CHROMATOGRAP

HY
DR. ANDREW.A.LAMARE
2ND YEAR PG
CHROMATOGRAPHY
• It is a technique used to separate and identify the components of a
mixture.

• Works by allowing the molecules present in the mixture to distribute

themselves between a stationary and a mobile medium.

• Chromatography is derived from two Greek words Chroma meaning

colour and Graphein meaning to write
PURPOSE OF CHROMATOGRAPHY
• ANALYTICAL

 Determine Chemical composition of a sample

• PREPARATIVE

 Used to purify sufficient quantities of a substance

TSWETT EXPERIMENT
• Tall glass open column filled with sand-like
particles

• Ground-up plant extracts

• Poured into the column and saw coloured

“bands” develop as the extract percolated
down through the column
CHROMATOGRAPHY TERMS
• CHROMATOGRAM - equipment that enables a sophisticated
separation.
e.g. Gas Chromatography/ Liquid Chromatography
• ELUENT - Fluid entering column/ solvent that carries the analyte.
• ELUATE - Mobile phase leaving the column
• STATIONARY PHASE – IMMOBILIZED PHASE
Immobilized on the support particles or on the inner wall of the
column tubing
e.g. Silica layer – Thin Layer Chromatography
CHROMATOGRAPHY TERMS
• MOBILE PHASE – Moves in a defined direction. Liquid (LC), Gas (GC).
The mobile phase moves through the chromatography column (the
stationary phase) where the sample interacts with the stationary
phase and is separated.
• RETENTION TIME – Time taken for a particular analyte to pass
through the system (from the column inlet to the detector) under set
conditions.
• SAMPLE (ANALYTE) – Substance analysed in chromatography
• SOLVENT – Any substance capable of solubilizing another substance.
CHROMATOGRAM
• Visual output of the chromatograph
• Separation – Different peaks or patterns on the chromatogram
corresponds to different components of the separated mixture.
• X-axis: Retention
time
• Y-axis: Signal
• Signal is
proportional to the
concentration of the
specific analyte
separated.
CHROMATOGRAPHY - PRINCIPLE
• It is based on the principle of the partition of the solute between two
phases/solvents. Chromatography usually consists of a mobile phase and a
stationary phase.
• Mobile phase usually refers to the mixture of the substances to be separated
dissolved in a liquid or a gas.
• Stationary phase is a porous solid matrix through which the sample
contained in the mobile phase percolates. The interaction between the
mobile and the stationary phases results in the separation of the
compounds from the mixture.
• These interactions include the physio- chemical principles such as the
adsorption, ion-exchange, molecular sieving and affinity.
CHROMATOGRAPHY
CLASSIFICATION
• Based on nature of stationary phase or mobile phase.
1. PLANAR – It may be PAPER or THIN LAYER
2. COLUMNAR – It may be GAS or LIQUID

Planar chromatography is a separation technique in which the

stationary phase is present as or on a plane.

Column chromatography is a separation technique in which the

stationary bed is within a tube.
• Based on interaction between sample component and stationary
phase:

i. PARTITION
ii. ADSORPTION
iii. ION-EXCHANGE
iv. GEL-FILTRATION
v. AFFINITY
vi. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
PARTITION CHROMATOGRAPHY
• It is used for the separation of mixture of amino acids and peptides.
The molecules of a mixture get partitioned between the stationary
and the mobile phase depending on the relative affinity of each one
of the phases.

• It is undertaken in two ways:

1) PAPER CHROMATOGRAPHY
2) THIN LAYER CHROMATOGRAPHY (TLC)
PAPER CHROMATOGRAPHY
• An analytical technique for the separation and identifying mixtures
that are either coloured or can be made coloured

• It is a liquid partition chromatography

• Used for separation of amino acids, sugars, sugar derivatives and

peptides
Cont…..
• The stationary phase is water held on a solid support of filter paper
(cellulose)

• The mobile phase is a mixture of immiscible solvents which are

mixtures of water, a non polar solvent and an acid or base.

E.g. Butanol, acetic acid water or phenol-water-ammonia.

Procedure
1) A small spot of sample is applied to a strip of chromatography paper
about 2cm away from the base of the plate. This sample is absorbed
onto the paper and may form interactions with it.
2) The paper is then dipped in the a suitable solvent, such as ethanol
or water, taking care that the spot is above the surface of the
solvent, and placed in a sealed container.
3) The solvent moves up the paper by capillary action and dissolves
the sample mixture, which will then travel up with the solvent
solute sample.
Cont….
• Different compounds in the
sample mixture travel at different
rates due to differences in
solubility in the solvent, and due
to differences in their attraction
to the fibres in the paper.

