CHROMATOGRAPHY
Subject: Modern Pharmaceutical Analytical Technique
Subject Code: MAT101T
Topic: Ion Exchange Chromatography and Column Chromatography
Prepared by: Garima Singh
M. Pharma
Ist SEM
Department Of Pharmaceutics
1Smt B.N.B Swaminarayan
Pharmacy Collage Salvav Vapi

Smt B.N.B Swaminarayan
Pharmacy Collage Salvav Vapi
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DEFINITION
• Ion chromatography or ion-exchange
chromatography) is a chromatography
process that separates ions and polar
molecules based on their affinity to the ion
exchange
3Smt B.N.B Swaminarayan
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PRINCIPLE
• Exchange of ions is the basic principle in this type of Chromatography. In this
process two types of exchangers i.e., cationic and anionic exchangers can be
used.
• Cationic exchangers possess negatively charged group and these will attract
positively charged cations. These exchangers are also called “Acidic ion
exchange” materials, because their negative charges result from the
ionization of acidic group.
• Anionic exchangers have positively charged groups that will attract negatively
charged anions. These are also called “Basic ion exchange” materials.
• Anionic exchange Chromatography should be carried out with cationic
buffers.
• Cationic exchange Chromatography should be carried out with anionic
buffers.
• The pK of the buffer should be as near as possible to the pH at which the
system is buffered. This result in high buffer capacity, which can with stand
the local changes of pH in the column easily.
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•
BUFFERS PH range
Ammonium acetate 4-6
Ammonium formate 3-5
Pyridiniumformate 3-6
Pyridinium acetate 4-6
Ammonium carbonate 8-10
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ION EXCHANGE MATERIAL
• •DEFINITION: A complex resin structure which is water insoluble & loaded
with ions which can easily replace the desired ion from the solution.
• •There are 3 types of ion exchange material
A) Synthetic inorganic ion exchangers-
• •These materials posses a relatively open three dimensional frame work
structure with channel and interconnecting cavities
• •E.g. alumina-silicates, TiO2, ThO2, zirconium oxide, phosphate etc. the
hydrous oxide of tri and tetravalent metals are useful as cation exchanger.
Besides phosphate, molybdate, tungsten etc. of some metal acts as cation
exchanger materials.
B) Natural organic ion exchangers-
• Substance like coal, paper, cotton etc can be converted into cation
exchange by reaction of sulphonation or phosphorylation.
• They act as cation exchanger since they carry sulphonic acid or carboxylic
group attached to them.
• These materials are less uniform in structure and get readily affected by
other chemicals.
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C) Synthetic organic ion exchangers-
• The synthetic organic ion exchanger resin are made of cross linked
polymer network to which are attached various functional group.
• The nature of functional group determines whether it is a
• Cation exchanger
• Anion exchanger
• In cation exchanger materials the acid group are sulphonic acid, carboxylic
acid or phenolic, while in anion exchanger resin the group are basic as
amine, quaternary ammonium.
• On the basis of strength of group, they are further divided into four
categories:
 Strongly acidic cation exchange resin: Sulphonic group
 Weakly acidic cation exchange resin: Carboxylic group
 Strongly basic anion exchange resin: Quaternary ammonium group
 Weakly basic anion exchange resin: Polystyrene, formaldehyde
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Requirements of ion exchange resin
• It should have a sufficient degree of cross liking for use in chromatography.
• It should be insoluble in common solvents.
• It should be sufficiently hydrophilic to permit diffusion of ions through its
structure at a constant and finite rate.
• It should have desired particle size and shape.
• It must be chemically stable.
• It must contain sufficient number of ion exchanger group.
• It must have the ability of regeneration and reuse.
• The swollen resin must be denser than water.
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METHODS OF ION CHROMATOGRAPHY
• 1.Batch Method-It involves single step equilibrium. The resin and the
solution are mixed in a vessel until equilibrium is attained.
• The solution is filtered off, and further fresh portion of
the resin is added to the solution. This process is known as batch operation,
but is very little important in quantitative analysis.
• Used for producing deionised and demineralised water and for softening of
water.
• 2.Column Method-It is better and efficient method. The resin is placed on
the top of a glass or wool plug or sintered glass disc in a vertical tube or
burette.
• •By passing sufficient concentration of solution through the resin column, a
strongly acidic cation exchange resin can be easily converted completely
into the desired ionic form.
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TECHNIQUES OF ION CHROMATOGRAPHY
• Ion exchange chromatography is performed on column with adsorbents,
exchangers and solution which is to be separated is poured on the column
then elute from the column is investigate
• [A] Column for Ion exchange chromatography
• They are designed in such a way that no disturbance occurs in flow of liquid
and all operation is carried out in downward direction.
• As the liquid moves down, ions comes in contact with untreated resin and
get completely exchanged with resin.
• Column should not be too wide nor should be too narrow because it will
produce uneven flow of liquid.
• Ion exchange chromatography is based on exchange of ion between solid
ion exchange and ions present in the solution.
