ion-exchange-chromatography-ppt_240129_121141.pdf

Introduction

1. Dr.D.Y.PatilACS College PimpriPune-
18
Chemistry Department
Mrs.TarannumS Attar

3. INTRODUCTION:-
“Ionexchangechromatographymaybedefinedasthereversibleexchangeof
ionsinthesolutionwithionselectrostaticallyboundtosomesortofinsoluble
matrixorastationaryphase.”
Thistechniqueisextremelyusefulintheseparationofchargecompoundslike
proteinsdifferingbyonlyonechargedaminoacid.
InIonexchangechromatographytechniqueonecanchoosewhethertobind
thesubstanceofinterestandallowthecontaminationtopassthroughthe
columnandviceversa.
Thistechniquehasbeendevelopingsince19thcenturywhichwasfirstlyused
forpurifyingthedrinkingwater.
Ionexchangechromatographyisadistinctprincipleofchromatography
performedinthecolumn

4. TYPES LABORATORY COMMERCIAL
AUTOMATED:-

5. PRINCIPLE:-
Ionexchangechromatographyreliesontheattractionbetweenoppositely
chargedstationaryphase,knownasanionexchanger,andanalyte.
Theionexchangerconsistsofaninertsupportmediumcoupledcovalently
topositive(anionexchanger)ornegative(cationexchanger)functional
groups.
Tothesecovalentlyboundfunctionalgroupstheoppositelychargedionsare
bounded(mobilecounterion),whichwillbeexchangedwithlikecharge
ionsinthesamplehavingchargemagnitudemorethantheionsboundedto
thematrix.
Thusifanionexchangechromatographyisperformed,negativelycharged
samplecomponentswillinteractmorewiththestationaryphaseandwillbe
exchangedforlikechargedionsalreadyboundedtothematrix

6. WORKING:-
→E
-
Y
+
X
+
E
-
X
+
Y
+
+
Desired bounded cation (X+ ) can now be eluted by either of the twoways;
1.By adding a component M+ having magnitude of charge more than thatof
X+ so that M+ will replace X+ and X+ will be elutingout.
2.By changing pH of the solvent (mobile phase so that X+ have no charge andis
then unbounded from the matrix and can be elutedout.
Consider a column having E
-
Y
+
cation exchanger in which E
-
isnegative
charged exchanger and Y
+
is the mobile counterion.
Let X
+
be the cation in the sample having charge greater than Y
+
.
The X
+
ion can exchange sites with the counter ion Y
+
with satisfying the
followingrelationship;
The remainingneutral
and negativelycharged
particles

8. WORKINGPRINCIPLE:-
The 4 basic steps of ion exchangechromatography:-
1.Equilibration
2.Sample application andwash
3.Elution
4.Regeneration

13. REQUIREMENT:-
1.Column:-
glass, stainless steel orpolymers
2.Packing the column:-
Wet packing method: A slurry is prepared of the eluent with the stationary phase
powder and then carefully poured into the column. Care must be taken toavoid
air bubbles.
3.Application of thesample:-
After packing, sample is added to the top of the stationary phase, use syringeor
pipette.
This layer is usually topped with a small layer of sand or with cotton or glass
wool to protect the shape of the organic layer from the velocity of newlyadded
eluent.
Continue...

14. 4.Mobile phase:-
Acids, alkalis,buffers.
5.Stationaryphase:-
The ionic compound consisting of the cationic species (M+) and theanionic
species(B-)
6.Elution:-
Components of mixture separate & move down the column atdifferent
rates depending upon the affinity of the ion for ionexchanger.
The eluates are collected at differentstages
7. Analysis of theeluate:-
Spectrophotometric, flame photometry polarographic,conductometri.

