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 Russian botanist
Mikhail Tsvet

 Chromatography
may be
preparative or
Chromatographic techniques involve the interaction of a mixture between a
stationary and a mobile phase. The stationary phase may be a solid, oil, or gel
while the mobile phase may be a liquid or a gas. The substances to be separated
move with the flow of the mobile phase. Depending upon their relative affinities
for each phase, the components of the mixture may move at different rates.

Chromatography basically involves the

separation of mixtures due to differences in
the distribution coefficient (equilibrium
distribution) of sample components between 2
different phases.
One of these phases is a mobile phase and
the other is a stationary phase.
Stationary Phase: Silica (SiO2)

Si O
Si O
Si O O
Si O O
O Si Si
Si O O O
Si O O
Si O
Distribution Coefficient (Equilibrium Distribution )


Concentration of component A in stationary phase

Concentration of component A in mobile phase

Different affinity of these 2 components to stationary

phase causes the separation.
Classification of Chromatography

• Paper chromatography
• Thin layer
• Column chromatography
• Gas chromatography
• Chromatography is the process use to separate
molecules based on SOME physical property of
the molecule:

• • Mass (i.e. size)

• • Charge
• • Affinity for ligands or substrates
• • Hydrophobic interactions
Types of Chromatography

1.Adsorption Chromatography
2.Partition Chromatography
3. Ion Exchange Chromatography
4. Molecular Exclusion Chromatography
5. Affinity Chromatography
Types of Chromatography
Adsorption Chromatography

• The equilibriation between the mobile and stationary

phase accounts for the separation of different solutes.
Partition Chromatography

• This form of chromatography

is based on a thin film formed
on the surface of a solid
support by a liquid stationary
• Solute equilibriates between
the mobile phase and the
stationary liquid.
Ion Exchange Chromatography

• In this type of
chromatography, the
use of a resin (the
stationary solid phase)
is used to covalently
attach anions or
cations onto it.
Molecular Exclusion Chromatography
• Also known as gel
permeation or gel
• No attractive
interaction between the
stationary phase and
• The liquid or gaseous (Smaller
phase passes through a Mols
porous gel which Trapped
separates the molecules By gel)
according to its size.
Affinity Chromatography
• This is the most
selective type of
• It utilizes the specific
interaction between
one kind of solute
molecule and a second
molecule that is
immobilized on a
stationary phase.
• For example, the
immobilized molecule
may be an antibody to
some specific protein.
Classification of chromatographic processes

Partition chromatography Adsorption Chromatography

• In partition Chromatography the mixture is separated by

means of partition between a mobile phase and a
stationary phase.The solute sample gets distributed in the
two immiscible liquids.
• In adsorption chromatography there is difference in the
adsorption behavior of sample between a mobile phase
and a stationary phase
Classification on the basis of types of MP and SP and the
mechanism of separation


Gas C
Liquid C


Paper chromatography

• Paper is made of cellulose, a polar substance

• More polar substances bond with the cellulose

paper more quickly, and therefore do not travel as
Thin layer chromatography

• Similar to PC.
• Stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert
• Compared to paper, it has the advantage of faster
runs, better separations, and the choice between
different adsorbents.
• For even better resolution and to allow for
quantification, HPTLC can be used.
• TLC is a simple, quick, and inexpensive procedure
Principle of TLC

• After the sample has been applied on the

plate, a solvent or solvent mixture (known as the
mobile phase) is drawn up the plate via capillary

• Because different analytes ascend the TLC

plate at different rates, separation is achieved
Thin-Layer Chromatography: A Two-
Component Mixture

solvent front

component B Less polar!

solvent front

component B

component A More polar!

