Separation Techniques

UNIT 2 BASIC CHROMATOGRAPHY

Structure

2.0 Introduction

2.1 Objectives

2.2 Classification of chromatographic techniques

2.3 Thin layer chromatography

2.4 Paper chromatography

2.5 Gas chromatography

2.6 Ion exchange chromatography

2.7 Size exclusion chromatography

2.8 Affinity chromatography

2.9 Let Us Sum Up

2.10 Terminal questions

2.0 INTRODUCTION
Chromatography is one of the most powerful and versatilelaboratorybased
analytical techniques used for the purification, identification and separation
of complex mixturesinto their individual components for quantitative and
qualitative examination. This technique was very first used by an Italianscientist
Mikhali Tsvet in the initial phase of twentieth century for the separation of
plant pigments.In comparison to the conventional methods such as
crystallization, distillation, sublimation chromatography is much more
beneficial owing to its advantagessuch as compounds in mg to kg range can
be easily purified without any loss in properties, each component of a mixture
can be isolated separately in pure form which is usually difficult by other
methods, minimum loss of compound, etc.

The underlying principle of this method is separation of the components of a

mixture based on the interaction (partitioning, adsorption and electrostatic
interactions) between the components, a stationary phase which may be solid
or liquid and a mobile phase which may be liquid or gas as well as on the
distribution of components between these two phases. Stationary phase adsorbs
the components of the mixture and mobile phase transports the components
on stationary phase while passing through it. During the purification process,
different components of the mixture get transported at different rate depending
upon their adsorption on stationary phasei.e. the strongly adsorbed components
move slowly whereas weakly adsorbed components move at relatively faster
rate along with the moving phase and get separated.

26
 Basic Chromatography
2.1 OBJECTIVES
After studying this unit you should able to

z Understand the significance of chromatographic techniques

z Define mobile phase and stationary phase

z Define retention factor

z Explain why chromatography is better than other separating process

z Prepare a TLC

z Classify the chromatographic technique based on mobile phase, stationary

phase and mechanism of separation

z To monitor the reaction profile using TLC

SAQ 1:
What is the difference between stationary phase and mobile phase?
...................................................................................................................
...................................................................................................................
..................................................................................................................

2.2 CLASSIFICATION OF
CHROMATOGRAPHIC TECHNIQUES
Chromatographic techniques can be classified broadly on the basis of:(i) nature
and physical state of mobile phase and stationary phase;(ii) nature of support
used for holding the stationary phase; (iii) mechanism of separation.

(i) Classification based on the nature and physical state of mobile phase
andstationary phase

27
Separation Techniques (ii) Classification based on the nature of support used for holding the stationary
phase

(iii) Classification based onmechanism of separation

Liquid-Liquid chromatography: In liquid-liquidchromatographic technique

both the phases are liquid and separation of the components of a mixture arises
from the distribution of the solutes between two immiscible liquids. The
stationary phase is composed of a liquid immobilized in the pores of a solid
support such as paper, glass or plastic.

Liquid-Solid Chromatography:Liquid-solid chromatography is also referred

as normal chromatography or adsorption chromatography. In this
chromatographic technique, the stationary phase is solid (SiO2, Al2O3, etc.)
and mobile phase is liquid.

Ion-Exchange chromatography:In this chromatographic technique the

stationary phase is solid consisting of charged ionizable functional groups and
ligands and mobile phase is liquid. This technique separates the charged
molecules based on their affinities towards an ion-exchanger.

Size-Exclusion Chromatography:Size-exclusion chromatography is also termed

as molecular sieves chromatography or gel filtration chromatography.It
separates the molecules on the basis of their size and shapes through exclusion
effect.

Gas-Liquid Chromatography: In this chromatography, separation of the

components follow the partition principle. The mobile phase is an unreactive
gas, such as nitrogen (the carrier gas) and the stationary phase consists of
small amount of non-volatile liquid held on a finely divided inert solid support.

Gas-Solid Chromatography:This technique is used for those applications which

are difficult to achieve with gas-liquid chromatography. The stationary phase
for this is a solid (charcoal or molecular sieves) and mobile phase is a gas. The
separation of components takes place through adsorption process.

28
Paper Chromatography: In this type of chromatography, the separation takes Basic Chromatography
place through the distribution or partition of the substances between stationary
phase and mobile phase. The stationary phase in paper chromatography is
water absorbed on cellulose of the Whatmann filter paper and mobile phase is
also a liquid (pure solvent or mixture of solvents).

