Gel chromatography Gel permeation Molecular sieving Size exclusion steric exclusion

1. Gel chromatographyGel permeationMolecular sievingSize exclusion= steric exclusion

2. Principle of separation It is a kind of chromatography technique based on the difference of molecular weight and is one of the effective and mild methods extensively used to isolate and analyze the biomacromocular substances. The stationary phase consists of beads containing pores that span a relatively narrow size range. when the gel is packed into a column and percolated with a solvent, it permits the large molecular weight compounds to pass rapidly without penetration of the pores Smaller molecules spend more time inside the beads than larger molecules and therefore elute later (after a larger volume of mobile phase has passed through the column).

5. Nature of the gel 1- chemically inert 2- mechanically stable 3- ideal porous structure Wide pore size give low resolution 4- uniform particle size Types of gel:

6. Types of gel: 1- Sephadex ? 1-6-polymer of glucose is prepared by microbial fermentation of sucrose (glucose + fructose) The resulting glucose provids the required a1-6 glucosan polymer called dextran The resulting dextran is treated with epichlorohydrin to give several types of crossed linked dextran (sephadex)

8. Sephadex is obtained in different degrees depending on the pore size High percentage of epichlorohydrin give high degree of cross linking (small pore size) Lower percentage produce sephadex with large pore size Characters of sephadex 1- highly stable gels 2- stable at PH 2-12 3- their particles are free from ions 4- insoluble in water and organic solvent 5- they swell in water and other hydrophillic solvent 6- they require bactericidal such as Hg acetate

9. 2- Agarose gel Obtained from agar and composed of alternating units of 1,3 linked �-D-gal and 1,4 linked 3,6-anhydro-a, L-galactose This was subjected to epichlorohydrin to give sepharose Characters: 1- it dissolves in H2O at 50 c and on cooling form gel 2- insoluble below 40 c 3- freezing destroys the gel

10. 3- Acrylamide gels 9synthetic gel) It is not dextran polymer It is polymerized acrylamide or methylen-bis-acrylamide

11. Column packing: 1- gel is mixed with solvent for 3 hrs to swell 2- pack the column 3- sample should be solution 4- Not to allow dry

12. Application of gel filtration chromatography 1- separation of large molecular weight compound as protein, carbohydrate, peptides, nucleic acids 2- desalting of colloids Small size of contaminating salt will allow them to diffuse inside the gel particles E.g. Desalting of albumin from 25% ammonium sulfate 3- molecular weight determination A linear relationship exists between the logarithm of the molecular weight and the elution volume

13. Ion exchange chromatography Mix-X- + R+Y- Y- + R+X- anionic exchange Mix-X+ + R-Y+ Y- + R-X+ cationic exchange The polymer matrix carries functional groups that carries a positive or negative charge (fixed charge), which is balanced by ions of opposite charge (counter ions) these counter ion is lossely attached to the matrix and can change places with ions similar charge in solution

14. Advantages: 1- separation of very pure compound from extract 2- require small amount of solvent 3- very useful in microbial fermentation for antibody production

15. Anaion exchange as: strong anion as quaternary ammonium form Matrix- (NR3)+ -Cl- - weak anion as Matrix-NH2(CH3)-Cl- Cation exchange as: sulfonic acid Matrix-(SO3)� H+ (strong). And Matrix-COO- H+ (weak) The stronger the charge on the sample, the stronger it will be attached to the ionic surface and thus the longer it will take to elute. The mobile phase is an aqueous buffer, where the PH is adjusted to control elution time

16. Sulphonic acid (Cation) Quaternary ammonium group (Anion)

17. Substance form ion in aqueous solution (carry charge) when they are brought into contact with the head of ion exchange interaction occurs . The ion exchange expel or repels ions carrying the same charge as the fixed charge and will bind ion of the opposite charge. The beads of the ion exchangers represent the stationary phase and the solution following through is the mobile phase.

19. Types and preparation of exchange material A) Ion Exchange Resins 1- Cross linked cation exchange resins: Condensation of polyhydric phenols with formaldehyde to give uni-functional resins charged by the exchangeable H+ of the phenolic OH Now it is prepared by copolymerization between styrene and divinyl benzene and then sulfonic acid groups were introduced by sulfonation

20. cation exchange Strong cation Weak cation exchange can be prepared by copolymerization of methacrylic acid with divinyl benzene

21. Weak cation resin 2- anion exchange resins: They are prepared in similar way to that of anion using cross linked polystyrene which is chloromethylated which is then treated with a secondary amine to give weakly basic tertiary amine resin or with primary amine to give weak anion exchanger

22. Strong basic quaternary amine resin Weak anion

23. B) ION EXCHANGE GELS Dextran (sephadex, cross linked dextran): inorganic unit introduced on the cross-linked dextran (sepahdex) by estrification of the hydroxyl groups by reagent contains terminal acid or base E.g. Sulphoxyl SO3H strong H+ Carbomethoxy CH2-CCO weak H+ Diethyl amino ethyl (DEAE) weak base C) ION EXCHANGE CELLULOSE

24. Factors affecting the exchange potential 1- the valence of the exchanging ion Ca more than Na 2- increase with atomic number Li less than Na , Ca 3-the exchange of H or OH depends on the strength of the acid or the base formed with the functional group of the resin The weaker the acid or base formed the greater the exchange potential

25. Ion exchange techniques 1- batch technique 2- column technique 1- Batch technique The resin is allowed to contact with the solution in a vessel and equilibrium is reached by shaking or stirring

26. 2- column technique The most common types 1- washing the resin is washed with mobile phase for the purification of degradation product from industry 2- swelling leave resin for 10-20 min in mobile phase to facilitate the softening of resin and facilitate penetration 3- sample application 5 g extract (in 20 ml solvent) added onto the top of a column then 0.5-1ml/min flow rate and collect fractions

27. The effect of the PH on the capacity of ion exchangers The capacity of the ion-exchanger resins is determined by the concentration of measurable ionic groups within the structure, The capacity of ion-exchangers is a function of PH Rcat.H ==========R(-ve)cat. + H+ve ��� Equation #1Ran.OH ========== R(-ve)an. + OH-ve��� Equation #2 Where Rcat. & Ran. are cation & anion exchangers, respectivelyIn equation #1 it is clear that the ionization of a cation exchange resin (Rcat.H) to produce the resin ion (R-ve cat.) & H+ is influenced by PH. Thus at low PH (high concentration of hydrogen ions), the ionization of the acidic resin is inhibited & the exchange capacity is decreasedIn equation #2, the ionization of the basic ion exchanger is inhibited at high PH, thereby reducing the exchange capacity of this resin So the PH will directly affect the ionization state of the resins either leading to increasing the resolution or decreasing it depending also on the functional groups & the chemical nature of the resin itself

28. Applications of IEC 1- analytical applications: -water softening , exchange of Ca, Mg, Pb and Hg by Na 2- determination of total salt concentration RH+ + salt (NaCl, unknown).RNa + HCl (titrated with N/2 NaOH) 3- separation of interfering ions or electrolyte 4- Ion exclusion (Donnan exclusion) separation of electrolytes from non electrolytes

29. Applications of IEC in the field of natural products and pharmacy 1- separation of antibiotics 2- separation of vitamins 3- separation of amino acids 4- separation and purification of alkaloids