• Paper chromatography takes

anywhere from several minutes
to several hours.
ASCENDING AND DESCENDING
PAPER CHROMATOGRAPHY
• ASCENDING CHROMATOGRAPHY
In this method, the solvent is in pool at the
bottom of the vessel which the paper is
supported. It rises up the paper by capillary
action against the force of gravity.
• DESCENDING CHROMATOGRAPHY
In this method, the solvent is kept in a trough
at the top of the chamber and is allowed to flow
down the paper. The liquid moves down by
capillary action as well as by the gravitational
force.
ANALYSIS
• After development, the spots corresponding to different compounds
may be located by their colour, ultraviolet light, ninhydrin or by
treatment with iodine vapours.
• The paper remaining after the experiment is known as the
Chromatogram.
• The components which have been separated differ in their Retention
Factor i.e. Ratio of distance travelled from the spot or origin by the
solute component to that of the distance travelled from the spot or
origin by the solvent.
Rf value
• If Rf value of a solution is 0,
the solute remains in the
stationary phase and thus it
is immobile.
• If the Rf value = 1, then the
solute has no affinity for
the stationary phase and
travels with the solvent
front.
TWO DIMENSIONAL
CHROMATOGRAPHY
• Sometimes it is difficult to separate a
complex mixture of substrates by a
single run with one solvent system. In
such a case, a second run is carried out
by a different solvent system, in a
direction perpendicular to the first run.
• This is referred to as a two dimensional
chromatography which enhances the
separation of a mixture in to individual
components.
SIGNIFICANCE OF PAPER
CHROMATOGRAPHY
1. Very easy, simple, rapid and highly efficient method
2. Can be applied even in microgram quantities of the sample
3. Can be also used for the separation of a wide variety of materials
like amino acids, oligopeptides, sugars, oligosaccharides, glycosides,
purines and pyrimidines, steroids, vitamins and some alkaloids like
penicillin, tetra cyclin and streptomycin
4. Not preferred for separating proteins because they are not soluble
in many of the solvent systems and are denatured by them.
5. Paper chromatography is inferior to TLC in resolving power.
 THIN LAYER CHROMATOGRAPHY
(TLC)
• Technique use to separate mixtures

• It involves a stationary phase consisting of a thin layer of adsorbent

material, usually silica gel, aluminium oxide or cellulose immobilized
onto a flat, inert carrier sheet.

• A liquid phase consisting of the solution to be separated which is

dissolved in an appropriate solvent and is drawn up the plate via
capillary action, separating the solution based on the polarity of the
components of the compound in question.
PROCEDURE
• Similar to paper chromatography with the advantage of faster runs,
better separations, and the choice between different stationary
phases

• A small spot of solution containing the sample is applied to the plate,

about 1cm from the base. The plate is then dipped in to a suitable
solvent, such as hexane or ethyl acetate, and placed in a sealed
container. The solvent moves up the plate by capillary action and
meets the sample mixture, which is dissolved and is carried up the
plate by the solvent.
TLC OVERVIEW
SIGNIFICANCE OF TLC
• Its wide range of uses include

 determination of the pigments a plant contains

Detection of pesticides or insecticides in food

Identifying compounds present in a given substrate

Monitoring organic reaction

ADVANTAGES OF TLC OVER PAPER
CHROMATOGRAPHY
1. It takes a shorter time for TLC to complete a run as compared to
paper chromatography
2. TLC can utilise corrosive reagents like sulphuric acid which pose a
limitation for paper chromatography
3. Easier to separate and visualise the components by this method
4. Capacity to analyse multiple samples in a single run
5. Relatively low cost
ADSORPTION CHROMATOGRAPHY
• In this technique the separation is based on differences in adsorption
at the surface of the solid stationary medium
• Adsorbents such as silica gel, charcoal powder and calcium
hydroxyapatite are packed in to a column in a glass tube. This serves
as the stationary phase
• The sample mixture in a solvent is loaded on this column
• The individual components get differentially adsorbed on to the
adsorbent
Adsorption chromatography- Elution
• The elution is carried out by a buffer system (mobile phase). The most
weakly held fraction moves fastest, followed by others, according to
the order of tightness in adsorption
• The individual compounds come out of the column at different rates
which may be separately collected and identified.
• Amino acids may be identified by Ninhydrin colorimetric method
• An automated column chromatography apparatus- fraction collector
is frequently used now a days
ION – EXCHANGE
CHROMATOGRAPHY
• It is a process that allows the separation of ions and polar molecules
based on the charge properties of the molecules
• It can be used for almost any kind of charged molecule including large
proteins, small nucleotides and amino acids
• The solution to be injected is usually called a sample, and the
individually separated components are called analytes
• It is often used in protein purification, water analysis and quality
control
• Ion-exchange chromatography retains molecules on coulombic (ionic)
interactions. The stationary phase surface displays ionic functional
groups that interact with analyte ions of opposite charge