Thin Layer Chromatography: The principle of thin layer chromatography (TLC)

is selective adsorption which involves the distribution of compounds between
a solid stationary phase and a liquid mobile phase. The stationary phase in
TLC technique is a thin layer of a solid adsorbent while the mobile phase is a
liquid (volatile pure organic solvent or mixture of solvents).

Column Chromatography:The mechanism of separation of column

chromatography is based on adsorption of the substances on the stationary
phase and affinity towards stationary phase as well as mobile phase. The
stationary phase is a solid adsorbent packed in a cylindrical tube and mobile
phase is liquid.

Adsorption Chromatography:This chromatography is based on the principle

of adsorption and separation depends on the selective adsorption of substances
on the surface of solid adsorbent. In this technique the stationary phase is
solid while mobile phase is liquid or gas.

Partition Chromatography:In partition chromatography, the separation of

components occurs due to the partition of the components between mobile
phase and stationary phase. The stationary phase is a liquid or a liquid supported
on an inert solid whereas mobile phase is a liquid or gas.

Gel Permeation Chromatography:In this type of chromatographic technique,

the separation of components of a mixture is based on their molecular sizes
which occurs by passing the mixture in solution form (dissolved in mobile
phase) through a porous column packing. The stationary phase for this is gel
and mobile phase is a solvent.

Electrophoresis:In this technique, the separation of constituents of a mixture

depends on the size and speed of the molecules when placed in an electric
field. This process is carried out using moistened filter paper strips between
the ends of which a potential difference is applied.

SAQ 2:
How is paper chromatography different from thin layer chromatography?
...................................................................................................................
...................................................................................................................
...................................................................................................................
...................................................................................................................
...................................................................................................................

29
Separation Techniques
Table 1. List of various important chromatographic techniques and
corresponding stationary phase, mobile phase and mechanism of separation.

Type of chromatography Stationary Mobile Mechanism of

technique phase phase separation

Thin layer chromatography Solid Liquid Adsorption

Paper chromatography Solid Liquid Partition

Gas chromatography Solid or Liquid Carrier Gas Adsorption

Ion-exchange Solid Liquid Ion-exchange

chromatography

Size exclusion Solid Liquid Exclusion

chromatography

Affinity chromatography Solid Liquid Interaction

Gas liquid chromatography Liquid or Solid Liquid Ion-exchange

High-performance Solid Liquid Modified

liquid chromatography partition

Supercritical fluid Solid Supercritical Modified

chromatography fluid partition

Gel filtration Solid Liquid Exclusion

chromatography

Gel permeation Solid Liquid Exclusion

chromatography

Chiral chromatography Solid Liquid Selective

adsorption

2.3 THIN LAYER CHROMATOGRAPHY

Thin layer chromatography (TLC) is a laboratory technique which is widely
used for analyzing the mixtures by separating the components of the mixture.
It is one of the fastest, simplest, inexpensive and sensitive techniques that can
be employed to analyze microgram quantities of the material. The stationary
phase in TLC is a thin layer (~0.15 mm to 2.0 mm thick) of adsorbent such as
silica gel, alumina,or cellulose on a flat and some inert substrate adsorbed on
a sheet of glass, metal foil or plastic and known as TLC plate or chromatoplate.
Commonly, silica gel (SiO2) or aluminum oxide (Al2O3) is used as solid
absorbent to prepare TLC plates. The choice of stationary phase depends on
the nature and properties of the compounds such as acidity, basicity. The mobile
phase includes a volatile pure organic solvent or mixture of solvents; however,
the choice of mobile phase is an important aspect and may require a degree of
trial and error. The principle of TLC is selective adsorption involving the
distribution of compounds between a solid stationary phase and a liquid mobile
phase.
30
Table 2. Few examples of mobile phase system used in TLC. Basic Chromatography

S. Mobile phase Ratio Polar Non-Polar

No. Solvent solvent

1 Hexane 100% - Hexane

2 Hexane:Ethyl acetate 90:10, 80:20, 50:50 Ethyl acetate Hexane

3 Hexane:Diethyl ether 90:10, 80:20 Diethyl ether Hexane

4 Chloroform:Methanol 98:2, 95:5, 90:10 Methanol Chloroform

5 Dichloromethane: 98:2, 95:5, 90:10 Methanol Dichloro-

methanol methane

6 Dichloromethane: 90:10, 80:20 Acetone Dichloro-

Acetone methane

7 Hexane: Chloroform 50:50 Chloroform Hexane

SAQ 3:
Name the two adsorbents commonly used for the reparation of TLC plates.
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

Functioning of TLC
The functioning of TLC majorly involves the following steps: (i) preparation
of TLC plate; (ii) spotting of the samples or mixtures on the plate; (iii)
development of TLC; (iv) visualization of spots on TLC; (v) calculation of
retention factor (Rf value).