component A

solvent front
origin origin

Increasing Development Time

Migration parameters

• Rf = distance traveled by solute

distance traveled by solvent

• Rx = distance traveled by substance

distance traveled by the standard substance
Depends on solvent, filter paper used, temperature and
type of technique-ascending, descending or radial
Interactions of the Compound and the
• The strength with which an organic compound
binds to an adsorbent is due to the following
types of interactions: ion-dipole, dipole-dipole,
hydrogen bonding, dipole induced dipole, and
van der Waals forces.
• Highly polar molecules interact fairly strongly with
the polar Si—O bonds of these adsorbents and
will tend to stick or adsorb onto the fine particles
of the adsorbent
• Weakly polar molecules are held less tightly.
Weakly polar molecules thus generally tend to
move through the adsorbent more rapidly than
the polar species.
As an example the chromatography of an extract
of green leaves (for example spinach) in 7 stages
of development. carotene elutes quickly and is
only visible until step 2. Chlorophyll A and B are
halfway in the final step and lutein the first
compound staining yellow.

•The compound runs as a streak rather than a spot

•No spots are seen on the plate

•The plate solvent front runs crookedly

•Many random spots are seen on the plate

•The sample runs as a downward/upward crescent (smear)

Advantages of TLC

• Simple
• Better separation than PC
• Less time
• Wide choice of adsorbent
Applications of TLC

• assaying the radiochemical purity of the

• determination of the pigments a plant contains
• detection of pesticides or insecticides in food
• analysing the dye composition of fibers in
• identifying compounds present in a given
• monitoring organic reactions
• Qualitative and quantitative analysis of organic
• High Performance Thin Layer Chromatography
is a sophisticated and automated form of TLC
Layer of Sorbent  100um  250um
 High due to smaller particle size
Efficiency • Less
Separations  3 - 5 cm • 10 - 15 cm

• Shorter migration distance and the

Analysis Time  Slower
analysis time is greatly reduced

 Wide choice of stationary phases like  Silica gel ,

Solid support silica gel for normal phase and C8 , Alumina &
C18 for reversed phase modes Kiesulguhr

 New type that require less amount of

Development chamber • More amount
mobile phase

Sample spotting  Auto sampler  Manual spotting

• Use of UV/ Visible/ Fluorescence

scanner scans the entire chromatogram
Scanning qualitatively and quantitatively and the • Not possible
scanner is an advanced type of
Column Chromatography

• In column chromatography, the stationary phase, a

solid adsorbent, is placed in a vertical glass (usually)
column and the mobile phase, a liquid, is added to
the top and flows down through the column (by
either gravity or external pressure).

• Column chromatography is generally used as a

purification technique: it isolates desired
compounds from a mixture
Types of columns

• Packed column: The particles of the solid

stationary phase or support coated with a liquid
stationary phase may fill the whole inside volume
of the tube.

• Open-tubular column: along the inside tube wall

leaving an open, unrestricted path for the mobile
phase in the middle part of the tube.
• An equilibrium is established between the solute
adsorbed on the adsorbent and the eluting
solvent flowing down through the column.
• Because the different components in the mixture
have different interactions with the stationary and
mobile phases, they will be carried along with the
mobile phase to varying degrees and a separation
will be achieved.
• The individual components, or elutants, are
collected as the solvent drips from the bottom of
the column.
Diagram of Simple Liquid Column Chromatography


Solvent(mobile or
moving phase)
OOOOOOOOOOO (packing material- OOOOOB OOOO

Eluant (eluate)
Reverse Phase
Column Chromatography
• The stationary phase (column packing) is now NON-
• Non-polar compounds will move more slowly
because they are attracted to the column packing.
• The more polar component moves more quickly
down the column.
• Polar solvents, such as water and methanol are
used in reverse phase chromatography
• Used mainly in columns, such as HPLC
Reverse phase chromatography
Silica is alkylated with long chain hydrocarbon
groups, using 18
carbons long. This is usually referred to as C-18
silica. CH 3

CH3 17 CH3
CH2 SiCH3)3 CH3
17 CH3
Si O
O Si
Si O
Si O
Si O O
Si O O
O Si
Si O
Si O
Si O O
Si O O
Si O
Types of CC

• gravity column chromatography - the solvent is

allowed to flow down the column by gravity, or

• flash chromatography-the solvent is forced down the

column by positive air pressure
The Adsorbent

•Silica and Alumina are two adsorbents commonly used.

