Basic Principles of Chromatography

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 Introduction
 Chromatography is combination of two Greek words chroma
"color" and graphein "to write“
 Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one
of which is stationary (stationary phase) while the other (the mobile
phase) moves in a definite direction.
 It is the collective term for a set of laboratory techniques for the
separation of mixtures
 It involves passing a mixture dissolved in a "mobile phase" through a
stationary phase, which separates the analyte to be measured from
other molecules in the mixture based on differential partitioning
between the mobile and stationary phases
 The differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus changing the
separation

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 Introduction
Chromatography terms
 The analyte is the substance to be separated during
chromatography
 A bonded phase is a stationary phase that is covalently bonded to
the support particles or to the inside wall of the column tubing
 A chromatogram is the visual output of the chromatograph. In
the case of an optimal separation, different peaks or patterns on
the chromatogram correspond to different components of the
separated mixture

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 Introduction
Chromatography terms
 The eluate is the mobile phase (solvent) leaving the column and
carry the analyte
 The mobile phase is the phase which moves in a definite
direction
 It may be a liquid (LC and CEC), a gas (GC), or a supercritical
fluid (supercritical-fluid chromatography, SFC)
 The mobile phase consists of the sample being separated/analyzed
and the solvent that moves the sample through the column
 In the case of HPLC the mobile phase consists of a non-polar
solvent e.g. hexane in normal phase or polar solvents in reverse
phase chromotagraphy
 The mobile phase moves through the chromatography column
(the stationary phase) where the sample interacts with the
stationary phase and is separated

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 Introduction
Chromatography terms
 The retention time is the characteristic time it takes for a
particular analyte to pass through the system (from the column
inlet to the detector) under set conditions
 The solute refers to the sample components in partition
chromatography.
 The solvent refers to any substance capable of solubilizing other
substance, and especially the liquid mobile phase in LC.
 An immobilized phase is a stationary phase which is
immobilized on the support particles, or on the inner wall of the
column tubing
 The stationary phase is the substance which is fixed in place for
the chromatography procedure e.g. silica layer in thin layer
chromatography

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Extraction
 Extraction is basic aspect of chromatography
 It refers to the transfer of a solute from one liquid phase to
another
 Extraction may be carried out by means of batch , continuous,
or countercurrent processes.

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 Extraction…
1. Batch Extraction
 The solute is extracted from one solvent by shaking it with a second,
immiscible solvent
 When equilibrium has been reached, the partition coefficient, K, is a
constant
2. Continuous Extraction
 Continuous liquid-liquid extraction requires special apparatus, but is more
efficient than batch separation
 One example is the use of a Soxhlet extractor for extracting materials from
solids
3. Countercurrent Extraction
 It refers to a serial extraction process
 It separates two or more solutes with different partition coefficients from
each other by a series of partitions between two immiscible liquid phases

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 Chromatography
 Historical Perspective
 Modem chromatography originated in the late nineteenth
and early twentieth centuries from independent work by
David T. Day a distinguished American geologist and
mining engineer, and Mikhail Tsvet a Ressian botanist
 The first publication on gas chromatography (GC)
appeared in 1952
 By the late 1960s GC developed into a sophisticated
instrumental technique
 Supercritical fluid chromatography (SFC), first
demonstrated in 1962, is finally gaining popularity

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Characteristics of different
chromatographic method

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 Basic principle Of
Chromatographic Process:
 It is based on the differential migration of the individual
components of a mixture through a stationary phase under the
influence of a moving phase.

