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Ion Exchange Chromatography
Ion Exchange Chromatography
Ion Exchange Chromatography
INTRODUCTION
lon exchange chromatography coined by Small, Stevens and R
fast, economical and versatile technique which can be used in thes
1ons, amino acids, peptides and nucleic acids. Ion ex«
xchange mayseparation
be of
a s the reversible interchange of ions of like sign between a defined
solution anddefined
a
in contact with it. The solid (ion solid
exchanger) mustsol
highly insoluble body
molecular structure so that ions
permeable an solvent
molecul have
an open
move freely in and out. lon exchange chromatography is carried
column packings that have charge-bearing functional groups
polymer matrix.
attached to a
ION EXCHANGERS
The ion exchanger is complex and
1s in fact polymeric. Ion exchange
resins are high molecular weight, cross-linked
polyelectrolytes that consist of
three dimensional polymeric hydrocarbon network to which are
bonded a large
number of electrically charged groups such as SOg or
aN(CH) First
synthetic resin, phenol formaldehyde condensate resin was applied by E.A.
Adams and E.L. Holmes (1935) although zeolites had been in use since
1900.
Ethylenie polymers led to a major switch from condensation polymers to
addition polymers. Ion exchange materials include ionic dyes, green sands,
zeolites, synthetic aluminates, phosphates, silicates, humic coals, glauconites
and ferrocyanides
which show selective sorptive properties.
Fundamental Requirements of a Resin.
A useful resin must be sufficiently hydrophilic to permit diffusion of
1ons through the structure at a finite and usable rate.
It must be
sufficiently cross iinked to have only a negligible solubility.
It must be chemically stable and must
contain sufficient number
accessible ion exchange groups.
Reversibility and no permanent change in structure.
When swollen it must be denser than water.
Properties of lon Exchangers.
lon exchangers complex, porous and polymeric in nature: with
are
They contain active or counter ions that will exchange reversiDiy ith
other ions in a liquid
phase.
Granular resins swell in water to
give a gel structure. Swelln
idhly
directly proportional to the percentage of
cross-linked resins are brittle, harder, cross-linklng
Divinyl benzene prevents resin from complex
and porou
swelling indefinitely
Types of lon Exchangers.
having
1. Synthetie Inorganic lon sieves
Exchangers.
regular structure and pore size act as ion Molecular
cular
sangersc
g e r sc a n
he prepared from zirconium and thorium oxides. Zirconyl phosphate and ammonium
12-molybdophosphate also possess useful ion exchange capabilities.
2. Natural Organic Ion Exchangers. Substances like cotton, paper, nut shell can be
converted into cation exchangers by sulphonation or phosphorylation process.
3. Synthetie Organie lon Exchangers. Groups like sulphonic acid and phosphorie
acid, when attached to the polymers give certain exchange propertie8.
4. Condensation Folymers. These are high molecular weight cross linked structures.
These are formed by splitting out such small molecules as water, alcohol and ammonia from
small polyfunctional monomers. Thus, phenol may condense with formaldehyde to give a crOss
linked polymer structure. The condensation polymers are easily broken up by hydrolysis and
oxidation.
OH OH OH OH
SOgH SOgH
Currently most of the common ion exchange products are built on the styrene divinyl
backbone because of their extreme stability. These polymers carry an electric charge that is
exactly neutralised by the charges on the counter ions. These active ions are cations in cation
exchanger and anions in anion exchanger. Thus, an anion exchanger contains polymerie
cationsand active anions while a cation exchanger consists of polymeric anions and active
cations.
CATION-EXCHANGE RESINS
Cation-exchange resins are high molecular weight, cross-linked polymers containing
phenolic, sulphonic, carboxylic and phosphorie groups as an integral part of the resin and an
equivalent number of cations.
1. Sulphonic Acid Exchangers. The sulphonic acid cation exchangers have received
attention mainly in water softening. Phenol-formaldehyde condensation polymers provide the
1110 SEPARATION TECHNIQUEODES
basis for more stable strong acid cation exchangers. Some of the cation exchangers cond
are obtained as follows:
xchangers containing
sulphonic or methyl sulphonic groups
C Na C"Na C"Nat
CH2 CH2
OH OH OH OH
H2SO4 HCHO
low pH
CH2 CH2
SOg H* SOg H t
SOg H
Phenol Phenol sulphonic acid
The advent of styrene and divinylbenzene has led to a family of durable and economic
sulphonic acid cation exchangers.
