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

6. Resins
Gels
Inorganic
exchanger
6

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

12. 12

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

14. 14

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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