•These adsorbents are sold in different mesh sizes, “silica gel 60” or
“silica gel 230-400” .
• This number refers to the mesh of the sieve used to size the silica,
specifically, the number of holes in the mesh or sieve through which the
crude silica particle mixture is passed in the manufacturing process.
•If there are more holes per unit area, those holes are smaller, thus
allowing only smaller silica particles go through the sieve.

Smaller particles (higher mesh values) are used for flash

chromatography, larger particles (lower mesh values) are used for gravity
chromatography. For example, 70–230 silica gel is used for gravity
columns and 230–400 mesh for flash columns.
The Solvent

A highly polar solvent will move even highly polar

molecules rapidly through the column. If a solvent is too
polar, movement becomes too rapid, and little or no
separation of the components of a mixture will result. If a
solvent is not polar enough, no compounds will elute from
the column.

Often a series of increasingly polar solvent systems are

used to elute a column. A non-polar solvent is first used to
elute a less-polar compound. Once the less-polar compound
is off the column, a more-polar solvent is added to the
column to elute the more-polar compound.

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol >


Non-polar Solvents

N-Decane > N-Hexane > N-Pentane >

Technique of CC
• Separating column – 20-50 cm
• Selection of adsorbent-alumina, silica, fullers
earth, polymers,etc.-uniform in size, inert, pure.
• Preparation of adsorption column
• Sample application
• Elution
• Detection-optical detectors or reagents-UV-Vis
Spectrophotometer or conductivity detectors
• Collection of components
• Analysis-spectroscopic methods-Qualitative and
•Separation occurs due to VARYING degrees of interaction of
sample with the stationary phase.

•Interaction of sample with stationary phase can be modulated

by changing the solvent conditions (i.e. pH, ionic strength,
competitive ligands, etc.).

•For column chromatography: stationary phase is referred to

as resin or gel or matrix.

•Three primary types of RESINS:

• Gel Filtration = Size Exclusion (SEC) =Molecular Sieve
• Ion Exchange (IEC)
• Affinity (ligands, substrates)
Applications of CC

• Qualitative analysis
• Separation of tautomers
• geometrical isomers
• Diastereomers
• racemates
• In 1978, W. C. Still introduced a modified version
of column chromatography called flash column
• the solvent is driven through the column by
applying positive pressure.
• This allowed most separations to be performed in
less than 20 minutes, with improved separations
compared to the old method.
• Modern flash chromatography systems are sold
as pre-packed plastic cartridges, and the solvent
is pumped through the cartridge.
• Systems may also be linked with detectors and
fraction collectors providing automation.
• The introduction of gradient pumps resulted in
quicker separations and less solvent usage

• High performance liquid chromatography is a

powerful tool in analysis

• High-performance liquid chromatography (or High

pressure liquid chromatography, HPLC) is a form
of Column Chromatography used frequently in
Biochemistry and analytical chemistry

• To separate, identify, and quantify compounds.

•HPLC utilizes a column that holds chromatographic packing
material (stationary phase),

• A pump that moves the mobile phase(s) through the column,

•With HPLC, a pump (rather than gravity) provides the higher

pressure required to propel the mobile phase and analyte
through the densely packed column and

•A detector that shows the retention times of the molecules.

•Retention time varies depending on the interactions between

the stationary phase, the molecules being analyzed, and the
solvent(s) used.


Instead of a solvent being allowed to drip through a

column under gravity, it is forced through under high
pressures of up to 400 atmospheres. That makes it much

It also allows you to use a very much smaller particle size

for the column packing material which gives a much
greater surface area for interactions. This allows a much
better separation of the components of the mixture.

