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1 INTRODUCTION 1
2 PRINCIPLE 2
5 EXPERIMENTAL TECHNIQUE 14
6 SEPARATION FACTOR 18
8 TECHNIQUES 25
10 APPLICATIONS 35
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INTRODUCTION
PRINCIPLE
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Ion Exchange Chromatography relies on charge-charge interactions between the
protiens in your sample and the charges immobilized on the resin of your
choice.Ion exchange chromatography can be sub divided into cation exchange
chromatography,in which postively charged ions bind to a negatively charged
resin;and anion exchange chromatography,in which the binding ions are
negative,and the immobilized functional group is postive.Once the solutes are
bound,the column is washed to equilibriate it in your starting buffer,which
should of low ionic strength,and then the bound molecules are eluted off using a
gradient of a second buffer,which steadily increases the ionic strength of the
eluent solution.Alternatively,the PH of the eluent buffer can be modified as to
give your protien or the matrix a charge at which they will not interact and your
molecule of interest elutes from the resin.If you know the PH ,you want to run at
and need to decide what type of ion exchange to use paste your protien sequence
into the titration curve generator.If it is negatively charged at the PH you
wish,use an anion exchanger.Ofcourse this means that your protien will be
bindind under the condition you choose.In many cases,it may be more
advantageous to actually select conditions at which your protien will flow through
while the conatminates will bind.This mode of binding is referred to as “flow
through mode”.This is a particularly good mode to use this type of mode to bind
up endotoxins or other highly negatively charged substances well at the same
time relatively simply flowing your protien through the matrix.
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Ion exchange resins are highly ionic, covalently cross linked,
insoluble polyelectrolytes supplied as beads. The beads have either
have a dense internal structure with no discrete pores or a porous
multichanneled structure. They are commonly prepared from styrene
a various level of cross linking agent divinyl benzene, which control
the porosity of the particles. Porous beads can also be made by
adding homopolystrene, which is soluble in precursor beads are post
functionalized to yield the finished resin Acrylic based; ion exchange
resins are also available. These ionic polymers contain two types of
ions, those which are bound within the structure and the oppositely
charged counter ions which are free. The property of ion exchange is
a consequence of Donnan exclusion-when the resins is immersed in
a solution in which it is soluble, the counter ions are mobile and can
be exchanged for other counter ions from the surrounding medium;
ions of the same type of charge as the bound ions do not have free
movement into and out of the polymer. Ion exchange resins have
been classified based on the charge on the exchangeable counter
ion and the ionic strength of the bound ion.
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Some ion exchange resins are prepared from chelating properties
making them highly selective towards certain ions. In addition to
there use in ion exchange, organic polymer supports, many of which
are based on PS-DVB resins, are being used as polymer catalysts in
the expanding research area involving heterogenization of
homogenous catalysts and as a polymeric supports and reagents in
combinatorial chemistry. The internal structure of the resin beads,
i.e whether micro porous or macroporous,is important in the
selection of an ion exchanger.Macroporous resins with their high
effective surface area, facilitate the ion exchange process. Also they
give access to the exchange sites for larger ions, can be used with
almost any solvent, irrespective of whether it is a good solvent for
the uncrossed linked polymer, and take up the solvent with little or
no change in volume. They make more rigid beads, facilitating ease
of removal from reaction system. In the case of micro porous resins
since they have no discrete pores, solute ions diffuse through the
particle to interact with exchange sites. Despite diffusional
limitations on reaction rates, these resins offer certain advantages:
they are less fragile, requiring less care handling, react faster in
funtionalization and application reactions, and possess loading
capacities. In addition to being functions of bead morphology, the
kinetics of exchange depend on the particle size distribution of resin.
It is enhanced by monodisperse resin.
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According to their origin both, organic and inorganic ion exchangers
may be natural or sythetic.For chromatographic purpose organic
synthetic ion exchangers, ion exchangers and ion exchange
derivatives of cellulose, polydetran and agarose are of the greatest
importances.
There are four basic types of resins which are commonly used in the
ion exchange chromatography.