2. Carboxylic Acid Exchangers. Carboxylic acid resins are of considerably weaker
acid strength than their sulphonic acid counterparts and are useful for the removal of cations
from basic solutions. Some of the ion exchange resins containing carboxylic acid functional
groups a r e :
- CH-CH2-
-CH-CH2 -CH-CH2
H20 HNO3
CHC
Chloromethylated
Hydrolysis
H.OH
CH2OH
Oxidation
cOOH
styrene-DVB copolymer
3. Phosphorus Containing Exchangers. Because of high capacity and novel selective
characteristics phosphorus containing cation exchangers offer interesting possibilities as
intermediate strength exchangers. Most important phosphorus containing cation-exchange
resins are
CH3 CH3
C=CH2 +DVB - -CH-CH2-
CH-CH2 ---CH-CH2 -CH-CH2
COOR COOR
and
PO(OH)2 CH2P(OH)>
Acrylic acid Aryl phosphoric Benzylic phosphoric
or its ester
-CHCH2-- acid resin acid exchanger
whieh are
Likephosphoric acid resins certain arsenic acid resins have been prepared
reported to have high affinity for uranium cations.
Action of a Cation Exchanger.
A cation exchange resin can be represented as (Res A) B* where Res is the basic polymer
of the resin to which is attached the anion A and mobile cation Bt. Thus (Res. SO°) OE
Na" is the sulphonated styrene resin in the hydrogen or sodium form. When a cation excha
in
resin is placed in a solution, the mobile or active cation of the resin is exchanged for cao
solution.
ON EXCHANGE CHROMATOGRAPHY
1111
(Res. A")B* + Ct
Cation exchange resin Cation in solvent (Res. A") Ct + B
The experimental conditions Resin Cation in solution
crcated in such a way that
are
displaced from left to right and cation C* from
the equilibrium is completeiy
exchanger. When a cation solution is completely fixed on the cation
exchanger is in equilibrium with a solution
or three metals (eg., C', D" and
E' etc.), the containing salts of two
This is due to the basic nature of exchanger may show different affinities for them.
cation exchange. Consider the
from Dowex-50, a sulphonated
polystyrene cation resin, by calcium displacement of mobile ions
ions.
(Res.SO3) H' + Na'Cl (soln.) (Res. SOg) Na' +H'C (soln.)
2 (Res.
S0g) Na' + Ca
(Res. SOg) Ca +2 Na
or (Res. SO3) Li + Na' (soln.) = (Res. Na' + Li' (soln.)
SOg)
The calcium or sodium ion from the
product can be easily removed by passing solution
containing sodium or lithium ions respectively in the above two reactions and the original
resins are thus restored. For the strongly acidic cation
linked polystyrene sulphonic acid resins, the
exchange resins such as the cross
Vmax as
max Vb (D, + B)
Note. These relationships are strictly applicable only when the loading of the columns
is less than 5% of its
capacity.
A recent development, is the use of micro computer controlled
elution e.g., for the
systems for gradient
separation of amino acids by IEC. The scope of
separation may be extended
by using (for elution) a solution capable of complexing the ions exchanged. Complexes of
different stabilities behave differently with selected eluants. The results
of ion exchange
separations may be influenced by varying the pH, solvent, eluant, temperature, particle size,
length of the column, rate of flow of eluant and nature of ion exchange resin.
ANION EXCHANGE RESINS
Anion exchange resins like cation exchangers are also cross linked high molecular weight
polymers, containing basic groups such as amino, substituted amino, or quaternary
ammonium groups. Condensation reaction between epichlorohydrins and amines, ammonia
and a variety of nitrogen bases yield weak base resins. Weak base anion exchangers based on
addition polymers are also known. Some of the weak base anion-exchange resins are
CH2-CHOH-CH2
CH2CHOH-CH2-N-CH2-CHOH-CH2-
-N-CH2-CHOH-CH2-N--
CH2-CHOH-CH2***
Condensation product of ammonia and epichlorohydrin.