Preparation of TLC plate

TLC plates are usually prepared by spreading a mixture of a suitable adsorbent
(like silica gel, alumia) with a small amount of inert binder (calcium sulfate)
and water on an unreactive carrier sheet. The mixture, also called slurry is
spread uniformly on the glass or plastic or thick aluminum foil in the form of
a thin aqueous paste to form a thin layer known as chromaplate. The formed
plate is dried and activated by heating at around 110 oC temperature for half an
hour in an oven. The binder helps in proper adherence of adsorbent onto the
plate and generally the thickness of the adsorbent layer is 0.25 mm.

31
Separation Techniques Spotting of the sample
For the spotting first the samples are dissolved in minimum amount of a volatile
solvent and then diluted to make about 1% solution. Then a small strip of TLC
plate is cut with the help of a knife and a straight line is drawn on one-end of
plate approximately 1 cm from the bottom and point few marks(point should
be far enough away from each other) using a pencil. Thereafter,using a micro
pipet, spots of the solution are applied on the marks and the solvent is allowed
to evaporate. Again, a spot is made at that point (note: repeat the spotting 2-3
times carefullyto get a concentrated spot. Too much spotting must be avoided.
The spot on TLC is thereafter checked using a short-wave UV; a purple spot
can be seen which is the analyzed).

Fig. 1.2: Spoting on TLC

Development of TLC
This process involve the placement of TLC after drying the marked spots of
solution, in a beaker or jar (developing chamber) preoccupied with small
amount of solvent or mixture of solvent (mobile phase). In order to develop a
perfect TLC the jar must be saturated with the vapor of developing solvent
before placing the TLC in it, the spots should not be dipped into the solvent,top
of the jar must be firmly closed and it should not be disturbed after placing the
TLC. As soon as the TLC is placed into the solvent, the solvent moves up the
plate by capillary action. After a very short period of time (within seconds),
the solvent meets with the spots of the mixture and carries them upwards.

Depending on the attraction or binding of components of the mixture with the

stationary phase and solubility in the solvent, different components travel at
different rates. Since the solid phase commonly is silica and it is highly polar,
therefore it holds the polar components very tightly and allows them to move
slowly. On the other hand, non-polar components moves at faster rate because
of their weak binding with stationary phase. When the mobile phase solvent
reaches close of the top of the stationary phase, the TLC plate is removed and
the solvent front is marked with a pencil.

Visualization of spots
The colored compound can be visualizevery easily as spots can be directly
observed or seen after development of TLC. Since most of the compounds are
colorless, these molecules cannot be visualized directly and hence other
32
visualization approaches are needed. The colorless molecules can be visualized Basic Chromatography
by shining ultraviolet (UV) light on the plate or by placing the TLC plate in a
chamber saturated with iodine vapour. Colourless compounds can also be seen
by spraying a reagent that can react with one or more components of the sample
for example, ninhydrin spray is used for the visualization of amino acids; ceric
ammonium nitrate spray can be used for alcohols, etc.

Retention factor (Rf value)

Retention factor provides a useful index for comparing two compounds and
can be defined as the ratio of the distance travelled by the component to that of
total distance traveled by solvent on the stationary phase. The distance travelled
by the pure solvent and the compounds can be measured by drawing
perpendicular lines from the point of spot to the final position of solvent front
or compound on TLC plate. Retention factor can never be greater than one as
compound cannot migrate faster than solvent.

Fig. 1.4: Determination of retention factor

Application of TLC
The common application of TLC are as follows:

1. To monitor the progress of organic chemical reactions and checking the

purity of the samples.

2. To determine the number of components of a mixture.

3. For assaying the purity of organic compounds in photochemistry and

biotechnology.

4. To determine the appropriate conditions for the separation of the

compounds through column chromatography and monitoring the fraction
obtained in column chromatography.

5. For the identification of active substances and their metabolites in biological

matrices, and in the diagnosis of metabolic disorders.

6. To detect the presence of fungicides and pesticides in drinking water.

7. For the qualitative analysis of alkaloids in control phase of both vegetable

drug and pharmaceutical formulation.