•
• RA + B+ → RB + A+
• [B-] / [A-] = KA,B[B+] / [A]
•
• Where B-and A-are concentration of ion in a solid phase and B+ and A+ are
concentration of ion in liquid phase and K is ion exchange equilibrium
constant. 10Smt B.N.B Swaminarayan
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• [B] Packing of column
• The resin is treated with the solvent and equilibriums are achieved before
packing the column.
• The slurry of the resin is poured in the column.
• The solvent which is to be used as an eluent should be used for making the
slurry.
• The slurry is added in several portions allowing the resin to settle down.
• When packing is complete the eluent is permitted to pass through the
column for certain time so to ensure uniform rate of flow over whole cross
section area of the column, and level of liquid is so adjusted that it remain
below the top of the resin bed.
• Now the column is ready for experiment and sample solution which is to be
separated is introduced or poured on top of the resin in column by using
micropipette.
• [C]Operation of the column
• There are three methods of operation-the basis of all of these operations is
that the substance in solution has some affinity for the substrate over which
it flows. The affinity is due to ion exchange properties of the column.
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• Displacement Analysis: Most strongly absorbed material displaces less strongly
absorbed material and each component in turn displaces less readily adsorbed
component.
• Frontal Analysis: If solution of mixture of ion having different affinity coefficient is
passed through a column of ion exchange resin in sufficient quantity to exceed
the exchange capacity of the resin then the least absorbed ion breaks first
through the column.
• Elution Analysis: Mixture to be separated is adsorbed in a narrow band at the
top of the resin column and is desorbed by passing down the column of a
solution of another ion which has a lower affinity coefficient then that of the
component of mixture & which is already absorbed on a major portion of the
column.
• [D]Analysis of eluent
• After flowing down the column the solution is passed through automatic fraction
collector for continuous determination of PH, refractive index etc.
• These reading are then plotted against the elute volume. Other methods are:
• Spectrophotometric method: used for direct analysis.
• Polarographic method: in this diffusion current under constant potential is
recorded as function of time & the amount of solute present in solution can be
calculated from area under the curve.
• Conductometric method: Electrical conductance of elute from the column is
recorded. 12Smt B.N.B Swaminarayan
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• [E] Regeneration of ion exchange resin:
• Regeneration refers to the replacement of the exchangeable cations or anion
present in the original resin.
• Hence the regeneration of the cation exchange resin is done by the changing the
column with strong acid like hydrochloric acid.
• Regeneration of anion exchange resin is done by using strong alkali like sodium
hydroxide.
 Factor affecting ion exchange separation:
• A)Nature & properties of ion exchange resins:cross linking & swelling is important
factor which depends on the proportion of cross linking agent &
polystyrene.When more cross linking agent is present they are more rigid but
swell less.When swelling is less separation of ions of different size is difficult as
they cannot pass through the pores present. B)Nature of exchanging ions:
• Valency of ions: at low concentrations & temperature exchange increase with
increase in valency.
• Size of ions: exchange increase with decrease in particle size.
• Concentration of solution: in dilute solution polyvalent anions are generally
adsorbed preferentially.
• Concentration & charge of ions: if the resin has higher positive charge & solution
has lower positive charge exchange is occur at higher concentration
13Smt B.N.B Swaminarayan
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ION EXCHANGE CHROMATOGRAPHY
Advantages
• Useful to resolve very complex
samples.
• Useful for separation of mixtures
of biological origin.
• Useful for detection of many
inorganic salts.
• Also used for detection of
organic ions with poor UV
absorptive.
Disadvantages
• Column efficiency is less.
• It is difficult to achieve control
over selectivity & resolution.
• Stability & reproducibility of the
column is difficult.
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APPLICATION OF ION
CHROMATOGRAPHY
• Softening of water: removal of monovalent & divalent ions likes sodium,
potassium, calcium, magnesium.
• Demineralisation of water: removal of different ions to get demineralised
water.
• Purification of solution
• Separation of inorganic ions.
• Biochemical separation: isolation of some drugs or metabolites from blood &
urine.
• Concentration of ionic solution: a cation or anion from a bulk of solution can
be absorbed onto ion exchange resin. After absorption it can be eluted by
using small volume of eluent.
• As ion exchange column in HPLC.
15Smt B.N.B Swaminarayan
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16Smt B.N.B Swaminarayan
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DEFINITION
• Column chromatography is a separation technique in which the components
of mixture is separated by using a glass column packed with S.P. & the
liquid M.P. is continuously flowing through the column.
• This is one of the most useful methods for the separation & purification of
both solid and liquid.
• This is a solid-liquid technique in which the S.P.is a solid & M.P. is a liquid.
PRINCIPLE
• It is based on the adsorption principle.
• Mixture of components dissolved in M.P. is introduced in to the column.
• Components moves depending on their relative affinities.
• The adsorbent is packed in a glass column & a solvent the M.P. is moves
slowly through the packed column.
• A compound attracted more strongly by the M.P. will move rapidly through
the column dissolved in the M.P.
• A compound more strongly attracted to the S.P. will move slowly through the
column.