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 Chromatography…
 Chromatographic techniques based on
Physicochemical Principles of Separation
1. Adsorption (Solid-Liquid) Chromatography

2. Partition (Liquid-Liquid) Chromatography

3. Ion Exchange Chromatography

4. Size Exclusion Chromatography

5. Affinity Chromatography

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1. Adsorption (Solid-Liquid) Chromatography
 Adsorption Chromatography is the oldest form of
chr0matography used by Tsvet in 1903 in the experiments that
eslablished modem chromatography
 In this chromatographic mode, the stationary phase is a finely
divided solid (to maximize the surface area) and the mobile
phase may be either a gas or a liquid
 The stationary phase (adsorbent) is chosen to permit
differential interaction with the components of the sample to
be resolved
 The intermolecular forces thought to be primarily responsible
for chromatographic adsorption include
 Vander waals forces □ Hydrogen bonds

 Electrostatic forces □ Hydrophobic interactions

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 1. Adsorption (Solid-Liquid) Chromatography
Application
1. Adsorption chromatography can be used in separation of
aromatic or aliphatic non polar compounds, such as lipids,
primarily according to the type and number of functional groups
present
2. The labile, fat-soluble chlorophyll and carotenoid pigments
from plants can be studied extensively by adsorption
chromatography
3. Adsorption chromatography also has been used for the analysis
of fat-soluble vitamins
4. Frequently, it is used as a batch procedure for removal of
impurities from samples prior to other analyses

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1. Adsorption (Solid-Liquid) Chromatography

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 2. Partition (Liquid-Liquid)
Chromatography
 In 1941, Martin and Svnge undertook an investigation of the
amino acid composition using a countercurrent extractor with
chloroform and water flowing in opposite directions
 The efficiency of the extraction process was improved
enormously when a column of finely divided inert support
material was used to hold one liquid phase (stationary phase)
immobile, while the second liquid, an immiscible solvent
(mobile phase) flowed over it, thus providing intimate contact
between the two phases
 Solutes partitioned between the two liquid phases takes place
according to their partition coefficients

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 2. Partition (Liquid-Liquid)
Chromatography
 A partition system is manipulated by changing
 The nature of the two liquid phases, usually by

combination of solvents
 pH adjustment of buffers

 Often, the more polar of the two liquids is held stationary

on the inert support and the less polar solvent is used to
elute the sample components
 Reversal of this arrangement, using a non polar stationary
phase and a polar mobile phase, has come to be known as
reversed-phase chromatography

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2. Partition (Liquid-Liquid)
Chromatography

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 3. Ion Exchange Chromatography
 IE is a separation/purification process occurring naturally
e.g. in soils and is utilized in water softener and de ionizers
 Three types of separation is achieved
1. Ionic from non-ionic
2. Cationic from an-ionic
3. Mixtures of similarly charged species
 It is a type of adsorption chromatography in which
interaction between solute and stationary phase primarily
electrostatic in nature
 The stationary phase contains fixes functional groups that are
either negatively or positively charged
 Cation exchangers contain covalently bound negatively
charged functional groups, where as
 Anion exchangers contain bound positively charged groups
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 3. Ion Exchange Chromatography…
Food-related applications of ion-exchange chromatography
 The separation of amino acids, sugars, alkaloids, and proteins
 Fractionation of amino acids in protein was initially carried
out by ion-exchange chromatography; automation of this
process led to the development of commercially produced
amino add analyzers
 Sugars, complexed with borate to form negatively charged
species can be separated on columns of strong anion
exchange resin in the borate form
 Many drugs, fatty acids, and the acids of fruit, can be
chromatographed in the ion-exchange mode

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 4. Size Exclusion Chromatography
 SEC is also known as molecular exclusion, gel
permeation and gel filtration chromatography
 In SEC, molecules are separated solely on the basis of
their size
 The stationary phase in SEC consists of column packing
material that contains pores
 Solutes travel with the mobile phase between the
particles out side the pores and largest molecules are
eluted first from an SEC column
 It is widely used in
 Biological sciences for the resolution of macromolecule, such
as protein and carbohydrates
 The fractionation and characterization of synthetic polymers

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4. Size Exclusion Chromatography

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 5. Affinity Chromatography

 In AC separation is based on the specific, reversible interaction

between a solute molecule and a ligand immobilized on the
chromatographic stationary phase
 It is the ultimate extension of adsorption chromatography and
termed as biospecific adsorption
 AC usually involves immobilized biological material as the
stationary phase e.g. antibodies, enzyme inhibitor, lectins etc.
that selectively and reversibly bind to complimentary analyte
molecule in the sample
 AC also encompasses the separation of small molecules