Resins in which liable ions are cations are known as cation exchange
resin. Thus a cation exchange resin is a high molecular weight, cross
linked polymer having acid mobile ions or functional groups such as
sulphonic, carboxylic, phenolic etc.In this cation exchanger the
hydrogen ions are mobile and exchangeable with other cations.The
anions remain attached to the resin network a widely used cation
exchange resin is obtained by copolymerization of styrene with little
of divinyl benzene followed by sulphonation.
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Cation exchange may be sub divided into:
• Strongly acidic.
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B) A weakly acidic cation exchanger mainly contain carboxylic
group or phenolic.Such as exchanger ionizes in alkaline media
only and should therefore be used at pH greater than 7.The
rate of exchange has found to increase with increase in pH.
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Resins in which ions are anions are known as anion exchange
resins. The anion exchange resin is a high molecular weight, cross
linked polymer containing basic functional group such as amino
groups, quaternary ammonium group, halides group etc.A widely
used anion exchange resin prepared by co-polymerization of
styrene and a little divinyl benzene followed by chromethylation
and interaction with base such as trimethyl amine.
AMPHOTERIC:-
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Amphoteric ion exchanger contains both cation exchanging and
anion exchanging groups in their matrix. These ion exchangers
are capable of forming internal salts which dissociates in contact
with electrolytes and bind both its components. However they can
be easily regenerated with water dipolar ion exchangers; are a
special kind of atmospheric ion exchangers. On the matrix are
bound amino acids which dipoles in aqueous solution. These types
of ion exchangers are suitable for the chromatography of
biopolymer with which the dipole interacts selectively.
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Resin type Chemical Usual Common Selectivity
constitution form of trade name
purchase Rohm DOWEX
& chemical
Hass
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PROPERTIES OF ION EXCHANGE RESINS:-
Ion exchange resin of different origin, structure and components will
have different properties. The most important properties of ion
exchange resin are color, density, mechanical strength, particle
strength andcapacity,selectivity,cross
linking,swelling,porosity,surface area and chemical resistant.
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TYPICAL ION EXCHANGE EXPERIMENT
The simplest apparatus for ion exchange analysis consist of a
burette provided with a glass- wool plug or sintered glass disc at the
lower end .A glass- wool pad may be placed at the top of the bed of
resin and the eluting agent is added on top of the bed of resin should
be of small particle size so as to provide a large surface of contact.
In all cases, the diameter of the resin bed should be less than one-
tenth of that of the column. The resin should be stirred with water in
an open beaker for several minutes, any fine particle if present, is
removed by decantation. The resin slurry is then transferred portion-
wise to the column previously filled with water. To ensure the
removal of trapped air bubbles or any particles or an uneven
distribution of resin granules, the column is backwashed before use.
A stream of distilled water or deionised water is run up through the
bed from the bottom at a sufficient flow rate to loosen and suspend
the exchanger granules. The excess of water is drained off. However,
the water level must never fall below the surface of resin throughout
the operation.
Procedure:-
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resin.Thus, the elute coming out last from the bottom of the column
is that which was most firmly held by the resin.
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EXPERIMENTAL TECHNIQUE
There are three ways of performing an ion exchange separation: by
column chromatography, by batch methods, and by expended bed
absorption. This section will mostly deal with column
chromatography.
Column chromatography:
COLUMN:-
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to settle down in between each addition. It is also advisable to bring
the resin in equilibrium with solvent before packing the column.
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dilute, otherwise a concentrated solution of HCl will obtained and
equilibrium would not favor the complete exchange of sodium for
hydrogen.
SAMPLE PREPARATION
Sample concentration:-
Sample composition:-
Sample volume:-
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normally chosen so that all important substances are adsorbed at
the top of the bed. In this case, the sample mass applied is of far
greater importance than the sample volume. This means that large
volumes of dilute solutions, such as pooled fractions from a
preceding gel filtration step or a cell culture supernatant can be
applied directly to the ion exchanger without prior concentration.
