1. Chapter 4-1
Chromatography
Is a technique used to separate and identify the components of a mixture.
Dr Gihan Gawish

2. Chromatography Application in Biomolecules
Before a protein or other biological macromolecule can be rigorously studied from a structural and functional basis, it must be purified.
The problems that can arise during protein purification become clear when one considers that a single protein has to be purified from a mixture of as many 10,000 proteins, each of which are made up of the same constituent amino acids.
Proteins differ in size (how many amino acids), charge (how many positively and negatively charged amino acids), and in sequence and presence of specific binding sites on the proteins.
Any technique that could be used to purify protein must be based on these inherent differences.
Dr Gihan Gawish
Chromatography Application in Biomolecules

3. General Principles of Chromatography
Separation of molecules by distribution between a stationary phase and a mobile phase.
A stationary phase can be solid, gel, or liquid. Also called matrix, resin, or beads.
The mobile phase is the solvent and it is usually a liquid, but may also be a gas. Also called eluting buffer
The compounds to be separated are considered solutes
Dr Gihan Gawish

4. Basis of Chromatography
Differential retardation of materials caused by interactions with the stationary phase:
Dr Gihan Gawish

5. Classification of Chromatography
by mobile phase:
Gas chromatography,
Liquid chromatography or its variations
by stationary phase or shape
Paper chromatography,
Thin-layer chromatography
Column chromatography
Dr Gihan Gawish

6. Classification by the Separation mode
The mechanism that causes the stationary phase to retard the movement of molecules
Sieve mechanism  separation according to size or MW.
(molecular sieve = gel filtration = size exclusion = gel permeation)
Charge interaction  separation based on net charge
Dr Gihan Gawish

7. Classification by the Separation mode
3. Solubility characteristics  separation based on polarity
Hydrophobic chromatography, reverse-phase chromatography, Adsorption or normal-phase chromatography
4. Biological or Specific interaction  capture any molecule that exhibit such property
affinity chromatography, dye-chromatography
Antibody-antigen: (Immuno precipitations and other forms)
Dr Gihan Gawish

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. Examples of Partition Chromatography
With adsorptive effect
Without adsorptive effect
1- Paper.
2- Thin layer
3- Gel
4- Gas liquid
Column
Support: sillica gel, cellulose powder or dextran (washed or suspended of solvent)
Dr Gihan Gawish

15. Adsorptive Chromatography (adsorptive effect)
A stationary phase solid (support; matrix) + liquid (adsorbent)
The mobile phase is the solvent
The relative movement of each molecule is a result of a balance between the driving force and retardation force.
Dr Gihan Gawish

16. Common Materials Used in Adsorption Chromatography in Biochemistry
Substance separated
Material
Small organic molecules, proteins
Alumina
Sterols, amino acids
Silica gel
Peptides, amino acids, carbohydrates
Activated carbon
Proteins, polynucleotide
Calcium phosphate gel
Nucleic acids
Hydroxyapatite
Dr Gihan Gawish