1. Chapter 4-3
Chromatography
Gel Chromatography
Dr Gihan Gawish

2. Gel Filtration Chromatography
also called "Molecular Sieve“ or "Size Exclusion“:
Stationary phase (column matrix) "beads" of hydrated, porous polymer.
Mobile phase: buffer or solvent.
Dr Gihan Gawish

3. Gel Filtration Chromatography
Concept of separation: molecules "filter" through the porous beads:
Large molecules can’t get through any pores in the beads and move more rapidly through the column, emerging (eluting) sooner.
Smaller molecules may enter some pores in the beads, and thus, have more space to "explore"  elute later.
Ions and small molecules can enter through all pores of the beads  elute last
Dr Gihan Gawish

4. Gel Filtration Chromatography
The porous beads represent a restricted space or volume
Molecules may diffuse to this space if permitted by pore size
Dr Gihan Gawish

5. Gel Filtration Chromatography
For every column, three volumes should be distinguished:
1. The void volume, Vo: which is the volume external to the beads
This space is accessible to all molecules entering the gel
Can be determined by the elution of HMW molecules
2. The total volume, Vt:
can be calculated from the dimension of the column
3. The elution volume, Ve, of molecules
All molecules must eluted between the void volume and total volume of the column
Dr Gihan Gawish

6. Gel Filtration Chromatography- Partition Coefficient
In classical and more rigorous science, elution position of any molecules should be reported as partition coefficient (Kav) rather than volume.
Dr Gihan Gawish

7. Gel Filtration Chromatography
Kav represents the fraction of bead-restricted volume into which a solute could partition
Kav ranges between 0 and 1 for all molecules
Kav allows comparison between columns of different dimensions and/or materials
Dr Gihan Gawish

8. Gel Filtration Resins Resins suitable for proteins and nucleic acids:
Dextran (e.g. Sephadex)
Inert, hydrophilic, not mechanically rigid, usually cross linked
Polyacrylamides (e.g. Biogel P)
strong, large selection of more uniform-pore sizes
Agarose (e.g. Sepharose Biogel A)
Higher porosity, suitable for high molecular weight proteins
Composite gels (e.g. Sephacryl, Ultragel AcA)
Quite strong mechanically, polyacrylamide with either dextran or agarose
Dr Gihan Gawish

9. Resins not suitable for protein products
Synthetic polystyrene, or porous Glass beads
Fine beads, uniform-size pores, highly rigid and very hydrophobic
Suitable for fractionation of ions and very small molecules
Used also for separation of organic-soluble polymers
Dr Gihan Gawish

10. Properties of Gel Filtration Resins
Some resins come in variety of shape (Granules, beads, fibers)
And variety of sizes (Course, fine, and superfine)
All consist of semi-permeable, porous gels of cross linked polymers with a range of pore sizes.
The degree of cross linking is controlled to yield a series of gels having different pore sizes
Dr Gihan Gawish

11. Properties of Gel Filtration Resins
All gels have their own fractionation range:
Upper and lower molecular weight range
Only molecules within that molecular weight range can be separated.
The ideal matrix should be: strong, stable, hydrophilic with proper fractionation range.
The matrix should not interact with solvent or solutes
Dr Gihan Gawish

12. Preparation of column for gel filtration
Equilibrate the resin.
Some resin come in a powder form. These must be hydrated first.
The resin must be equilibrated with the desired buffer
Pack the column
Make a slur of gel plus buffer and pour it into column
Wash the resin
After packing, pass several column volumes of the buffer through the column.
Equilibration is the first step for good separation.
Dr Gihan Gawish

13. Preparation of column for gel filtration
The column is now ready for experiment
Second step: loading the sample onto the column (sample must enter the resin).
Third step: eluting the sample
Fractions are collected as the sample elutes from the column.
Final step: Regeneration of column by washing with appropriate solvent
Dr Gihan Gawish

