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P SE E B
RE NT ND Y:
K AM I
.SH IL
1702-13-886-005
GUIDE B Dr.H
D Y:
RIDAY B RA
E

1
W
hat Is Ion E
xchange Chromatography?
P
rinciple
Ion E
xchangers
P
rocedure
F
actors Affecting Ion E
xchange Chromatography
Advantages & Disadvantages
Applications
References
2
Ion-exchange chromatography (or ion
chromatography) is a process that allows the
separation of ions and polar molecules based on their
charge.
Ion exchange chromatography is coined
by SM L ST VE
AL , E NS, B
AUM
ANN.
It can be used for almost any kind
of charged molecule including large
proteins, small nucleotides and
amino acids.

3
Ion-E
xchange chromatography retains analyte
molecules on the column based on coulombic (ionic)
interactions.
T stationary phase surface displays ionic functional
he
groups (R-X) that interact with analyte ions of opposite
charge.
T type of chromatography is further subdivided
his
into:
Cation exchange chromatography
Anion exchange chromatography
T ionic compound consisting of the cationic species
he
M and the anionic species B can be retained by the
+
4
stationary phase.
Cation exchange chromatography retains
positively charged cations because the stationary
phase displays a negatively charged functional
group
Anion exchange chromatography retains anions
using positively charged functional group

5
Resins

Gels

Inorganic
exchanger

6
Resins are amorphous particles of organic materials
Ion exchange resins are used for the separation of small
molecules.
Strongly acidic cation exchanger -sulphonic acid groups
attached to styrene and di vinyl benzene copolymer.
W
eakly acidic cation exchanger-carboxylic acid groups
attached to acrylic and divinyl benzene co-polymer
Strongly basic anion exchanger-quaternary ammonium groups
attached to styrene and divinyl benzene co-polymer
W
eakly basic anion exchanger-poly alkyl amine groups
attached to styrene and divinyl benzene co-polymer
7
Cellulose and dextran ion exchangers , which are
polymers of the sugar glucose , posses larger pore sizes
and lower charge densities.
B
ecause they are much softer than polystyrene
resins , dextran and its relatives are called gels .

8
Column : glass, stainless steel or polymers
L
ength: diameter ratio 20:100 to 100:1
Packing the column : W packing method
et
Application of the sample : After packing, sample is added to
the top of the column, use syringe or pipette
M
obile phase : Acids, alkalis, buffers…
E
lution : Components of mixture separate & move down the
column at different rates depending upon the affinity of the ion
for ion exchanger.
T elutes are collected at different stages
he
Analysis of the elute : spectrophotometric, flame
photometry
9
A sample is introduced, either manually or with
an autosampler, into a sample loop of known volume.
T mobile phase (buffered aqueous solution) carries the
he
sample from the loop onto a column that contains some form
of stationary phase material.
Stationary phase material is a resin or gel matrix consisting
of agarose or cellulose beads with covalently bonded
charged functional groups.
T target analytes (anions or cations) are retained on the
he
stationary phase but can be eluted by increasing the
concentration of a similarly charged species that will displace
the analyte ions from the stationary phase.
10
F example, in cation exchange chromatography,
or
the positively charged analyte could be
displaced by the addition of positively charged
sodium ions.
T analytes of interest must then be detected
he
by some means, typically by conductivity or
UV/
Visible light absorbance.
A chromatography data system (CDS)
usually needed to control an IC.

is
11
12
•
•
•
•

Nature & properties of ion exchange resins
Cross linking & swelling is important
If more cross linking , they are more rigid, but
swelling is less
swells less → separation of ions of different sizes is
difficult
Nature of exchanging ions
valency of ions
Size of ions
P
olarizability
Concentration of solution
13
14
Ion exchange chromatography is used to convert one salt to
other.
E we can prepare tetra propyl ammonium hydroxide from a
g;
tetra propyl salt of some other anion.
Ion exchange is used to prepare de-ionized water
Separation of similar ions
A mixture of sodium, hydrogen and potassium can be separated
using cation exchanger resin.
A mixture of Chloride, bromide, and iodide can be separated
using basic anion exchange resin.
M T OD: M
EH
ixture of chloride, bromide & iodide is passed
through basic anion exchanger using 0.5M sodium nitrate as
eluant. Chloride will first elute. Raise the conc of Sodium Nitrate,
B
romide will elute, raise the conc of Sodium Nitrate further,
15
Removal of interfering radicals: P
hosphate ion is the interfering
with the calcium & barium ions. Phosphate is removed using
sulphonic acid cation exchanger.
+
Calcium & barium ions exchanged with H ions while
phosphate ion pass through the column.
Softening of hard water: H
ardness of water due to cal, mg and other
divalent ions. T water is passed through cation exchanger charged
his
with the sodium ions. Ca & M ions retained in the column while
g
sodium is exchanged.
Complete demineralization of water: Removal of both cations &
anions.
Step A) H
ard water is first passed through an acidic cation
+
exchanger- Ca, M & Na are exchanged by H ions.
g
Step B T
) his water is then passed thro a basic anion
exchanger – Cl, NO2, SO4- are exchanged by OH ions of the
exchanger.
Separation of L
anthanides- L Y, Ce, Rb etc
a,

