Chapter 4-3 Chromatography Gel Chromatography Dr Gihan Gawish

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Practical Considerations 1 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