Major improvement over column chromatography - the

detection methods - highly automated and extremely
• A typical column has an internal diameter of 4.6
mm (and may be less than that), and a length of
150 to 250 mm.
• Polar compounds in the mixture being passed
through the column will stick longer to the polar
silica than non-polar compounds. The non-polar
ones will therefore pass more quickly through
the column.
• Highly improved form of column chromatography
HPLC Columns

Syringe for HPLC/GC

HPLC Operation

• The sample to be analyzed is introduced in small

volume to the stream of mobile phase.
• The analyte's (organic molecules, biomolecules,
ions and polymers) motion through the column is
slowed by specific chemical or physical
interactions with the stationary phase as it
traverses the length of the column.
• The amount of retardation depends on the nature
of the analyte, stationary phase and mobile phase
• The time at which a specific analyte elutes (comes
out at the end of the column) is called the
retention time; considered a reasonably unique
identifying characteristic of a given analyte.
•The use of smaller particle size column packing
(which creates higher backpressure) increases the
linear velocity giving the components less time to
diffuse within the column, leading to improved
resolution in the resulting chromatogram.

•Common solvents used include any miscible

combination of water or various organic
liquids (the most common are methanol and
Instrumentation of HPLC
• Solvent selection-methanol,THF,CCl4
• Solvent delivery system-high press pump-400-
1500 psi at the rate 1-5 ml/min
• Sample injection system-syringe
• Column-porous silica particles (surface area-
400m2/g),porous layer beads made of silica or
glass with dia 30-55 micrometers or macroporous
• Detectors-spectrophotometer, conductometer,
• Amplifiers connected to microprocessor or
The Liquid Chromatograph

• The heart of a HPLC system is the column.

• The mixture to be separated is injected into the
flowing mobile phase by an injector.
• When the mobile phase passes through the
column that contains the stationary phase, the
molecules that adsorbs most to the stationary
phase migrates slowest through the column.
• When the mobile phase has passed through the
column it enters into the detector that detects
the different molecules as they have pass through
• A signal goes from the detector to a printer
that presents the separation graphically.
Solvents must be degassed to eliminate formation of

The pumps provide a steady high pressure with no

pulsating, and can be programmed to vary the
composition of the solvent during the course of the

Detectors rely on a change in refractive index, UV-VIS

absorption, or fluorescence after excitation with a
suitable wavelength.
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Photodiode 1

Photodiode 2

Refractive Index Detector

Conventional UV-Vis Absorption Detector

Conductivity Detector
Applications of HPLC
• Qualitative and quantitative analysis
• Pollutants, drugs, insecticides, organic
compounds,polymers, amino acids-
• Quality control of the industrial products
Gas Chromatography
• The mobile phase is a gas
• Types-GSC and GLC
• Principle-when vapors of sample in gas stream are
allowed to pass through a column containing
stationary liquid or solid phase, components get
separated due to difference in B.P’s, solubility or
• GSC- the principle involved is adsorption
chromatography-the rate of movement of solute is
determined by the relative polarities of the solute
and solid SP.
• GLC-The principle involved is partition
chromatography-the relative solubility of solute in
both the phases determines the rate of movement of
the solute.when solute is gas then movement is
determined by volatility.
Some terminologies
• The analyte is the substance that is to be separated
during chromatography
• 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
• 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.
• D = (Solute)sp
(D-Distribution ratio)
tR = length of the column
linear flow rate of solute migration

(tR – retention time)

Chromatogram-gives different peaks for
different components
• Number of peaks-complexity of the sample
• Qualitative analysis-comparing peak postns
with stds
• Quantitative analysis-concs detd


Detector response
Rf = Distance the solute moves
Distance the solvent front moves
Effect of temperature
• For speed, higher temp may be desired but at
the expense of resolution
• At lower temp resolution may be better, but
time required may be very long.
Regulator, injection port, glass or fused quartz
column, thermostat detector and recorder.
• Feed injection-The feed is injected into the
mobile phase. Through the pump the mobile
phase flows through the system
• Separation in the column- components having
weaker attraction for the stationary phase move
• Elution from the column- components elute out
at different time
• Detection-measures the concentration and
characteristic chemical composition
• Chemically inert carrier gas employed is He,
N2, Ar or H2.
• Temperature of the column is equal or slightly
above the B.P of the least volatile component
of the sample.