adition product of
Addition product 2-methyl
henzyl chloride is as follows 5-vinyl pyridine 1113
and DVB
which is then
en alkylated
alkylated with
wi
-CH-CH2-CH-CH-
N RCI
Diphenyl ether is first
CH
presence of
prese FeCl3 catalyst. chloromethylated,
It 1s
reacted with then the product
below
(CH3)3K to give anionundergoes
exchangecondensation
resin
in
as shoWn
[ CH2
CH2
0-
CHN (CH,)3C
Diphenyl ether is the basic structural unit for CHN (CH,)hC
exchange resins. These have unusual a wide
variety of recently developed anion
A widely used anion chemical stability.
exchange resin is prepared by
little divinyl benzene followed copolymerisation
by chloromethylation (introduction of styrene and a
position) and interaction with a base such as
of -CH,CI
grouping in para
a
polystyrene anion exchange resin is shown trimethylamine.
as follows
A hypothetical structure of
such
CH-CH2-CH-CH2- -CH-CH2
CH2NMe"Cr CH CHNMetC
H2C CH2
- H2C-CH
CH-CH2
H,NMe°CI CHNMe'cr
The introduction of macroreticular reSins with pore diameter of 130 nm allows
Complete removal of high molecular weight 1ons. Macroporous resins are well suited far
and are better than gel-type resins. Pellicular resins
on-aqueous
and microparticle packingsapplications
ion exchange have been currently developed to meet the specific needs of HPLC
The diameter) and narrow distribution of silica based
ne small particle size (5-10 um column efficiencies and
provide high-resolution anale
xChange packings provide high nucleotides etc. Some ot the commercially availahlo
dmino acids, proteins, peptides, in Table 1
CXChange resins are summarised
1114
SEPARATION TECHNIOUES
Table 1. Commerclal lon exchange resins.
Type of Functional Nature of pl Commercial
resin group resin range name
Applications
Cation Exchange Resins
Strongly CH,SOgH' Sulphonated 1-14 Amberlite
|Inorganic
acidic polystyrene IR-100
IR-105 separations,
IR-109
fractionation of
cations
-
Strongly-CHNR,OH
basic
Quaternary 0-12 IRA-410 Halogens, fatty acids,|
polystyrene
-CHN Ammonium 0-12 Alkaloids, vitamin B
polystyrene complex
-(CH,COOH)2 Chelating amino 0-12 Dowex A-1 Fractionation of
diacetic acid anions
ilibrium
of the reaction;
Res. N(CH3)l + H,0
|Res. NH(CH3}I OH
is always to
wards left so that the weakly basie anion exchange resin is always present in the
amine form
in basic solutions. They can, however, be used in acidic or weakly asic media (pH,
yield highly ionised salt form
They
0-9).
N(CH3)gl + H'CI Res.|Res. NH(CH,)al' C
Res.
+CI (Soln)
[Res. NH(CH3)2l' Cl NOg(Soln) IRes. NH(CH3)h| NOg
+
be tDytincreasing
h e 1on of higher charge is in
solution,
lower charge in is in the exchanger and
of lower charge favoured
dilutions.
Ifthe ion will be
by high
Solution, exchange
1116 PARATION TECHNIQUES
2. Nature of Ion Exchange Resin. The relative adsorption of ions usuallv ION
the presence of functional groups and the degree of cross-linking in the resin. The naton
the functional groups largely determines the type of the exchange phenomenon a n o f SuP
and
applications. The increase in the degree of cross-linking makes the resin more their zero
towards ions of different sizes. Ion with the smaller hydrated volume will be adove
conv
preferentially. instr
expressed as millimoles per gram of exchanger. The capacities of weakly acidic and we
Pac
basic ion exchangers are functions of pH. The moderately constant values of pH
ove9 are ah
and
below 5 respectively. Values for the capacities (TEC) are
total exchange expressed 100-
m mol
g of dry resin. The TEC of wet resin is about 1/3 to 1/2 m of dry resin.
molg The unif
capacity of a cation exchange resin is evaluated by measuring the number of milligram
to ti
equivalent of sodium ion taken up by 1 gram of dry resin in the hydrogen h. Similarlythe The
capacity of an anion resin is measured by determining the number of milliequivalents of in
chloride ions adsorbed by 1 gram of dry resin in the hydroxide form. The ion exchange thrc
capacity
for some resins is given in m mol g of resin.
i) De-Acidite FF (CI form) 4-0, (ii) Amberlite IR - 120 (Na' form) 4-44 The
(ii) De-Acidite G (CI form), 9.0 (iv) Amberlite IR - 50 (H* form) 10-0 sol
elu=
(v) Zeo-Karb 225 (Na' form), 4-5-5-0,
(vi) Zeo-Karb 226 (H form) 4-0. The values of ion-exchange capacity help by
estimating the amount of dry resin required in a determination.
