySteric exclusion of Valencia orange, packing Bio-Beads SX-2, mobile phase chloroform, flow rate 1.5 ml/min, UV 254 nm.

Partition Chromatography, Packing ODS, mobile phase, linear gradient from 5 % methanol in water to 100 % methanol, 1.5 ml/min

giving chromatogram as below

Size-Exclusion ChromatographyAlso called GEL PERMEATION CHROMATOGRAPHY

Separation principle

Also used for: De-salting or buffer exchange of, eg, protein solutions Determination of Molecular mass of biological macromolecules - calibrate column with similar molecules of known molecular mass

Quaternary structure usually remains intactTypes of matrix for forming stationary phase: Cross-linked dextran polymer (Sephadex G-10 to G-200) Cross-linked polyacrylamide (Biogel P-2 to P-300) Agarose - the largest pore size