Injector Amplifier

P gauge
P reg

F Thermostat
M chamber
Carrier gas Capillary
supply column
• Thermally stable volatile samples are introduced into
the gas streams via an injection port.
• Different constituents migrate at different rates due
to their different distribution ratios between MP and
• Components get eluted in the column and then pass
through Detector and reach the recorder
• Thermally stable and chemically inert SP

• Detectors-TCD, FID or ECD have good

precision and accuracy, high signal to noise
ratio, non-destructive and exhibit good
response over a wide range of temperature.
• TCD-Filament within the cavity of a brass block, heated by
DC supply.
• When an analyte elutes, the thermal conductivity of the
column effluent is reduced and produces a detectable
• Amount of heat lost by conduction to detector walls
depends upon the thermal conductivity of the gas phase.
• Org compds decrease thermal conductivity.

A TCD detector consists of an electrically-heated wire or

thermistor. The temperature of the sensing element
depends on the thermal conductivity of the gas flowing
around it. Changes in thermal conductivity, such as when
organic molecules displace some of the carrier gas, cause
a temperature rise in the element which is sensed as a
change in resistance
- +
Brass block

Gas flow


Gas from
• The TCD is based on the principle of thermal
conductivity which depends upon the
composition of the gas.
• The sample components in the carrier gas pass
into the measuring channel.
• A second channel serves as a reference channel
where only pure carrier gas flows. Electrically
heated resistance wires are located in both
• The difference in thermal conductivity between
the column effluent flow (sample components in
carrier gas) and the reference flow of carrier gas
alone, produces a voltage signal proportional to
this difference.
• The signal is proportional to the concentration of
the sample components
• The Thermal Conductivity Detector (TCD) is truly a
universal detector because it responds to all
compounds and can detect air, hydrogen, CO, Ar, O2,
N2, CO2, sulfur oxide, inorganic gases and many other
• The TCD is a non-specific and non-destructive
detector. For most organic molecules, the sensitivity
of the TCD is lower compared to the Flame Ionization
Detector (FID.)
• Chemically active compounds like acids and
halogenated compounds should be avoided when using
TCD since they can attack the filament (wires) and thereby
change the resistance and permanently reduce the
detector sensitivity.
• Oxidizing substances, such as oxygen, can also damage
the filament, and a leak free environment should be
• For an optimal and proper response of the TCD, there
are a couple of critical factors:
• temperature of the detector block
• flow rate of the carrier gas and the reference gas
• resistance of the filaments
All these factors must be optimal to obtain a
representative TCD response.
• ECD-The gases from column exit pass
between two electrodes. One of the
electrodes on its surface has a radioisotope
that emits electrons c/a thermal electrons
the compound absorb these electrons and
produce negative ions. Decrease in thermal
electrons shows dec in detector current.
Radioactive foil

e- e-

Ceramic insulator

(Pulsed mode or
DC mode) Electron collector

Exit gas from column


• The ECD uses a radioactive beta particle

(electron ) emitter, a metal foil holding
radionuclide nickel-63. the electrons formed
by collision with nitrogen carrier gas (easy to
remove electrons from N2/Ar/CH4).The e
attracted to anode generating a steady
current. The analyte molecules captures the
electrons and reduce current between
collector anode and a cathode.
• It is 10-1000 times more sensitive than a FID
and one million times more sensitive than a
• Application mostly for detecting halogenated
compounds (electronegative compds) such as
pesticides and CFC’S in ppt, thus
revolutionizing our understanding of the
atmosphere and pollutants
The ECD uses a radioactive Beta Schematic of an ECD
emitter (electrons) to ionize some
of the carrier gas and produce a
current between a biased pair of
electrodes. When organic
molecules that contain
electronegative functional groups,
such as halogens, phosphorous, and
nitro groups pass by the detector,
they capture some of the electrons
and reduce the current measured
between the electrodes. The ECD is
as sensitive as the FID but has a
limited dynamic range and finds
its greatest application in analysis
of halogenated compounds
FID cannot detect compounds which do not contain
carbon-hydrogen bonds.
Vapors of compound from column exit are ionised
by a high temp flame of H2/Air .ions and electrons
formed between parallel plate electrodes increase
conductance.increased current means inc in conc
of component which is amplified and sent to
The eluent exits the GC column (A) and enters
the FID detector’s oven (B). As the eluent
travels up the FID, it is first mixed with the
hydrogen fuel (C) and then with the oxidant (D).
The effluent/fuel/oxidant mixture continues to
travel up to the nozzle head into the flame (F)
pyrolyzing the eluent. The ions are repelled up
toward the collector plates (G) which are
connected to a very sensitive ammeter, which
detects the ions hitting the plates, then feeds that
signal to an amplifier, integrator, and display
system. The products of the flame are finally
vented out of the detector through the exhaust
port (J).
An FID consists of a
hydrogen/air flame and a
collector plate. The effluent Schematic of FID
from the GC column passes
through the flame, which
breaks down organic molecules
and produces ions. The ions are
collected on a biased electrode
and produce an electrical