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 5. Affinity Chromatography…
 A ligand chosen because of the specificity and strength of
interaction between itself and the molecule to be isolated
(analyte) is immobilized on a suitable support material
 As the sample is passed through this column, molecules that
are complementary to the bound ligand are adsorbed while
other sample components are eluted
 In addition to protein purification, affinity chromatography
may be used to separate supramolecular structures such as
cells, organelles, and viruses
 It has been useful especially in the separation and purification
of enzymes and glycoproteins
 In the case of the latter, carbohydrate derivatized adsorbents
are used to isolate specific lectins, such as concanavalin A,
and lentil or wheat-germ lectin

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Scheme for subdividing the field of chromatography,
according to various applied techniques

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Chromatographic Techniques

 Paper Chromatography (PC)

 Thin layer Chromatography (TLC)
 Column Liquid Chromatography (CLC)
 Supercritical Fluid Chromatography (SFC)
 High Performance Liquid Chromatography
(HPLC)
 Gas Chromatography (GC)

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 Paper Chromatography

 PC was introduced in 1944

 Although adsorption by the
paper itself has been utilized,
paper generally serves only as
a support for the liquid
stationary phase.
 Stationary phase in PC is
usually water

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 Thin Layer Chromatography
 TLC, first introduced in 1938 has
largely replaced paper
chromatography (PC) because,
this technique is
 faster

 more sensitive

 more reproducible

 The resolution is TLC is greater

than in PC because the particles
on the plate are smaller and more
regular than paper fibers

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TLC…

 TLC utilizes a thin layer of stationary phase (sorbent)

bound to an inert support in a planar configuration
 The support is often a glass plate but plastic sheets
and aluminum foil also used
 Four frequently used sorbents are silica gel, alumina,
diatomaceous earth and cellulose

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 Factors Affecting Thin-Layer Separations. In
 both planar and column liquid
chromatography, the nature of the
compounds to be separated determines what
type of stationary phase
 is used. Separation can occur by adsorption,
 partition, ion-exchange, size-exclusion, or
multiple mechanisms.

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Thin-layer chromatography sorbents
and mode of separation

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Application of TLC
 In food industry, TLC may be used for quality control.
e.g.
 Corn and peanuts are tested for aflatoxins /mycotoxin prior
to their processing into corn meal and peanut butter,
respectively.
 TLC is also applied in many fields including
environmental, clinical, pharmaceutical, food, flavors
and cosmetics
 Also used for analysis of variety of compounds
including lipids, carbohydrates, vitamins, amino acids
and natural pigments.

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Column Liquid Chromatography
 CLC is the most useful method of separating
compound in a mixture.
 Stationary phase hydrated and preswelled in
mobile phase is packed in glass column
 As sorbent settles, excess solvent is drained
off and additional slurry is added.
 Sample to be fractionated, dissolved in a
minimum volume of mobile phase is applied
in layer at the top of column.

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Column Liquid
Chromatography…

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Supercritical Fluid Chromatography
 A supercritical fluid is one that is above both its critical
pressure and critical temperature.
 Supercritical fluid can be formed from
 a conventional gas by increasing a pressure

 a conventional liquid by raising temperature

 CO2 is frequently used as a mobile phase for SFC

 Other supercritical fluids that have been used in food
applications include
 nitrous oxide,

 trifluoro-methane,

 sulphur hexafluoride,

 pentane

 ammonia

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Supercritical Fluid
Chromatography…
 SFC can be performed using either packed column or

capillaries
 In packed column, small particle, porous, high surface area,
hydrated silica may serve as stationary phase itself.
 Capillaries are generally coated with a polysiloxane film,
which is then cross linked to form a polymeric stationary
phase that can’t be washed off by the mobile phase.

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Supercritical Fluid
Chromatography…

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