• It is further subdivided into Cation exchange chromatography and

Anion exchange chromatography
i. Cation exchange retains Positively charged cations because the
stationary phase displays a negatively charged functional group
ii. Anion exchange retains Negatively charged anions using positively
charged functional group
Procedure
• A mixture of amino acids or proteins can be conveniently separated
by this method. The amino acid mixture (pH 3.0) is passed through a
cation exchanger and the individual amino acid can be eluted by using
buffers of different pH.

• Suppose a mixture of Arginine and Aspartic acid is passed through a

cation exchange column. Arginine has extra positive charge and so
adheres to the column. But negatively charged Aspartic acid
molecules will not adhere and come out first from the column
• When a weak NaOH is passed, the positively charge of Arginine is
neutralised. Na+ will replace Arginine in the column, thus Arginine is
eluted finally.

• The various fractions eluted, containing individual amino acids, are

allowed to react with Ninhydrin reagent to form coloured complexes

• This is continuously monitored for qualitative and quantitively

identification of amino acids.
TYPES OF ION EXCHANGRES RESINS
• CATION EXCHANGE RESINS- Polystyrene sulfonate resin, CM-
Sephadex gel, CM- cellulose.
These bear acidic groups and immobilise cations from adjacent
solutions

• ANION EXCHANGE RESINS- DEAE cellulose, Trimethyl amino

Polystyrene, DEAE- Sephadex.
All these bear basic groups ionizing into fixed positions and immobilize
anions from neighbouring solutions
GEL FILTRATION
CHROMATOGRAPHY/ MOLECULAR
SIEVE CHROMATOGRAPHY
• This is extremely useful in separating ribosomes, viruses, nucleic acids
and proteins depending on their particle sizes and shapes
• In Gel Filtration Chromatography, the separation of the particles is
based on their size, shape and molecular weight
• The apparatus consist of a column packed with sponge like gel beads
(usually cross-linked polysaccharides) containing pores.
• The gel serves as molecular sieves for the separation of smaller and
bigger molecules
• The solution mixture containing molecules of different sizes is applied
to the column and eluted with a buffer

• The large molecules cannot pass through the pores of a gel and
therefore move faster

• The smaller molecules enter the gel beads and are left behind which
comes out slowly. By selecting the gel beads of different porosity, the
molecules can be separated.
AFFINITY CHROMATOGRAPHY
• Principle is based on the property of specific and non-covalent
binding of proteins to other molecules, referred to as substrates or
cofactors
• The technique involves the use of ligands covalently attached to an
inert and porous matrix column. The immobilised ligands act as
molecules hooks to selectively pick up the desired proteins while the
remaining proteins pass through the column. The captured proteins
cab be eluted using free ligand molecules.
• Alternatively, some reagents that can break protein ligand interactions
can also be employed for the separation
SIGNIFICANCE
1. It is useful for the purification of enzymes, vitamins, nucleic acids,
drugs, hormone receptors, antibodies etc.