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Requirement of column chromatography
1)Selection of S.P.
• Success of chromatography depends upon proper selection of S.P.
• It can depends on following
 Removal of impurity
 Number of components to be separated
 Length of column used
 Affinity difference between components
 Quantity of adsorbent used
2)Selection of M.P.
• They act as a solvent, developer& eluent.
• The function of M.P. are oAs developing agent oTo introduce the mixture
into the column oTo developing agent oTo remove pure components out of
the column
3)Column characteristics
• The main function of the entire column is to support the S.P.
• The material of the column is of good quality so it should not affected by the
solvents.
• Better separation will be obtained with a long narrow column because
number of plates will be more.
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• The length of column is depends on
 Number of components to be separated
 Type of adsorbent used
 Quantity of sample
 Affinity of components towards adsorbent used
4)Preparation of the column
• It consists of a glass tube with bottom portion of the column packed with glass wool/cotton
above which the adsorbent is packed.
• After the packing a paper disc kept on the top so that the adsorbent layer is not distributed
during the introduction of sample or M.P.
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5)Packaging technique The column should be free from impurity before using.
• It should be washed properly & dry it.
• Before filling the column with S.P. cotton/glass wool is kept.
• It should be uniformly filled.
• There are 2 types of packaging
• Dry packaging:adsorbent is packed in the column in the dry form.Fill the
solvent till equilibrium is achieved.
• Wet packaging: the material is slurred with solvent & generally added to the
column.The S.P. is settled uniformly in the column & there is no entrapment
of air bubbles.There will be not any crack in the column of the adsorbent.
• 6)Introduction of the sample
• The sample usually a mixture of components is dissolved in minimum
quantity of the M.P.
• The entire sample is introduced into the column at once & gets adsorbed on
the top portion of the column.
• From the zone individual sample can be separated by a process of elution.
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• 7)Development technique
• By elution technique the individual components are separated out from the
column.
• There are 2 techniques of elution
• Isocratic elution technique: in this technique same solvent composition or
solvent of same polarity is used throughout the process of separation.
• Example: chloroform
• Gradient elution technique: solvents of gradually increase polarity or
increase elution strength are used during the process of separation.
• Example: benzene – chloroform – ethyl acetate – chloroform
• 8)Detection of components
• If the compounds separated in a column chromatography procedure are
coloured, the progress of the separation can simply be monitored visually.
• If the compounds to be isolated from columnchromatography are colourless.
• In this case small fractions of the eluents are collected subsequently in
labelled tubes & the composition of each fraction is analyted by TLC.
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• Factors affecting on column efficiency
• Dimension of the column: column efficiency has been improved by
increasing length/width of the column.
• Particle size of the column packaging: separation is improved by decreasing
the particle size of the adsorbent.
• Activity of the adsorbent
• Temperature of the column: the speed of the elution increases at higher
temperature.
• Packaging of the column
• Quality of solvents: solvents having low viscosities are giving better results.
22Smt B.N.B Swaminarayan
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COLOUMN CHROMATOGRAPHY
ADVANTAGES
• Any type of mixture can be
separated •Any quantity of
mixture can be separated
• Wider choice of M.P.
• Automation is possible
• In preparative type the
sample can be separated &
reused.
DISADVANTAGES
• Time consuming
• More amount of M.P. are
required
• Automation makes the
techniques more complicated
& expensive
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APPLICATION OF COLUMN CHROMATOGRAPHY
• Isolation of active: from the plant extracts, from formulations or other crude
extracts, active constituents or required constituents can be isolated.
• Determination of primary & secondary glycosides in digitalis leaf.
• Purification process
• Estimation of drugs in formulation.
• Isolation & purification of vitamins & hormones.
• Examination of vegetable oils.
• In the separation of components after organic synthesis to obtain desired molecule.
• Removal of impurities or purification process: impurities present in a compound can
be removed by using appropriate S.P. & M.P.
• Isolation of metabolites from biological fluids: e.g. 17ketosteroids from urine,
cortisol, other drugs etc from biological fluids like blood, urine & serum.
• Separation of mixture components: column chromatography can be used for the
separation of several classes of drugs & constituents like alkaloids, glycosides,
amino acids, plant extracts, drugs & formulation.
• Determination of phytomenadione in injection & tablets.
• Separation of diastereomers.
• Separation of inorganic ions like copper, cobalt, nickel etc.
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REFERENCES:
Ion Exchange Chromatography
• Principles of Instrumental Analysis, Skoog, 6th edition , pg.no.641-647.
• Instrumental Method of Analysis by willard, dean,merit,settle,7th edition,
page.no.633-641.
• Instrumental Method of Chemical Analysis, Gurudeep R. Chatwal , Sham
,k.Anand, page.no.2.662-2.672.
Column Chromatography
• a.H.BECKETT & J.B. STENLAKE, Practical Pharmaceutical Chemistry,
4thedition, part II, page.no.86-105.
• Ashutoshkar, Pharmaceutical Analysis-II, page.no.161-181.
• Vogel's, Text book of quantitative analysis, page.no.289-314.
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