The FID is extremely sensitive

with a large dynamic range, its
only disadvantage is that it
destroys the sample.
Adsorption columns
• Packed column-stainless steel/ glass, length 3
mts, inner diameter 1.6-1.9 mm, bent or
coiled,solid-silica, polymer, fullers earth
• Open tubular column-inner diameter 1mm,
fused silica, coil, 30-90 mts having an open
free path for flow of MP with analyte.
Working of gas chromatography
• Pass carrier gas only to get base line in the recorder
• Inject sample at the same conditions of temp and
pressure as that of the carrier gas
• The components migrate with different retention
times . This gives both qual and quant estimation
• For comparison and identification use standard
compounds to be run in the GC under the same
Selectivity of mobile phase
• In chromatography, the selectivity of a phase system refers to
its capacity to retain certain types of solutes to a significantly
greater extent than others.
• For example, a gas chromatographic system, employing a
long chain hydrocarbon as the stationary phase, would retain
hydrocarbons very strongly, due to their mutual, strong
dispersive interactions. In contrast, short chain alcohols
would elute very rapidly due to the relatively small dispersive
interactions between the alcohols and the hydrocarbon
stationary phase.
Selectivity of mobile phase
• However, if the stationary phase was polyethylene glycol, the
short chained alcohols would be retained very strongly due to
the strong polar interactions between the hydroxy groups on
the solute and those of the stationary phase. In contrast, due
to the small dispersive capability of the polyethylene glycol,
hydrocarbons (including long chained hydrocarbons) would
interact very weakly with the stationary phase and would
only be slightly retained.
The selectivity factor (a)
• The selectivity factor, a, can also be manipulated to
improve separations. When a is close to unity it
gives good separation in a reasonable time. a is
increased by one of the following procedures:
• Changing mobile phase composition
• Changing column temperature
• Changing composition of stationary phase
• Using special chemical effects (such as incorporating
a species which complexes with one of the solutes
into the stationary phase)
• Gas chromatograph coupled with IR
spectrometer or a mass spectrometer
further enhances its usefulness
• Gas Chromatography is a unique and
versatile technique.
• It is an Analytical technique (Qual & Quan).
Applications of gas
• Separation of organic and inorganic compounds
• Study structure of compounds
• Determine mechanism and kinetics of reactions
• Elemental analysis
• Adsorption studies
• On an industrial scale can be used for process
More applications
• Food industry-determination of antioxidants, food
preservatives, decomposition prods, contamination
and adulterants can be routinely done.
• Drugs and pharmaceuticals-quality control, analysis
and monitoring of metabolites in biol.sys
• Environmental studies-air sample analysis
• Petroleum industry-components in pet. products
• Clinical chemistry-proteins, fats ,carbs,
Advantages of GC
• Versatile-solids, liquids and gases
• Convenient-routine analysis
• Cost effective-compared to other instruments
• Minimum analysis time-few seconds-30 mins
• High sensitivity-micro liters of sample
• High precision-2-5%
• Automated-automatic monitoring
• High resolution-phy and chem similar mols,
azeotropes can also be separated
Limitations of GC
• Samples must be volatile and thermally stable
below 400oC
• Detectors are non-selective
• Published retention data are not always
reliable for qualitative analysis