16. A . Ion ExchangeResin:
The swelling factor and cross linking is important for the effective separation. The cross
linking should be controlled as its affects the exchanger’s capacity. Swelling helps in
proper exposure of charged functional groups for exchange ofions.
swells less → separation of ions of different sizes isdifficult.
B. Nature of exchangingions:
1.valency ofions.
2.Size of ions
3.Polarizability
4.Concentration of solution.
5.Concentration & charge ofions
C . pH of the mobilephase
D . Ionicstrength
E . Mobile phasemodifiers
F .Temperature
G. Buffer: The pH of the buffer should impart the same charge to the sample ions aspresent
in the Column. Anionic Exchange Chromatography should be carried out with cationic
buffers and vice versa because buffer ion will indulge in ion exchange, which will be of
nouse.

17. IONEXCHANGERS:-
There are three classes of ionexchangers
1.Resins:-Ion exchange resins are used for the separation of smallmolecules
2.Gels:-Ionexchangegelsareusedfortheseparationoflargemoleculeslikeproteins
,nucleicacids
3.Inorganicexchangers:-Separationsinvolvingharshchemicalconditions(high
temperature,highradiationlevels,stronglybasicsolutionsorpowerfuloxidizing
agents)employinorganicionexchangers

18. CLASSIFICATION OF ION EXCHANGE RESINS:-
According to the chemical nature they classifiedas-
Strongly acidic cationexchanger:-
sulphonic acid groups attached to styrene and divinyl-benzenecopolymer.
Weakly acidic cationexchanger:-
carboxylic acid groups attached to acrylic and divinyl-benzene co-polymer.
Strongly basic anionexchanger:-
quaternary ammonium groups attached to styrene and divinyl-benzeneco-polymer.
Weakly basic anionexchanger:-
poly alkyl amine groups attached to styrene and divinyl benzene co-polymer.

19. ACCORDING TO THESOURCE:-
Natural resins:
Cation -Zeolytes,Clay
Anion -Dolomite
Synthetic resins: Inorganic & Organicresins
Organic resins:-are polymeric resinmatrix.

20. ION EXCHANGERESIN:-
Resins are amorphous particles of organicmaterials
Polystyrene resins for ion exchange are made by co-polymerizationof
styrene anddivinyl-benzene.
Divinyl-benzene content is varied from 1 to 16 percent to increasethe
extent of crosslinking.
Benzene groups are modified to produce cation exchange resin andanion
exchangeresin
Resin made from both of these materials differ in their flow properties,ion
accessibility, and chemical and mechanicalstability.
Selection of one or the other type of resin is done on the basisof
compounds beingseparated

21. POLYSTYRENE RESINS:-
Polystyrene resins are prepared by polymerisation reaction of styreneand
divinyl-benzene.
Higher concentration of divinyl-benzene produces higher crosslinkages.
Polystyrene resin are very useful for separating small molecular weight
compounds, however, unsatisfactory for the separation ofmacromolecules
styrene divinylbenzene

22. ION EXCHANGEGELS:-
Cellulose and dextran ion exchangers , which are polymers of thesugar
glucose , posses larger pore sizes and lower chargedensities.
Because they are much softer than polystyrene resins , dextran andits
relatives are called gels.

23. CELLULOSE:-
•Cellulose is a high molecular weight compound which can bereadily
obtained in a high purestate.
•Cellulose has much greater permeability tomacromolecules

24. EXCHANGE MEDIUM
•The choice of ion exchangers depends upon the stability, molecularweight,
and ionic strength of the samplecomponents.
•The volume of exchanger used for separation is usually 2.5 fold greaterthan
to exchange with the ion in thesample.
•The ion exchanger are packed in column having suitablebuffer.
•The ion exchangers are of twotypes;
CationExchangers
AnionExchangers

27. Anion and cationExchangers

28. Costeffective
Re-usable
Easilycollectable
Low maintenancecost
Efficienttechnique
Quickseparation
ADVANTAGES

29. Softening of hardwater
Demineralization ofwater
To analyze base composition of nucleicacid
To concentrate the metal ions in thesample
To measure the additives in food and drugsample
To separate protein mixtures
For extraction of enzymes fromtissues.
Purification of solutions free from ionicimpurities.
Separation of inorganicions.
Separation of sugars, amino acids andproteins.
Ion exchange column inHPLC.
APPLICATIONS

30. THANK YOU