2. It is also widely used for the estimation of Glycated Hb.

Normal Hb does not bind and comes out first, while glycated Hb
binds with the Boronic acid used as a ligand. Sorbitol is then added to
elute the Glycated Hb which can be quantitated.
GAS LIQUID CHROMATOGRAPHY
• method of choice for the separation of volatile substances or volatile
derivatives of certain in volatile substances
• In GLC, the stationary phase is an inert solid material (diatomaceous
earth or powdered firebrick), impregnated with a non volatile liquid
(silicon or polyethylene glycol)
• This is packed in a narrow column and maintained at high
temperature (around 200˚C)
• A mixture of volatile material is injected in to the column along with
the mobile phase, which is an inert gas (argon, helium or nitrogen)
• The separation of the volatile material is based on the partition of the
components between the mobile phase (gas) and the stationary
phase (liquid), hence the name gas liquid chromatography

• The separated compounds can be identified and quantified by a

detector. Gas liquid chromatography is sensitive, rapid and reliable. It
is frequently used for the quantitative estimations of biological
materials such as lipids, drugs and vitamins
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
• It is used to separate, identify and quantify components of liquid
samples
• Relies on high pressure to pass the liquid solvent (sample mixture),
through a column filled with a solid adsorbent material (stationary
phase)
• Different adsorption of components cause differences in their flow
rates which leads to their separation as they flow through the column
• Detector displays the result in the form of a chromatogram, which in
turn can be used for analysis and interpretation
PRINCIPLE
• All chromatographic separations, including HPLC operate under the
same principle; every compound interacts with other chemical
species in a characteristic manner

• Chromatography separates a sample into its constituent parts

because of the difference in the relative affinities of different
molecules for the mobile phase and the stationary phase used in the
separation
SIGNIFICANCE OF HPLC
• HPLC can be used in both qualitative and quantitative analysis
• Can be used to effectively separate similar simple and aromatic
hydrocarbons, even those that differ only by a single methylene group
• It effectively separates simple amines, sugars, lipids and even
pharmaceutically active compounds
• Special continuous flow detectors can handle small flow rates and
detect very small amounts
• It provides rapid analysis
INSTRUMENTATION
COMPONENT FUNCTION/FEATURE
SOLVENT The mobile phase is usually a mixture of
polar and non-polar liquid components
whose respective concentrations are
varied depending on the composition of
the sample

PUMP To ensure an adjustable and constant

flow of approx. 1.0 ml/min for analytical
columns

AUTO-SAMPLER Large numbers of samples can be

automatically injected onto an HPLC
system, by the use of HPLC auto-sampler
COMPONENT FUNCTION/FEATURES
COLUMN To reach optimum analyte separation
per unit of time at an acceptable
pressure drop and at acceptable column
life time.
Columns can be packed with solids as
silica or alumina

DETECTOR Highly sensitive, low detection limit, and

long term stability

DATA ACQUISITION/ EVALUATION To require and store data and perform

calculations, to control and adjust
operational and control system
parameters and separation conditions
WORKING
• A cartridge or column is packed with a sorbent (stationary phase)
• A liquid (mobile phase) is passed through the packed column
• A dissolved sample (in a liquid) is injected into the flow path of the
mobile phase
• The sample band separates into individual analyte bands as it passes
through the HPLC column
• Analyte bands are detected
• A chromatogram is generated, analyte band are seen as “peaks”
• Peaks are quantitated
TYPES OF HPLC
• There are following variants of HPLC, depending upon the phase
system (stationary) in the process:

1. NORMAL PHASE HPLC

2. REVERSE PHASE HPLC
3. SIZE-EXCLUSION HPLC
4. ION-EXCHANGE HPLC
NORMAL PHASE HPLC
• Separates analytes on the basis of polarity
• NP-HPLC uses polar (hydrophilic) stationary phase and non-polar
(hydrophobic) mobile phase
• The stationary phase is usually silica and typical mobile phases are
hexane, methylene chloride, chloroform, diethyl ether and mixtures
of these
• Polar samples are thus retained on the polar surface of the column
packing longer than less polar materials
REVERSE PHASE HPLC

• The stationary phase is nonpolar (hydrophobic) in nature, while the

mobile phase is polar liquid (hydrophilic), such as mixtures of water
and methanol or acetonitrile

• Works on the principle of hydrophobic interactions hence the more

nonpolar the material is, the longer it will be retained
SIZE-EXCLUSION HPLC
• The column is filled with material having precisely controlled pore
sizes, and the particles are separated according to their molecular size

• Larger molecules are rapidly washed through the column, smaller

molecules penetrate inside the pores of the packing particles and
elute later
ION-EXCHANGE HPLC
• The stationary phase has an ionically charged surface of opposite
charge to the sample ions

• Technique is used almost exclusively with ionic or ionizable samples

• The stronger the charge on the sample, the stronger it will be

attracted to the ionic surface and thus, the longer it will take to elute

• The mobile phase is an aqueous buffer, where both pH and ionic

strength are used to control elution time
THE DETECTOR
• There are several ways of detecting when a substance has passed
through the column