33
Separation Techniques
SAQ 4:
During the development of TLC Dr. X observed that the compound travels
0.75 cm on the TLC whereas the solvent front is 1.2 cm away from the
marked spot. What will be the retention factor for his TLC?
....................................................................................................................
....................................................................................................................
....................................................................................................................
....................................................................................................................

2.4 PAPER CHROMATOGRAPHY

Paper chromatography is an analytical technique which runs on a piece of a
specialized paper (Whatmann filter paper) and can be used to separate the
coloured chemicals or substances. The stationary phase for the paper
chromatography is a uniform thin film of liquid (water) adsorbed on an inert
support (cellulose of the Whatmann filter paper)while the mobile phase is also
a liquid (pure solvent or mixture of solvents), known as irrigant.

The separation of components through this technique is based on theprinciple

of partition chromatography wherein the substances are distributed or
partitioned between both the phases. The components get separated because
of their affinity towards water present in the pores of the filter paper (stationary
phase) and mobile phase during the movement of mobile phase because to the
capillary action. On the basis of procedure of development of chromatogram
the paper chromatography is further classified into five types chromatography:
(i) ascending paper chromatography; (ii) descending paper chromatography;
(iii) circular (radial) paper chromatography; (iv) ascending-descending paper
chromatography; (v) 2-dimensonal paper chromatography

Ascending paper chromatography

In ascending chromatography solvent moves upwards along the
chromatographic paper and therefore called as ascending chromatography. In
this technique small spots of the solution containing the components are put
on the paper strip slightly above the edge of paper as in TLC. The paper strip
is placed in a glass jar with the help
of hook in such a way that the
lower edge of the paper strip dips
into the solvent taken in jar but the
spots are above the solvent. When
solvent rises upwards through
capillary action on the filter paper,
the components of the mixture
move up at different rates and get
separated.

34
Descending paper chromatography Basic Chromatography

In this type of technique, solvent moves down

along the paper and therefore called as descending
chromatography. The mobile phase (solvent) is
placed in a boat-shaped container kept at the top
of a covered jar and spotted paper is hanged in jar
taking care that onlypaper end dips in the solvent
but not the spots. When solvent moves down on
the filter paper, the components of the mixture Fig. 1.6: Descending paper
move up at different rates and get separated. chromatography

Circular (radial) paper chromatography

In this chromatographic technique, solvent moves from the centre of circular
Whatmann filter paper towards its circumference horizontally. The sample is
spotted at the centre of a circular filter paper with the help of a fine capillary
and paper is placed on a petri dish (petri dish should be smaller than paper)
containing the solvent. A strip of filter paper is cut from the centre of the filter
paper (or a cotton wick) and dipped in the irrigant. The petri dish is covered
with a glass plate (lid) and solvent is allowed to move on the paper without
any disturbance.

Fig. 1.7: Circular (radial) paper chromatography; (A) Top view and (B) Side view.

Ascending-Descending paper chromatography:

This paper chromatographic
technique is hybrid of ascending
paper chromatography and
descending paper chromato-
graphy. This technique involves
the movement of solvents in two
directions, solvent first travels
upwards then after a particular
Fig. 1.8: Ascending-Descending paper chroma-
point travelsdownwards on the tography.
paper.

2-Dimensonal paper chromatography

The separation of components using this method of chromatography occurs in
two- directions at right angles. The samples are spotted on one corner of a
rectangular paper and paper is placed in a jar filled with small volume of
solvent. The solvent moves upwards very slowly and after few hours the filter
35
Separation Techniques paper is turned at right angle clockwise and the jar is filled with a different
type of solvent to separate the components of the mixture. Unfortunately, if
there is no pronounced separation then the filter paper is again turned at right
angle clockwise and another solvent is used. Substances having same retention
factor (Rf value) can be separated with the help of this technique.

2.5 GAS CHROMATOGRAPHY (GC)

Gas chromatography is a commonly used analytical technique which is applied
to separate and analyse the components of a mixture that can be vaporized
without decomposition. The substances of mixture separates into its constituents

Fig. 1.9: 2-Dimensonal paper chromatography.

as per their interaction with stationary phase by passing a moving gas phase.
Stronger is the interaction between a component and stationary phase, more
time will be required to migrate through the column. The stationary phase is a
solid or liquid in this chromatographic technique and mobile phase is an inert
or unreacted carrier gas such as helium, nitrogen, hydrogen etc. The mechanism
of separation of gas chromatography is based on adsorption process or partition
process and both of these mechanisms are extensively employed in gas
chromatography. On the basis of stationary phases, originally two types of gas
chromatography were described, gas-liquid chromatography (GLC) and gas-
solid chromatography (GSC) and later replaced by a simpler and more
satisfactory term gas chromatography (GC). The instrument used to perform
the gas chromatography is known a gas chromatograph which has the following
main components:

(i) Carrier gas supply system

(ii) Sample injection port
(iii) Column and thermal compartment (oven)
(iv) Detector
(v) Recorder fordata analysis

Fig. 1.10: Gas Chromatograph.

