Transcript 474 PHG 6

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).
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:
Types of gel:
1- Sephadex
 Α 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)
Sucrose
Microbial fermentation
Glucose + fructose
Specific PH
Dertan (a-1-6 glucosan polymer)
Cross linking
Gl-Gl-Gl
Gl-Gl-GlOH
O
+
CH2Cl
CH2
HOHC
H
Gl-Gl-GlOH
CH2Cl
C
Sephadex
OH
CH2
O
Gl-Gl-Gl
n
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
 2- Agarose gel
 Obtained from agar and composed of alternating
units of 1,3 linked β-D-gal and 1,4 linked 3,6anhydro-α, 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
3- Acrylamide gels 9synthetic gel)
 It is not dextran polymer
 It is polymerized acrylamide or methylen-bisacrylamide
O
O
O
NH
NH
NH
H
N
NH
NH
O
O
O
n
 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
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
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
 Advantages:
1- separation of very pure compound from extract
2- require small amount of solvent
3- 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)-ClCation 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)
Martix- SO3-
Fixed charge
H+
counter charge
 Quaternary ammonium group (Anion)
CH3
Martix- CH2-N+-CH3
Cl-
CH3
Fixed charge
counter charge
 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 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
 cation exchange
 Strong cation
HC
CH2
HC
CH2
Sulfonation
HC
Styrene
SO3H cationic resin
CH2
Divinyl benzene
 Weak cation exchange can be prepared by
copolymerization of methacrylic acid with divinyl
benzene
 Weak cation resin
CH3
*
H2C
H
C
CH2
C
CH2
COOH
CH3
*
 2- anion exchange resins:
H2C
C
H
H2
C
CH2
C
CH2
*
COOH
n
 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
 Strong basic quaternary amine resin
H
CH
C
2
2
CH2N+(CH3)3Cl-
n
 Weak anion
*
H
C
CH2
3
CH2N+H2CH3Cl-
H
C
n
4
CH2
CH2N+ H(CH3)2Cl-
n
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
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
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
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
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 #1
Ran.OH ========== R(-ve)an. + OH-ve Equation #2
 Where Rcat. & Ran. are cation & anion exchangers, respectively
In 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 decreased
In 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 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
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