Sample viscosity :-
Sample preparation:-
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or centrifugation before being applied to the column. This
requirement is particularly crucial when working with small particle
matrices, such as Minibeads(3µm), MonoBeads(10µm),SOURCE(15
and 30µm) and sepharose high performance (34µm).The “grade” of
filter required for sample preparation depends on the particle size of
the ion exchange matrix which will be used. Samples which are to be
separated on a 90 µm medium can be filtered using a 1µm filter. For
3,10,15,30 and 34µm media, samples should be filtered through a
0.45µm filter. When sterile filtration or extract clean samples are
required, a 0.22µm filter is appropriate. Samples should be clear
after filtration and free from visible contamination by lipids. If turbid
solutions are injected onto the column, the column lifetime,
resolution and capacity can be reduced. Centrifugation at 10000g for
15 minutes can also be used to prepare samples. This is not the
ideal method of sample preparation but may be appropriate if
samples are of very small volume or adsorb nonspecifically to filters.
SEPARATION FACTOR:-
Ion exchange chromatography is based on the exchange of ions
between a solid ion exchanger and ions present in the solution. The
exchange of ions obey the law of mass action. For example, the
equilibrium between the ion exchanger and the solution can be
represented as,
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The conditional ion exchange equilibrium constant in the first
approximation can be expressed as
Where [B-] and [A-] are the concentrations of the ions in the solid
phase and [B+] and [A+] are the concentrations of ions in the liquid
phase. KAB is conditional ion exchange equilibrium constant. It should
be noted that the sorption of ions depend on the following factors:
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M1 and M2 = First and second exchanging ions of metals
A = Anion of resin
K = [M2AZ2] Z1
[M1Z1+] Z2
[M1AZ1]Z2 [M2Z2+] Z1
OR
[M2AZ2]1/Z2 = Kc C21/ Z2
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The adsorptive effect of an ion is characterized by the DISTRIBUTION
COEFFICIENTS.
D = Cr.m
Cs.V
α = DA
DB
• pH
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• Tendency of ion to hydrate
Ion exchange constant vary with the cations involved and are
markedly different from unity. The rates of the movements of the
ions through a layer of an ion exchanger are inversely proportional
to the values of ion exchange constant. In this way, a number of
mixtures of a complex composition can be separated into their
components.
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constant but present in a high concentration. Example of such ion is
H+ ions. For this purpose HCL solution are commonly employed. It
should be noted that efficient separation of ion can be achieved only
when their is a considerable difference in ion exchange constants.
Li Na k Rb Cs
Mg Ca Sr Ba
Lu Yb Tu Er Ho Dy Tb Gb Eu Sm
Pm
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anions and cations.Columns of greater length is needed for
separation of cations.
CHOICE OF BUFFER : -
As with the choice of an ion exchanger, there are a number of
variables which have to be considered. These include:
BUFFERS : -
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starting ionic strength of at least 10mM is required to ensure
adequate buffering capacity. Salt also play a role in stabilizing
protein structures in solution and so it is important that the ionic
strength should not be so low that protein denaturation or
precipitation occur. A major advantage of using Amersham
Bioscience ion exchangers is that they have excellent capacities and
so the initial ionic strength of the buffer can be quiet high with out
significant affecting capacity for sample 77
TECHNIQUES
Recent advances in ion chromatography are mostly related to highly
selective separation and to extremely low detection limits. Through
the choice of stationary phase and the variation of eluent
composition, higher separation selectively can be obatined.Analyzing
the samples in which analytes are presented at low concentration in
the presence of high ionic strength or extreme pH matrix, the major
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matrix ions would displace the adsorbed analyte ions on the
stationary phase. The result would be band broadening and poor
separation because the extreme pH of sample causes disturbance to
the equilibria in the eluent.Therefore, technique relating to sample
preparation and separation need to make progress.
The first used suppressor was an ion exchange column in the form of
H+ or OH-,2 but this technique is restricted by the limited capacity
of the suppressor, which must be regenerated periodically off-line.
This problem has been overcome with the introduction of the hollow-
fiber membrane suppressor.However; a problem of this kind of
suppressor is the low surface area of the fiber, resulting in low ion
exchange and suppression capacity. The appearance of the micro
membrane suppressor, in which two flats of membrane were
inserted into three ion exchange screens as a sandwich, solved this
problem.