36
(i) Carrier Gas Supply System:A carrier gas should be in highly pure form, Basic Chromatography
inert, cheap and easily available. Usually He, N2, H2 are used as carrier
gases in gas chromatography. Due to high thermal conductivity of helium
relative to vapours of most organic compounds it is preferred when thermal
conductivity detectors are used. The flow of carrier gas from tank is
controlled by regulators and flowmeters; the operating efficiency of the
gas chromatograph depends directly on the maintenance of constant flow
of carrier gas. However, the contamination in the carrier gas may affect
the column performance and detector response.

(ii) Sample Injection Port:Several devices have been developed for

introducing the sample since major applications involve liquid samples
and introduced using a microsyringe. Usually in GC the liquid samples
are injected using a microliter syringe of 1 to 0.5 millilitres (500 micro
litters) capacity. The sample is injected with the help of needle of syringe
through a self-sealing silicone rubber septum into a heated metal block of
the column. The insertion of the sample is the most challenging problem
and insertion, injection and withdrawal of needle should be accomplished
in a proper manner without any loss of the sample. Gaseous samples are
injected in a similar fashion by a gas tight syringe or through by-pass
loops and valves.

(iii) Column and Thermal Compartment (Oven):The actual separation of

sample components is effected in the column. Basically, the
chromatographic column is the heart of the gas chromatography and is
made up of metals or glass. The gas chromatography columns are either
in U-shape or coiled into an open spiral or a flat pan cake type. Various
sizes of columns are used depending upon the requirements. The nature
of stationary phase, type and amount of liquid phase, packing method,
column length and temperature are important parameters in obtaining the
desired resolution. To keep the temperature constant the column is enclosed
in a thermostatically controlled oven. The operating temperature may vary
from ambient temperature to 400 oC and for isothermal operations
temperature is kept constant during the separation process. The oven is
programmed in a way that the temperature of the oven (and column) can
be increased from a chosen initial temperature to a final temperature at a
controlled heating rate in the range of 1oC per minute to even 100 oC per
minute for performing the programed temperature gas chromatography.

(iv) Detector:In gas chromatograph the detector is situated at the exit point of
the separation column. The function of detector is to sense and measure
the amounts of separated components present in the carrier gas stream
leaving the column and provide a signal. The choice of detector and
intensity of signals will depend on the concentration, mass and nature of
the separated components. The main detectors mostly used in gas
chromatography are the flame ionization detector (FID), electron capture
detector (ECD), and thermal conductivity detector (TCD).

(v) Recorder for Data Analysis: Final, the data is recorded in a computer for
the analysis.
37
Separation Techniques Gas chromatography has found the extensive applications in the following
fields

9 In pharmaceutical industry GC is used for the separation of compounds in

complex mixture based on the polarity, to analyse the residual solvents in
row material and products.
9 In food industry it is used for the quantity and quality analysis of food
composition, food additives, flavor and aroma components, natural
products and contaminates which includes pesticides, environmental
pollutants, natural toxins etc.
9 GC is also used in determination of product contents, purity, monitoring
production processes in a chemical industry. The other application includes
natural gas analysis, gasoline characterization and fraction quantitation,
quantification of drugs and their metabolites in blood and urine.

SAQ 5
Why helium gas is preferred in gas chromatograph when thermal
conductivity detectors are used?
....................................................................................................................
....................................................................................................................
....................................................................................................................
....................................................................................................................