INSTRUMENTATION FOR ION-EXCHANGE CHROMATOGRAPHY
Separator Column for lon Exchange.
The ion-exchange chromatography is carried out in a chromatographic column which
usually consists of a burette provided with a sintered glass disc or glass wool plug at the lower
and
end. The top of the bed resin may be covered with a glass wool pad. The column is never
allowed to drain out. The column length depends on the
separation factor. The degree of separation required Solution containing say
determines the length of the column (Fig. 1). Ca2 100 twie
equ
Column Dimensions. Resin
to e
The ratio of the height to diameter of the column is rins
Column
usually 10: 1 or 20: 1. Too narrow or too wide columns give Solution containing per-
uneven flow of liquid and sometimes poor separation. Now Ca2 0 it
low-capacity anion exchangers have been prepared by a
surface agglomeration method in which finely divided anion FPlug
exchange resin is contacted with surface sulphonated
effl=
styrene-divinyl benzene copolymer. The small particles of
the
anion exchanger are held on the oppositely charged surface off
sulphonated beads. These resins are stable over a wide range
col
of pH and are superior to glass or silica-based pellicular Eluate the
resins. 1. Apparatus for
Fig.
ion-exchange.
ONEXCHANGE ANGE CHROMATO
OGRAPHHY
is
e
con
r
verted
o
and a
olution containi
solution
containing
the right in the following
the right: reaction
polystyrene displaced to
is
Columonated equilibrium
n of cation exchanger.
er. The
components.
iv) Radiochemical method. Radioactivity in the cluate is automatically recordet
with the help of Geiger Muller counter. The method is used in the separation of fission
products.
LIQUID ION EXCHANGER
The ion exchange processes involving exchange resins occur between a solid and a liquid
phase whereas in case of liquid ion exchangers the process takes place between two immiscible
solutions. Liquid ion exchangers (LIE) consists of high molecular weight acids and bases whieh
have low solubility in water and high solubility in water-immiscible solvents. Thus, a solution
of anacid insoluble in water acts as cation exchanger for ions in aqueous solution whilea
solution of base insoluble in water acts as anion exchanger. Theliquid anion exchangers are
based on primary, secondary and tertiary aliphatic amines. These' are best employed as
solutions (2-5 to 12-5% v/v) in an inert organic solvent like benzene, toluene, ether, octane,
cyclohexane etc. Liquid cation exchangers are alkyl and dialkyl phosphoric acids, carboxylic
acids, alkyl sulphonic acid.
Operation of Liquid lon Exchangers (LIE).
and an immiscible
LIE involves the selective transfer ofa solute between aqueous phase
molecular weight amines in acid
organie phase containing the liquid exchangers. Hence high
ion pairs) with various
solution yield large cations capable of forming extractable species (e.g.
identical to that used in
anions. The technique employed for separations using LIE is thus
of both 1on
solvent extraction separations. These exchangers offer many advantages the
which arises with LIE is due to
exchange and solvent extraction. The main difficulty
formation of emulsions between organic and aqueous phases. Another disadvantage in the
use of liquid exchangers is that it is necessary to back-extract the required species from tne
organic phase into an aqueous phase prior to completing the determination. Sometimes tne
organic phase is used directly for determination of extracted species, by aspirating
direcuy
into a flame and estimating extracted metal ions by flame emission or atomic absorptio
spectroscopy. The extraction of metals by liquid amines depends on the formation of anion
solution.
complexes of metals in aqueous
APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY
ique
Ion exchange chromatography is a powerful but cumbersome technique. This techn
is used for the separation of rare earths, amino acids, carbohydrates, proteins, transi
elements, complex ions and even isotopes. Some of its applications are listed below.
ON EXCHANGE CHROMATOGRAPHY
1119
1. Removal of Interfering Radicals. Phosphate ion nterferes in the estimation of
or Ba 2 ions by oxalate method. Removal of phosphate ion is achieved by passing a
ca" orcCa2
solution o fCa2+ or Ba4 ions through
a sulphonic acid cation exchanger. The Ca2 or Ba ions
the resin will
re be removed by
using suitable eluent and estimated by usual
held by
ng
Softening of Hard Water. Ca2*, Mg2
and other divalent ions are removed b
methods.