• Detectors for HPLC

1. UV-Vis
2. FLUORESCENCE
3. REFRACTIVE INDEX
• When UV light is allowed to pass through the stream of liquid coming
out of the column, a UV detector on the opposite side of the stream,
can get a direct reading of how much light is absorbed by the liquid

• The amount of light absorbed will depend on the amount of particular

compound that is passing through the beam at the time

• The result is displayed in the form of a chromatogram

 CHROMATOGRAM
• A chromatogram is a pictorial record of the detector response as a function of
elution volume or retention time
• It consist of a series of peaks, ideally symmetrical in shape, representing the elution
of individual analytes
• It is a 2D plot with the ordinate axis giving concentrations in terms of detector
response (AU) and the abscissa represents the time (t)
• The base line represents any time period during which only mobile phase is passing
through the detector
Chromatographic peaks should possess Guassian profiles in the
optimal case and are characterised by:

 RETENTION TIME Tr

 PEAK HEIGHT h

 PEAK SYMMETRY b/a

 THEORATICAL PLATE HEIGHT H

(peak width)
CHROMATOGRAPHIC PARAMETERS
• RETENTION TIME

• RESOLUTION

• SELECTIVITY

• RETENTION FACTOR
RETENTION TIME
• The time taken for a particular compound to travel through the column to the detector is
known as its retention time
• This time is measured from the time at which the sample is injected to the point at which
the display shows a maximum peak height for that compound
• Different compounds have different retention times. For a particular compound retention
time may vary depending on
i. Pressure used
ii. Nature of the stationary phase
iii. Exact composition of the solvent
iv. Temperature of the column
• That , means that conditions have to be carefully
controlled if one has to use retention times as a
way of identifying compounds
 RESOLUTION
• The most important thing in HPLC is to
obtain resolution in the minimum time

• Resolution (Rs) is defined as the distance

between peak maxima compared with the
average base width of the two peaks
• A resolution value of 1.5 or greater
between two peaks will ensure that
the sample components are well
separated to a degree at which the
area or height of each peak me be
accurately measured
• Resolution depends on
1. SELECIVITY
2. EFFICIENCY
3. RETENTION
SELECTIVITY
• Selectivity of the system is a measure of its inherent ability to
discriminate between two analytes
• It is usually measured as a ratio of the retention factor (k) of the two
peaks and can be visualised as the distance between the apices of the
two peaks
• Some chromatographic mechanisms are inherently highly selective, like
affinity chromatography
EFFICIENCY
• The efficiency of a chromatographic peak is a
measure of the dispersion of the analyte band as
it travels through the HPLC system and column
• In an ideal world, chromatographic peaks would
be pencil thin lines, however, due to dispersion
effects the peak take on their familiar ‘Guassian’
shape
• Efficiency can be increased by increasing the
column length, reducing the column internal
diameter
RETENTION FACTOR
• It is simply the additional time that the analyte takes to elute from the
column relative to an unretained or excluded analyte that does not interact
with the stationary phase and which, by definition, has a k value of 0
• If an analyte has a k of 4, it follows that there will be four times the amount
of analyte in the stationary phase than in the mobile phase at any point in
the column at any time
• Retention factor for an analyte will increase with both the distribution
coefficient between the two phases and the volume of the stationary phase
• Values of k normally range from 1 to 10
AN EXAMPLE OF A
HPLC REPORT
• The area under the peak is proportional to the amount which has passed the
detector, and this area can be calculated automatically by the computer
linked to the display
• If the solution of the desired component was less concentrated, the area
under the peak would be less – although the retention time will still be the
same
• The standard for the desired component (retention time and peak area) can
be used to prepare the calibration curve
• With the help of calibration curve we can find concentration of the desired
component in the test sample
Problems that arise in chromatogram
• Some problems can arise either from the characteristics of the detector
or from the efficiency of the separation process. Problems that are
attributed to the detector are:
1. Baseline drift- where the detector signal gradually changes with time
2. Baseline noise- which is a series of rapid minor fluctuations in
detector signal, commonly the result of the operator using too high a
detector sensitivity or possibly an electronic fault
• Problems in chromatogram-
1. Peak broadening
2. Asymmetric peaks
3. Resolution