2.6 ION EXCHANGE CHROMATOGRAPHY

(IEC)
Ion-exchange chromatography is a part of ion chromatography in which
separation depends upon the reversible adsorption of charged molecules to
immobilized ion exchange groups of opposite charge fixed to the solid phase
(ion-exchange material) without any permanent change in the structure of the
molecules. Electrostatic interactions between ionic and polar molecules present
in the liquid phase (eluent) and ionic functional groups present on
chromatographic supportas well as repulsion between similarly charged ions
play an important role in the separation using IEC. Most of the ion exchange
processes involve five main stages.First stage involves the equilibration of
appropriate ion exchange resin to starting state in terms of pH and ionic
strength.Second stage is the insertion of sample and adsorption process, where
molecules present in solute displace counter-ions and bind reversibly to the
resin and unbound substances are washed out. In third stage,components of
the mixture are removed from the column by altering the undesirable elution
conditions such as increase in ionic strength of the eluting buffer or change in
its pH for ionic bonding of solute molecules. The next two stages, fourth and
fifth stages are the removal of substances from the column (not eluent) under
the previous experimental conditions and re-equilibration of the ion exchange
resin for the next purification cycle.
38
Ion exchange resin:Ion exchange resin is a high molecular weight, cross-linked, Basic Chromatography
insoluble, long chain organic polymers with porous structure. The covalently
bonded characteristic functional groups present on the polymeric chain are
responsible for ion exchange process. Usuallypolymer base of most of the
resins consist of styrene-divinylbenzene copolymer. Other polymers such as
phenol-formaldehyde polymers or methacrylic acid-divinylbenzene are also
used. An ideal and strong ion exchange resin has the following properties:

9 The sample loading capacity of the resin does not decrease with the change
in the pH values due to loss of its charge.

9 The mechanism of interaction between resin and solute is very simple.

9 Since the charge characteristics of the media do not change with the changes
in pH therefore ion exchange experiments are more controllable.

The functional groups attached to the resin may be acidic or basic and therefore
can be classified as cation exchange resins and anion exchange resins.

Cation Exchange Resins:Cation exchange resins are those resins which are
capable of exchanging the cation of the solutions with their H+ ions. These are
usually styrene-divinylbenzene copolymers containing acidic groups like
carboxylic group (COOH) or sulphonic group (SO3H). Various synthetic cation
exchanger resins which contain sulphonated phenolic or aromatic hydrocarbons
have been prepared and known as H-form cation exchanger. These H-form
cation exchangers can be converted to Na-form exchangers with the help of
sodium chloride (NaCl). The cation exchange resins can be represented as
RH+.

Fig.1.11: Cation exchange resin representation.

Resins with carboxylic group (COOH) are known as weakly acidic cation
exchange resins whereas resins containing sulphonic group (SO3H) are termed
as strongly acidic resins. When a cation exchange resin is immersed in an
aqueous solution containing positively charged ions (Mn+) the following
exchange equilibrium is quickly established.

39
Separation Techniques Anion Exchange Resins:Resins bearing basic functional groups such as amine,
substituted amine or quaternary ammonium groups as their hydroxide salts
are known as anion exchange resins. Anion exchange resins are capable of
exchanging the anion present in the solutions with their anions like hydroxide
ions (-OH).These resins are represented as R(-OH).

Fig.1.12: Anion exchange resin representation.

Resins having quaternary ammonium salts are strongly basic whereas resins
having amine (-NH2) or imine (=NH) group are weakly basic anion exchange
resins. When an anion exchange resin is immersed in an aqueous solution
containing negatively charged ions (An-) the following exchange equilibrium
is quickly established.

The ion-exchange chromatography has the following important applications:

9 It is used for the analysis of amino acids and to determine the base
composition of nucleic acids.

9 It is one of the most effective techniques used for water softening.

9 This chromatographic technique is also used for the separation of proteins,

vitamins and other biological amines, organic acids and bases.

The separation of substances using IEC is obtained because different substances

have different ability to interact with the ion exchanger due to differences in
their charges, charge densities and distribution of charge on their surface which
can be controlled by varying ionic strength and pH of solute. Ion-exchange
separation may be carried out in a column or by a batch process or by expanded
bed adsorption.

2.7 SIZE EXCLUSION CHROMATOGRAPHY (SEC)

Size exclusion chromatography was developed in 1955 by Lathe and Ruthven.
Thisis a technique which separates the components according to the difference
in their size, shape and in some cases molecular weight. The stationary phase
in size exclusion chromatography is made up of silica (~ 10 mM in size) or
consists of polymer particles holding a network of uniform pores into which
solute and solvent molecules can diffuse.
40
Molecules with the diameter larger than the largest pores of stationary phase Basic Chromatography
are unable to enter the particles. Therefore these molecules are able to access
the smallest volume within the column and elute first. Whereas, the molecules
smaller than the pores of resin passes through the large accessible volume
within the column anddepending upon their size eluted later as per their
retention time in column and decrease in size and molecular weight. Smaller
molecules are more strongly retained and are last to eluted. This technique is
commonly used for the separation of large molecules or macromolecules such
as proteins, biopolymers and synthetic polymers etc.

