CHROMATOGRAPHIC TECHNIQUES

Submitted by:
ANJALI
ESEM 1st SEMESTER

Under the guidance of :

Dr. SUDESH CHOUDHARY

WHAT IS
CHROMATOGRAPHY?

A technique for separating &/or identifying the

components in a mixture
The basic principle is that components in a
mixture have different tendencies to adsorb
onto a surface or dissolve in a solvent.
It is a powerful method in industry, where it is
used on a large scale to separate & purify the
intermediates & products in various
syntheses.
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TYPES OF CHROMATOGRAPHY

Gas chromatography
Paper chromatography
Thin layer chromatography (TLC)
High performance liquid chromatography
(HPLC)

GAS
CHROMATOGRAPHY

Used in analytical chemistry for separating &

analyzing compounds that can be vaporized
without decomposition.
In this, the mobile phase is a carrier gas,
usually an inert gas such as helium or an
unreactive gas such as nitrogen.
The stationary phase is a microscopic layer of
liquid or polymer on an inert solid support,
inside a piece of glass or metal tubing called a
column.
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PRINCIPLE

The process of separating the compounds in a

mixture is carried out between a liquid
stationary phase & a gas mobile phase.
The column through which the gas phase
passes is located in an oven where the
temperature of the gas can be controlled.
The concentration of a compound in the gas
phase is solely a function of the vapor
pressure of the gas.
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FLOW SCHEME FOR GC

Sample injector

Flow controller

column

Waste

Detector

Carrier
gas
Column oven

APPLICATIONS

In testing the purity of a particular substance, or

separating the different components of a mixture.
In the analysis of various classes of persistent organic
contaminants in air, water, soils, sediments &biota.
Substances that vaporize below 300C can be measured
quantitatively.
In forensic science.
Disciplines as diverse as solid drug dose identification &
quantification , arson investigation, paint chip analysis,
& toxicology cases, employ GC to identify & quantify
various biological specimens & crime-scene evidence.
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ADVANTAGES &
DISADVANTAGES

High resolution power.

High sensitivity when
used with thermal
detectors.
Gives relatively good
accuracy & precision.
Separation & analysis of
sample very quickly.
Sample with less
quantity is also
separated.

Only volatile samples or

the sample which can be
made volatile are
separated.
During injection of the
gaseous sample proper
attention is required.
The sample of gas which
is about to inject must be
thermally stable so that it
does not get degraded
when heated.
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PAPER CHROMATOGRAPHY

In this, the stationary phase is a very uniform absorbent

paper. The mobile phase is a suitable liquid solvent or
mixture of solvents.
The paper is suspended in a container with a shallow
layer of a suitable solvent or mixture of solvents in it. It
is important that the solvent level is below the line with
the spots on it.
The reason for covering the container is to make sure
that the atmosphere in the beaker is saturated with
solvent from evaporating as it rises up the paper.
The distance travelled relative to the solvent is called
the Rf value.
Rf = distance travelled by compound/ distance travelled
by solvent

TYPES

DESCENDING PC
In this type, development of the chromatogram is done by allowing
the solvent to travel down the paper. Mobile phase is present in the
upper portion.
ASCENDING PC
Here the solvent travel upward direction of the chromatographic
paper.
ASCENDING-DESCENDING PC
It is the hybrid of both the above technique. The upper part of the
ascending chromatography can be folded over a rod & allowing the
paper to become descending after crossing the rod.
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 HORIZONTAL PC
Here a circular paper is taken & the sample is
given at the center of the paper. After drying the
spot the filter paper tied horizontally on a
petridish containing solvent. So that wick of the
paper is dipped inside the solvent. The solvent
rises through the wick & the component get
separated in form of concentrate circular zone.
TWO DIMENSIONAL PC
In this technique a square or rectangular paper is
used. Here the sample is applied to one of the
corners & development is performed at right
angle to the direction of the first run.

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APPLICATIONS

Specially used for separation of mixture

having polar & non polar compounds.
For separation of amino acids.
Used to determine organic compounds,
biochemicals in urine etc.
In pharma sector for determination of
hormones, drugs.
Sometimes used for evaluation of inorganic
compounds like salts & complexes.
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ADVANTAGES &
DISADVANTAGES

Requires very less

quantitative material.
Cheaper compared to
other chromatographic
methods.
Both unknown inorganic
as well as organic
compounds can be
identified.
Do not occupy much
space.