16
P
rinciples of instrumental analysis , skoog , latest edition,
pno. 641-647
en.wikipedia.org/
wiki/
Ion_chrom
atography
www.separations.us.tosohbioscience.com .../ rinciplesofChrom
/ P
at
ography
https:/www.gelifesciences.com .../
/
/ litdoc11000421_201310032330
16.pdf
www.authorstream
.com P
/ resentation/
shreekanta-1507297-ionexchange
17
18

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ion exchange chromatography

  • 1. P SE E B RE NT ND Y: K AM I .SH IL 1702-13-886-005 GUIDE B Dr.H D Y: RIDAY B RA E 1
  • 2. W hat Is Ion E xchange Chromatography? P rinciple Ion E xchangers P rocedure F actors Affecting Ion E xchange Chromatography Advantages & Disadvantages Applications References 2
  • 3. Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their charge. Ion exchange chromatography is coined by SM L ST VE AL , E NS, B AUM ANN. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. 3
  • 4. Ion-E xchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. T stationary phase surface displays ionic functional he groups (R-X) that interact with analyte ions of opposite charge. T type of chromatography is further subdivided his into: Cation exchange chromatography Anion exchange chromatography T ionic compound consisting of the cationic species he M and the anionic species B can be retained by the + 4 stationary phase.
  • 5. Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group Anion exchange chromatography retains anions using positively charged functional group 5
  • 7. Resins are amorphous particles of organic materials Ion exchange resins are used for the separation of small molecules. Strongly acidic cation exchanger -sulphonic acid groups attached to styrene and di vinyl benzene copolymer. W eakly acidic cation exchanger-carboxylic acid groups attached to acrylic and divinyl benzene co-polymer Strongly basic anion exchanger-quaternary ammonium groups attached to styrene and divinyl benzene co-polymer W eakly basic anion exchanger-poly alkyl amine groups attached to styrene and divinyl benzene co-polymer 7
  • 8. Cellulose and dextran ion exchangers , which are polymers of the sugar glucose , posses larger pore sizes and lower charge densities. B ecause they are much softer than polystyrene resins , dextran and its relatives are called gels . 8
  • 9. Column : glass, stainless steel or polymers L ength: diameter ratio 20:100 to 100:1 Packing the column : W packing method et Application of the sample : After packing, sample is added to the top of the column, use syringe or pipette M obile phase : Acids, alkalis, buffers… E lution : Components of mixture separate & move down the column at different rates depending upon the affinity of the ion for ion exchanger. T elutes are collected at different stages he Analysis of the elute : spectrophotometric, flame photometry 9
  • 10. A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. T mobile phase (buffered aqueous solution) carries the he sample from the loop onto a column that contains some form of stationary phase material. Stationary phase material is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. T target analytes (anions or cations) are retained on the he stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. 10
  • 11. F example, in cation exchange chromatography, or the positively charged analyte could be displaced by the addition of positively charged sodium ions. T analytes of interest must then be detected he by some means, typically by conductivity or UV/ Visible light absorbance. A chromatography data system (CDS) usually needed to control an IC. is 11
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  • 13. • • • • Nature & properties of ion exchange resins Cross linking & swelling is important If more cross linking , they are more rigid, but swelling is less swells less → separation of ions of different sizes is difficult Nature of exchanging ions valency of ions Size of ions P olarizability Concentration of solution 13
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  • 15. Ion exchange chromatography is used to convert one salt to other. E we can prepare tetra propyl ammonium hydroxide from a g; tetra propyl salt of some other anion. Ion exchange is used to prepare de-ionized water Separation of similar ions A mixture of sodium, hydrogen and potassium can be separated using cation exchanger resin. A mixture of Chloride, bromide, and iodide can be separated using basic anion exchange resin. M T OD: M EH ixture of chloride, bromide & iodide is passed through basic anion exchanger using 0.5M sodium nitrate as eluant. Chloride will first elute. Raise the conc of Sodium Nitrate, B romide will elute, raise the conc of Sodium Nitrate further, 15
  • 16. Removal of interfering radicals: P hosphate ion is the interfering with the calcium & barium ions. Phosphate is removed using sulphonic acid cation exchanger. + Calcium & barium ions exchanged with H ions while phosphate ion pass through the column. Softening of hard water: H ardness of water due to cal, mg and other divalent ions. T water is passed through cation exchanger charged his with the sodium ions. Ca & M ions retained in the column while g sodium is exchanged. Complete demineralization of water: Removal of both cations & anions. Step A) H ard water is first passed through an acidic cation + exchanger- Ca, M & Na are exchanged by H ions. g Step B T ) his water is then passed thro a basic anion exchanger – Cl, NO2, SO4- are exchanged by OH ions of the exchanger. Separation of L anthanides- L Y, Ce, Rb etc a, 16
  • 17. P rinciples of instrumental analysis , skoog , latest edition, pno. 641-647 en.wikipedia.org/ wiki/ Ion_chrom atography www.separations.us.tosohbioscience.com .../ rinciplesofChrom / P at ography https:/www.gelifesciences.com .../ / / litdoc11000421_201310032330 16.pdf www.authorstream .com P / resentation/ shreekanta-1507297-ionexchange 17
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