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were used to allow the generation of H+ or OH- necessary from
water. In this device, no external regenerate is needed. In analysis,
an anion SRS is used and H+ ions produced from anode migrate
across the cation-exchange membrane to transfer OH- ions into
water and meanwhile counter-cations in the eluent are driven by
electric field to cathode. Gas waste from electrolysis reaction and
liquid waste will move out of the regenerant chamber.Cation analysis
will operate in the same way except that OH- instead of H+ will
migrate across the anion exchange membrane.
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could be circumvented if an inert gas was used to supplement the
effluent flow from detector. This mode called “gas assisted recycle
mode” speeds up the removal of eluent counter-ions and electrolysis
products, hence leads to a performance comparable to “external
water mode”.
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occurs, the suppression capacity of this device is higher and
background.
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anions are carried through the detector. The equivalent conductive
value of sodium, potassium, calcium, magnesium and other common
cations are significantly lower than that of the cations (H-) in the
eluent.In this instance a negative peak is detected as the cations are
carried through the detected.
1. Conductivity detection.
2. Electrochemical detection
3. Potentiometric detection.
4. Spectroscopic detection.
Conductivity detection :-
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principle of operation and performance characteristics, modes
of detection, cell design post column signal enhancement i.e.
suppression and applications.
Principle of operation:
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ions is low, then an increase in conductance occurs when the
solute enters the detections cell, due to higher conductance.
In general this detection mode is referred to as direct. On the
other hand, when the mobile phase ions are highly conducting,
a decrease in conductance occurs when the solute enters the
detection cell, due to lower conductance. This mode of
detection is referred to as indirect.
Electrochemical detection :-
Voltametery:-
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Voltametery is a well-established technique in which a
changing potential is applied to a working electrode with
respect to a reference electrode. The current resulting from
the reaction of analyzed species at the working electrode is
measured. The key factor is that the applied potential is varied
over the course of the measurement.
Spectroscopic Methods :-
Molecular Spectroscopy :-
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a) UV-VIS Absorption :
b) Fluorescence:
c) Refractive index(RI):
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Atomic spectroscopy :- The combination of HPLC separation with
various forms of atomic spectrometry gives a method of great
sensitivity as well as a time resolved detection of species.
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APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY :-
1) Demineralization of water:-
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2) Determination of sodium and potassium in the mixture :-
According to beulankamp and Reimen,the separation of potassium
and sodium ions in a mixture can be done by introducing the sample
at the top of a containing a sulphonic acid resin saturated with H+
ions. The column is then eluted with a solution of 0.7M HCl at a flow
rate of 0.6mL/sq.min.Sodium ions, being the less strongly held, move
down the column more rapidly and can then be collected before
potassium ions appear in the effluent. The solutions are evaporated
to dryness and the alkali chlorides weighed. Divalent ions, if present
do not come out of the cation exchange resin until all the potassium
ions have appeared in the effluent.Partion of the sodium ions in each
theoretical plate column involves,
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top.Elutionwas carried out by successively more dilute solution of
HCl.The Ni was not retained at all even in the presence of
concentrated HCl.When the HCl was diluted to 6M,the acid caused
the elution of Mn;Co came out at 4M,Cu at 2.5M,and Fe at 0.5M and
Zn at 0.0005M HCl.This type of elution indicates the relative
stabilities of the complex chloride anions and varying affinity of the
resin and of water for these ions as chloride ions.
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(6)Concentration of traces of an electrolyte:- Concentration of
traces of an ion form a very dilute solution can also be carried by
making use of ion exchanger resins. For example, traces of metallic
elements can be collected from larger volumes of natural waters by
making use of cation exchange resins. The resin is first treated with
HCl to liberate the ions .As a result the solution becomes more
concentrated for further analysis.
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hydroxides while the amphoteric metals are eluted as AlO 2- ,Zn2- ions
etc.
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chromatographs for the analysis of amino acid mixtures are also
available commercially.
REFERENCES
Instrumental methods of chemical analysis : By B.K.Sharma.
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