Large quantity of
sample cannot be
applied.
In quantitative
analysis it is not
effective.
Complex mixture
cannot be
separated.
Less accurate.
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THIN LAYER
CHROMATOGRAPHY

Used to separate non-volatile mixtures

Performed on a sheet of glass, plastic or
aluminium foil, which is coated with a thin
layer of adsorbent material, usually silica gel,
aluminium oxide, or cellulose. This layer of
adsorbent is known as the stationary phase.
After the sample has been applied on the
plate, a solvent or solvent mixture is drawn up
the plate via capillary action.
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FLOW DIAGRAM FOR TLC

Watch glass

Thin layer
chromatography
plate

Beaker

Pencil line

Spot of mixture

Solvent

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PRINCIPLE

Different compounds in the sample mixture

travel at different rates due to the differences
in their attraction to the stationary phase, &
because of differences in solubility in the
solvent.
By changing the solvent, or perhaps using a
mixture, the separation of components can be
adjusted.
Separation of compounds is based on the
competition of the solute & the mobile phase
for binding places on the stationary phase.

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APPLICATIONS

Used to monitor the progress of a reaction, identify

compounds present in a given mixture, & determine
the purity of a substance.
For analyzing ceramides & fatty acids, detection of
pesticides or insecticides in food & water, analyzing
the dye composition of fibres in forensics, assaying
the radiochemical purity of radiopharmaceuticals, or
identification of medicinal plants & their constituets.
A simple, quick, & inexpensive procedure that gives
the chemist a quick answer as to how many
components are in a mixture.
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ADVANTAGES &
DISADVANTAGES

Very simple to use &

inexpensive
More than one compound
can be separated
The solvents for the TLC
plate can be changed
easily.
Identification of most
compounds can be done
simply by checking Rf
literature values.

Do not have long

stationary phases.
The length of separation
is limited.
The detection limit is a lot
higher.
Operates on an open
system, so factors such as
humidity & temperature
can be consequences to
the results of your
chromatograph.
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HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

Is a technique in analytic chemistry used to

separate the components in a mixture, to identify
each component, & to quantify each component.
It relies on pumps to pass a pressurized liquid
solvent containing the sample mixture through a
column field with a solid adsorbent material.
Its composition & temperature play a major role in
the separation process by influencing the
interactions taking place between sample
components and sorbent. These interactions are
physical in nature.
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FLOW SCHEME FOR HPLC

Solvent
reservoir
Processing unit
& display

Pump to
produce
high
pressure

Sample
injectio
n

Signal to processor

HPLC tube

Detector

waste

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TYPES

ION EXCHANGE CHROMATOGRAPHY

An ion exchange mechanism to separate analytes based on
their respective charges.
Usually performed in columns but can also be useful in planar
mode.
Uses a charged stationary phase to separate charged
compounds including anions, cations, amino acids, peptides, &
proteins.
Types of ion exchangers include polystyrene resins, cellulose &
dextran, controlled pore glass or porous silica.
Used in water purification, preconcentration of trace
components, ligand exchange chromatography, ion exchange
chromatography of proteins, etc.
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 NORMAL PHASE CHROMATOGRAPHY

Separates analytes based on their affinity for a polar stationary surface such as
silica, hence it is based on analyte ability to engage in polar interactions with the
sorbent surface.
Uses a non-polar, non aqueous mobile phase ,& works effectively for separating
analytes readily soluble in non polar solvents. The analyte associates with & is
retained by the polar stationary phase.
Adsorption strengths increase with increased analyte polarity.
SIZE EXCLUSION CHROMATOGRAPHY
Separates molecules according to their size
Smaller molecules are able to enter the pores of the media, & therefore molecules
are trapped & removed from the flow of the mobile phase.
The average residence time depends upon the effective size of the molecules .
However, molecules that are larger than the average pore size of the packing are
excluded & thus suffer essentially no retention; such species are the first to be
eluted.
It is a low resolution chromatography technique & thus it is often reserved for the
final , polishing step of a purification.
REVERSED PHASE CHROMATOGRAPHY
Is a liquid chromatography procedure in which the mobile phase is significantly more
polar than the stationary phase.
Hydrophobic molecules in the mobile phase tend to absorb to the relatively
hydrophobic stationary phase. Hydrophilic molecules in the mobile phase tend to
elute first . Separating columns typically comprise a C8 or C18 carbon chain bonded 26
to a silica particle substrate.

APPLICATIONS

For medical purposes (e.g. detecting vitamin D

level in blood serum)
Legal purposes (e.g. detecting performance
enhancement drugs in urine)
Research purposes (e.g. separating the
components of a complex biological sample,
or of similar synthetic chemicals from each
other)
Manufacturing purposes (e.g. during the
production process of pharmaceutical &
biological products)

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ADVANTAGES &
DISADVANTAGES

Speed (minutes)
High resolution
Sensitivity
Reproducibility of +/1%
Accuracy
automation

Cost
Complexity
Low sensitivity for
some compounds
Irreversibly adsorbed
compounds not
detected.
Coeluction difficult
to detect
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THANK YOU
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