Principle of separationIt 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).

Nature of the gel1- chemically inert2- mechanically stable3- ideal porous structure Wide pore size give low resolution4- uniform particle sizeTypes of gel:

Types of gel:1- SephadexΑ 1-6-polymer of glucose is prepared by

microbial fermentation of sucrose (glucose + fructose)

The resulting glucose provids the required α1-6 glucosan polymer called dextran

The resulting dextran is treated with epichlorohydrin to give several types of crossed linked dextran (sephadex)

Gl-Gl-Gl

O

CH2

CH

CH2

OH

O

Gl-Gl-Gl n

Gl-Gl-Gl-

OH

Gl-Gl-Gl-

OH

+CH2Cl

HOHC

CH2ClSephadex

Sucrose Microbial fermentation

Specific PH

Glucose + fructose

Dertan (a-1-6 glucosan polymer)

Cross linking

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 sephadex1- highly stable gels2- stable at PH 2-123- their particles are free from ions4- insoluble in water and organic solvent5- they swell in water and other hydrophillic

solvent6- they require bactericidal such as Hg acetate

2- Agarose gel Obtained from agar and composed of

alternating units of 1,3 linked β-D-gal and 1,4 linked 3,6-anhydro-α, L-galactose

This was subjected to epichlorohydrin to give sepharose

Characters:1- it dissolves in H2O at 50 c and on

cooling form gel2- insoluble below 40 c3- freezing destroys the gel

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

methylen-bis-acrylamide

NH

O O

NH

O

NHNH

O

NH

O

HN

O

n

Column packing:1- gel is mixed with solvent for 3 hrs to

swell2- pack the column3- sample should be solution 4- Not to allow dry

Application of gel filtration chromatography1- separation of large molecular weight

compound as protein, carbohydrate, peptides, nucleic acids

2- desalting of colloidsSmall size of contaminating salt will allow

them to diffuse inside the gel particlesE.g. Desalting of albumin from 25%

ammonium sulfate3- molecular weight determinationA linear relationship exists between the

logarithm of the molecular weight and the elution volume

Ion exchange chromatographyMix-X- + R+Y- Y- + R+X- anionic

exchangeMix-X+ + R-Y+ Y- + R-X+ cationic

exchangeThe 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

Advantages:1- separation of very pure compound from

extract2- require small amount of solvent3- very useful in microbial fermentation for

antibody production

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

Sulphonic acid (Cation)

Quaternary ammonium group (Anion)

Martix- SO3- H+

Fixed charge counter charge

Martix- CH2-N+-CH3 Cl-

Fixed charge counter charge

CH3

CH3

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.

Martix- COO- H+

Frame work matrix Fixed charge counter charge

Martix- N+R3- H-

Frame work matrix Fixed charge counter charge

Resin-COO-H++ Na+Cl- Resin-COO-Na+ + H+Cl-

Types and preparation of exchange materialA) Ion Exchange Resins1- 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

cation exchangeStrong cation

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

HC CH2HC CH2

HC CH2

Styrene Divinyl benzene

SulfonationSO3H cationic resin

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

HC

CH

H2C

H2C* CH2 C

CH3

COOH

CH2

H2C CH2 C

CH3

COOH

CH2 **

n

Strong basic quaternary amine resin

Weak anion

HC

CH2N+H2CH3Cl-

* CH2

n

3

HC

CH2N+(CH3)3Cl-

CH2

n

2

HC

CH2N+ H(CH3)2Cl-

CH2

n

4

B) ION EXCHANGE GELSDextran (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 baseC) ION EXCHANGE CELLULOSE

Factors affecting the exchange potential1- the valence of the exchanging ion Ca

more than Na2- increase with atomic numberLi less than Na , Ca3-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

Ion exchange techniques1- batch technique2- column technique

1- Batch techniqueThe resin is allowed to contact with the

solution in a vessel and equilibrium is reached by shaking or stirring

2- column techniqueThe 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 application5 g extract (in 20 ml solvent) added onto

the top of a column then 0.5-1ml/min flow rate and collect fractions

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

Applications of IEC1- analytical applications:-water softening , exchange of Ca, Mg, Pb

and Hg by Na2- determination of total salt concentrationRH+ + 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

Applications of IEC in the field of natural products and pharmacy1- separation of antibiotics2- separation of vitamins3- separation of amino acids4- separation and purification of

alkaloids