Size Exclusion chromatography can be categorized into two types: (i) Gel
filtration chromatography (GFC), (ii) Gel permeation chromatography (GPC).

Gel filtration chromatography

The chromatography based on hydrophilic packing in column is called gel
filtration chromatography and used to separate polar species such as salts.
This technique is also used for the rapid removal of reagents to terminate a
reaction and also for the buffer exchange before and after different
chromatography techniques.

Gel permeation chromatography

The chromatography based on hydrophobic packing in column is called gel
permeation chromatography and used to separate non-polar species. This
technique is often used for the characterization of polymers, to find the relative
molecular weight of polymer and distribution of molecular weights.

2.8 AFFINITY CHROMATOGRAPHY

Affinity chromatography is a widely applicable separating technique used for
the separation of biochemical mixture. First time this technique was used by
Lerman in 1953 during the isolation of enzymesin. Later onin 1968, this
technique was uncovered by Pedro Cuatecasa, Chris Anfinsen and Meir
Wilchek in an article that briefly described an enzyme purification technique
through an immobilized substrates and inhibitors. In this technique, the
separation of the components is based on the reversible interaction between
the proteins need to be purified and the affinity ligand immobilized or coupled
to a solid support. Most of the proteins have an intrinsic recognition spotby
which proteins interact selectively to a specific affinity ligand in reversible
manner. Therefore, it can be stated that affinity chromatography exploits the
differences in the interaction strength between the various biomolecules within
a mobile phase and the stationary phase and, principally is based on the
molecular recognition of a target molecule by a molecule bound to a column
(stationary phase). Affinity chromatography is a unique refining technique
since it separates the biomolecules on the basis of their individual chemical
structure orbiological function.

Purification of biomolecules using affinity chromatography involves mainly

the following three steps:
41
Separation Techniques (i) Development of a crude sample with the solid support to allow the target
molecule in the sample to bind to the immobilized ligand.

(ii) Washing of non-bound sample components from the support.

(iii) Elution (dissociation and recovery) of the target molecule from the
immobilized ligand by changing the buffer conditions to avoid the further
binding interaction.

Using this chromatographic technique, significant time savings and several

hundred-fold or higher purification can be achieved. Since success may vary
depending on the used method therefore it is important to optimize the
purification process to attain maximum recovery of the purified material.
Moreover, purification that would be time-consuming or impossible with other
techniques can be easily accomplished with affinity chromatography. Affinity
chromatography has played an essential role in many “Omics” technologies
such as genomics, metabolomics and proteomics and is still developing. The
technique can be used successfully to separate active biomolecules from
denatured or functionality different forms, to remove specific contaminants
and also to isolate pure substances present at low concentration in large volume
of crude samples.

SAQ 6
Why the proteins are able to interact selectively with a specific ligand?
...................................................................................................................
...................................................................................................................
...................................................................................................................
...................................................................................................................

2.9 LET US SUM UP

In view of the significance of the chromatographic techniques in separation,
identification and purification of mixtures, it has been extensively employed
in industries especially in the food industry for analyzing additives, vitamins,
preservatives, proteins and amino acids. Initially, chromatography was used
for the separation of components of herbal pigments based on their colours.
Later on, it got acceptance for widespread applications. Primarily, three
components form the basis of this technique which include (i) stationary phase
comprising of a solid phase or a layer of a liquid adsorbed on the surface a
solid support (ii) mobile phase which is composed of a “liquid” or a “gaseous
component” (iii) separated molecules. The type of interaction between these
three components would govern the effective separation of the components of
a mixture. Till now, various chromatographic techniques have been developed
including column chromatography, thin-layer chromatography (TLC), paper
chromatography, gas chromatography, ion exchange chromatography, gel
permeation chromatography, size exclusion chromatography and affinity
chromatography etc. The need for increasing adoption of these techniques in
environmental analyses, polymer characterizations, toxicological investigations,
42
chemical syntheses, pharmaceutical etc. can be realized considering their Basic Chromatography
advantages of increased resolution, molecular structure elucidation, sensitivity,
and separation power, in addition to decreased analysis times and detection
limits. Currently, efforts are being devoted towards using hyphenated techniques
that is coupling chromatography with other methods for obtaining better results
and hence future of chromatography is promising.

2.10 TERMINAL QUESTIONS

1. Why chromatography is considered better separating technique than other
conventional methods?

2. Classify the chromatographic techniques on the basis of nature and physical

state of mobile phase and stationary phase.