14. Advantages of Gel Filtration
Suitable environment for all biomolecules.
Separations can be performed under various conditions:
In the presence or the absence of essential ions or co-factors
In the presence of denaturants e.g. detergents, urea, guanidine hydrochloride
At high or low ionic strength
At any temperature according to the requirements of the experiment.
Dr Gihan Gawish

15. Applications of Gel Filtration
The most common application of gel filtration in biochemistry are:
Molecular weight determination
Fractionation of macromolecules
Desalting
Equilibrium binding, buffer exchange, or dialysis
Dr Gihan Gawish

16. 1.Molecular Weight Determination
Principle: For spherical proteins, its elution volume, Ve is proportional to log of molecular weight
Procedure:
Calibrate the column with a series of proteins with known molecular weights
Determine elution volumes of each protein including the unknown
Dr Gihan Gawish

17. 1.Molecular Weight Determination
Construct a calibration curve relating (known) molecular weight to (measured) elution volume specifically for that column.
Use this calibration curve to estimate the molecular weight of the unknown protein, based on its elution volume.
Dr Gihan Gawish

18. 2. Fractionation of macromolecules
Biological macromolecules are extracted as a mixture of several components
When scientists study a macromolecule (protein, antibody, hormone, enzyme, or DNA) they need to separate it from the mixture
These macromolecules are of different sizes and shapes, and thus, can be separated on gel filtration column.
Dr Gihan Gawish

19. 3. Desalting
Biological samples may contain unwanted substances such as:
salts,
buffer components
small ligands
cofactors
precipitating reagents
Gel filtration offers simple, rapid, and inexpensive method for removal of these substances
Dr Gihan Gawish

20. 3. Desalting
Since the solutes and contaminants vary greatly in molecular weight  Use Gel with low exclusion limit
Macromolecules will elute with little dilution in the void volume, while salts will elute near Vt of the column.
Short column and high-flow rate can be used
Such a strategy can be used to separate any two molecules differing greatly in molecular weight
Dr Gihan Gawish

21. Specialized Examples of GC in Purification
1- It is necessary to separate the product from the reactants:
For example;
It is used in preparing fluorescent antibodies by reacting antibody with fluorescein isothyocyanate.
The conjugated protein must be separated from unreacted dye.
This can be done with sephadex
Dr Gihan Gawish

22. Specialized Examples of GC in Purification
2. In the assay of enzymes or the determination of cofactor requirements:
The enzyme preparation sometimes contains inhibitors of small molecular size or the cofactor themselves.
Such small molecules are easily removed with the dextran or polyacrylamide gels
Dr Gihan Gawish

23. Specialized Examples of GC in Purification
3. In the assay of small molecules in mixture contaminants of large molecular size:
The small pore dextran are useful in such cases.
Also, proteins must often be free of nucleic acids by using an agarose gel.
Dr Gihan Gawish

24. Specialized Examples of GC in Purification
4. For most physical analysis of nucleic acids
The preparation of DNA from crude cell extracts for electron microscopic analysis must be free of protein.
Dr Gihan Gawish

25. Specialized Examples of GC in Purification
5. Protein Purifications :
To purify a protein from a cell extract, it is usually necessary to use a sequence of separation procedures based on such parameters;
Solubility in certain solutions
Molecular weight
Charge
Dr Gihan Gawish

26. GC is also valuable an analytical tool
In studying RNA metabolism, various fractions of RNA are usually distinguished by zone centrifugation or even better by ployacrylamide and agarose.
Plasma protein fraction must often be determined quantitatively in the diagnosis of certain human diseases .this can be done directly with dextran gels
Dr Gihan Gawish

27. Practical Considerations1
Gels of various pore size are available  choose the one with the best fractionation range for your sample 2
Gel filtration usually performed as the last purification step 3
Good separation usually require long columns and slow flow rate 4
Many other factors affect resolution or good separation of components e.g. temperature, pH, viscosity of solvent, and volume of the applied sample. 5
The shape of the macromolecule (not only its size) also affect its elution position 6
Gel filtration is NOT recommended for separating proteins with only a small difference in molecular weight.
Dr Gihan Gawish