2.
hard
passing h a r d water through cation exchangers charged with Na'. Ca2+ and Mg ions are
r e t a i n e d
in the column whereas Na ions pass into the solution.
3. Demineralisation of Water. Water is first passed through an acidic cation
T r when the cations like Na", Ca*, Mg* are exchanged by H* ions. It is then passed
basic anio
through basic anion exchanger to exchange Cl, SO by OH ions of the exchanger. Generally
nhonic acid resin is employed as the cation exchanger while a strong basic resin is used as
the anion exchanger.
of Anions.
4. Determination
Ion chromatography is used for the separation and determination of anions
height
2). Measure the 10
each anion and find the anion ncentration from
from
concentration
Volume, cm3
calibration graph. Fig. 2. Separation of anions.
SEPARATION TECHNIQUESS
1120
by Gradient Elution. K. A. Kra.
Metal lons
5. Separation of
Transition
transition metal ions, eg., Mn(11), Ni(II), Co(II). CCu(II), and g
E. Moore have separated basic anion
Fe(I),
exchange resin in
using strong
Cu(l1) and Zn(l1) by chloride form Dowex-1. The
the
column was first filled
with 12 M HCI and
THc AM 25MH
712M HCI
HCI Cu() 0.5MHCHO.005MHCT
packed
allowed to percolate through the column. Co(1l) Fe(1) Zn(1)
sample
transition metal ions
formed negatively
The Mn(1)
chloro complexes and adsorbed by
the
charged dilute
resin. Elution was then carried out using
HCI solutions. On elution with 12 M HCI, Ni(II) Ni(1)
moved out of the column band and the eluate was
collected. Other cations were eluted using varying
concentrations of HCI as illustrated in Fig. 3. The
relative stability of the chloro complexes of the
o 10 20 30 40 50 60 70 80 90 100 110 120
transition metal ions has been used for the Eluate volume,cm3
qualitative analysis o f the cations
(Zn, Fe, Mn, Co, Ni, Al and Cr by Arbeilate IRA 400 Fig. 3. Ion exchange separation of
(chloride form). The elution is done as described transition metal ions on an anion
above. exchange resin.
6. Separation of Halide Ions Anion
anion exchange resin in chloride form
on an
Exchanger.
The column is filled with
(De-Acidite FF). This is converted into
nitrate form by
passing concentrated solution of sodium nitrate. On
bromide ions, the nitrate ions of the resin passing a solution
containing chloride and
are rapidly
band at the top of column. After a exchanged
known volume of the mixture
with halide ions and form a
the column, it is eluted with a solution has passed
solution of NaNO3. Chloride ions are through
column band followed by bromide first eluted from the
ions and the eluate is titrated with standard
solution.
7.
AgNOg
Separation of Lanthanides and
Actinides. The solution
or actinides in aqueous solution containing lanthanides
undergo a
organie polymer (such as Dowex 50 in simple chemical ion exchange
NH{ form) is stirred into the reaction when an
3
[RSOI NH +M`* (soln) solution,
where M'*
represent
[Rsoglg M* (solid) +3 NH; (Soln)
cation exchange resin tripositive actinide or lanthanide
and the solid material, ions. A column is filled with some
column. It is eluted with (RSO3)M* is then
such ions as
a-hydroxy isobutyric acid, EDTA chloride, sulphate, nitrate, and placed the top of the
at
of affinity of the cations to (sodium salt) etc. The elution especially citrate, lactate,
order depends
the resin and
upon the degree
and actinides strength
agent. Both the lanthanides of the complex ion
the element with
the highest atomic have been found to formed with the eluting
elute in the reverse
successively lower atomic numbers. The number is eluted first followed order. Thus
can also be
achieved by ion by the
separation of actinide group and elements with
exchange technique. lanthanide group
EXPERIMENTAL ION EXCHANGE
Experiment 1. To
CHROMATOGRAPHY
Resin. Cation separate sodium and potassium
Flom Rate. 0-6exchange resin, Dowex-50 from the mixture.
cm°/min.
Eluant. 0-7 M HCI. cm. (particle size 300
mesh).
Procedure. The Time for complete elution is 5
J.Benkerkamp. separation of sodium and
Sodium and
hours.
potassium emergepotassium
from the
was
performed by W. Rieman
column quite anuis
separately.
NGECHPOMATOGRAP
l o r rate.