3. How does the TLC work? Explain its applications.

4. How will you distinguish descending paper chromatography from

ascending paper chromatography?

5. Explain the various applications of gas chromatography.

6. What is the ion exchange resin?

7. Why the larger molecules is get eluted before small molecules in size
exclusion chromatography?

ANSWERS
Self-Assessment Questions

1. Stationary phase is a solid adsorbent which adsorbed the components of

the mixture and mobile phase is a pure solvent or gas or mixture of solvents
which transports the components on stationary phase while passing through
it.

2. The stationary phase in paper chromatography is water absorbed on

cellulose of the Whatmann filter paper whereas the stationary phase in
TLC technique is a thin layer of a solid adsorbent.

3. Silica gel (SiO2) and aluminum oxide (Al2O3).

4. Retention factor is 0.62.

5. Due to high thermal conductivity of helium relative to vapours of most

organic compounds it is preferred when thermal conductivity detectors
are used

TERMINAL QUESTIONS

1. Chromatography is considered better separating technique than other

conventional methods such as crystallization, distillation, sublimation
chromatography because using chromatography compounds in mg to kg
range can be easily purified without any loss in properties, each component
43
Separation Techniques of a mixture can be isolated separately in pure form which is usually
difficult by other methods, minimum loss of compound, etc.

2. On the basis of nature and physical state of mobile phase and stationary
phase the chromatography can be classified as follows:

3. The working of TLC majorly involves the following steps: (i) preparation
of TLC plate; (ii) spotting of the samples or mixtures on the plate; (iii)
development of TLC; (iv) visualization of spots on TLC; (v) calculation
of retention factor (Rf value).

The common application of TLC are as follows:

1. To monitor the progress of organic chemical reactions and checking the

purity of the samples.

2. To determine the number of components of a mixture.

3. For assaying the purity of organic compounds in photochemistry and

biotechnology.

4. To determine the appropriate conditions for the separation of the

compounds through column chromatography and monitoring the fraction
obtained in column chromatography.

5. For the identification of active substances and their metabolites in biological

matrices, and in the diagnosis of metabolic disorders.

6. To detect the presence of fungicides and pesticides in drinking water.

7. For the qualitative analysis of alkaloids in control phase of both vegetable

drug and pharmaceutical formulation.

4. Descending chromatography is a technique in which solvent moves down

along the paper and therefore called as descending chromatography. The
mobile phase (solvent) is placed in a boat-shaped container kept at the top
of a covered jar and spotted paper is hanged in jar taking care that only
paper end dips in the solvent but not the spots. When solvent moves down
on the filter paper, the components of the mixture move up at different
rates and get separated.
44
 In ascending chromatography solvent moves upwards along the Basic Chromatography
chromatographic paper and therefore called as ascending chromatography.
In this technique small spots of the solution containing the components
are put on the paper strip slightly above the edge of paper as in TLC. The
paper strip is placed in a glass jar with the help of hook in such a way that
the lower edge of the paper strip dips into the solvent taken in jar but the
spots are above the solvent. When solvent rises up by capillary action on
the filter paper, the components of the mixture move up at different rates
and get separated.

5. Gas chromatography has found the extensive applications in the following

fields

9 In pharmaceutical industry GC is used for the separation of compounds

in complex mixture based on the polarity, to analyse the residual
solvents in row material and products.

9 In food industry it is used for the quantity and quality analysis of food
composition, food additives, flavor and aroma components, natural
products and contaminates which includes pesticides, environmental
pollutants, natural toxins etc.

9 GC is also used in determination of product contents, purity, monitoring

production processes in a chemical industry. The other application
includes natural gas analysis, gasoline characterization and fraction
quantitation, quantification of drugs and their metabolites in blood
and urine.

6. Ion exchange resin is a high molecular weight, cross-linked, insoluble,

long chain organic polymers with porous structure. The covalently bonded
characteristic functional groups present on the polymeric chain are
responsible for ion exchange process. Usually polymer base of most of
the resins consist of styrene-divinylbenzene copolymer. The functional
groups attached to the resin may be acidic or basic and therefore can be
classified as cation exchange resins and anion exchange resins.

7. Molecules with the diameter larger than the largest pores of stationary
phase are unable to enter the particles. Therefore these molecules are able
to access the smallest volume within the column and elute first. Whereas,
the molecules smaller than the pores of resin passes through the large
accessible volume within the column and depending upon their size eluted
later as per their retention time in column and decrease in size and molecular
weight. Smaller molecules are more strongly retained and are last to eluted.

45