Chromatography

 The term chromatography means writing in color (in Greek:

Chroma-colour, and graphein- To write).
 Discovered by Mikhail S Tsvet, a Russian botanist in 1906
for separation of colored plant pigments of chloropyll into
individual pigments.
 A physical technique used for the separation, purification
and identification of the colored as well as colorless
mixture.
Principle: Based on the distribution of the components of the
mixture between two phases. Stationary phase and mobile
phase.
 Stationary (Fixed) phase: The phase which does not move
with the sample. It may be solid or liquid.
 Mobile (Moving) phase: Phase that moves with the
sample.​ ​ It may be liquid or gas.
 Chromatography………
Basic Principle:
 The sample is applied onto a stationary phase and sample
loaded stationary phase is allow to come in contact with
mobile phase. “The mobile phase (solvent) moves through
the stationary phase and as it moves, it carries the
component of the mixture along with it.”.
 Components of the mixture moves with mobile phase
through the stationary phase at different speeds. Hence
they are separated from one another.
 eg.​ In Paper Chromatography:​ Paper strip acts as "stationary
phase" while solvent act as "mobile phase“.
 Chromatogram: A visible record (such as a graph) showing
the separation of the components of a mixture
by chromatography.
 Chromatograph: Instrument used.
 Chromatography….

On basis of interaction of solute to the stationary phase: two

types:

Partition chromatography: Adsorption Chromatography

A type of chromatography in A type of chromatography in
which separation is based on which separation occurs based
partition (division). The on adsorption. The stationary
stationary phase and mobile phase is a solid state and mobile
phase, both are in liquid state. phase is liquid or gas. Adsorption
eg. Paper chromatography. takes place in stationary phase.
eg. Thin layer chromatography.
Adsorption: the deposition of molecular species onto the surface.
Adsorbent: material on which adsorption takes place.
Adsorbate: substance which is adsorbed on the adsorbent.

Chalk= Adsorbent
Ink = Adsorbate
 Paper chromatography Thin layer chromatography

(Statio
nary)

(Mobile)

Separation of the components of Separation of analyte is

mixture occurs by the partition
based on the adsorption
between the stationary phase
process (Interaction of
(filter paper), and mobile phase
(solvent) the adsorbate with the
adsorbent).
 Types of chromatography
 Based on the phase, primarily, there are four different
types of chromatography:
1. Paper chromatography: The stationary phase is moisture
present in paper.
2. Thin-layer chromatography (TLC): A thin layer of
stationary phase on a solid support is used.
3. Gas chromatography: The mobile phase exists in the
gaseous state.
4. High-performance liquid chromatography (HPLC): It uses
liquid (solvent) mobile phase, that pushes the sample
through the machine using a high-pressure pump.
 Primary types of Chromatography
Types of chromatography Types of phase

Stationary Mobile
Adsorption Chromatography Solid Liquid

High performance liquid Liquid Solid

chromatography (HPLC)
Thin layer chromatography (TLC) Solid/Liquid Liquid

Gas-liquid chromatography (GLC) Liquid Gas

Paper chromatography Liquid Liquid

 Paper Chromatography
 Type of chromatography where paper is used as support
for the stationary phase.
 Stationary phase: Liquid (usually water), deposited in
the network of cellulose fiber. eg filter paper. Mobile
phase travels though it.
 Mobile phase: Liquid solvent eg alcohol (propanol,
butanol).
 It is a liquid-liquid chromatography.
 It is a partition chromatography. The components of the
mixture distributed or partitioned themselves the
mobile phase (solvent) and stationary phase (moisture
of filter paper).
 An analytical method used to separate colored chemicals
or substances.
 Principle
 The sample is applied onto a moisture of paper (stationary
phase). Paper (Stationary phase) with loaded sample is
then placed in a suitable solvent (mobile phase).
 The mobile phase (solvent) moves up the stationary phase
(paper) by capillary action, and as it moves, it carries the
component of the mixture along with it.
 The movement of the different components is based on
their solubility in the stationary phase. The component,
more soluble in stationary phase moves slowly while the
component which is less soluble in stationary phase move
fast up the paper.
 As a result of this differential movement, the components
from the mixture is separated.
Differential coefficient = Solute in stationary phase
Solute in mobile phase
A measure of speed of a solute in a given system (Phase
and support) and under given condition (temperature) is
retardation factor or retention ratio.

Rf = distance travel by solute (analyte)

distance travel by solvent
Rf value is always less than one because solutes must have
more affinity for stationary phases hence, move slowly while
the solvent always travels fast than the solute.
Rf values can be used to identify unknown chemicals if they
can be compared to a range of reference substances.
 Procedure of Paper Chromatography
Steps involved:
 Step 1 : Prepare the Stationary Phase
It is preferable to use a rectangular filter paper with dimension
(14cm*30 cm or different per requirement). A pencil line is
drawn above 2.5 cm from one end. However small dimension
of filter paper piece can also be prepared.
A drop of 1-2 µL. Of solution of sample is spotted from a
capillary tip. On the same lines, other standard references can
be spotted for the comparative study. The spot should be small
and concentrated. The spot should be allowed to dry
completely before proceeding.
 Step 3 : Preparing the Mobile Phase
Prepare the mobile phase by pouring a small amount of the
solvent into a container. The level of the solvent should be
below the spot on the filter paper.
Steps involved…
 Step 4 : Placing the Paper in the Container
Place the filter paper in the container with the solvent. The
paper should be held in place so that it does not move.
 Step 5 : Developing the Chromatogram
Allow the solvent to move up the paper by capillary action.
The solvent will carry the different components of the mixture
along with it. Once the solvent front reaches a desired height
(usually around three-fourths of the paper strip), remove the paper
from the container and mark the solvent front with a pencil line.
Allow the paper to air dry.
 Step 6 : Analyzing the Chromatogram
The chromatogram can be analyzed visually or using other
methods such as UV or fluorescence spectroscopy.
The different components of the mixture will appear as spots
on the paper. Retention factor (Rf) is measured and Compare
the obtained chromatogram with known standards or
reference samples to identify the components in the mixture.
Chromatographic experiment is basically a three-step process:
1. Application of the sample,
2. Developing the chromatogram by allowing the mobile phase
to move up the paper,
3. Calculating Rf (Retardation factor) values and making
conclusions
14
Rf value and is calculated in the following manner.
Distance traveled by substance
Rf =
Distance traveled by solvent
 Applications
 Separation of Amino Acids
To separate amino acids. Amino acids can be identified by
their characteristic Rf values.
 Forensic Analysis
In forensic analysis to identify drugs, poisons, and other
substances. The characteristic spots on the chromatogram
can be used to identify the substance in question.
 Food Analysis
In the food industry to analyze food additives and identify
any contaminants present in food. This technique is also
used to identify the different pigments present in foods
such as fruits and vegetables.
 Environmental Analysis
To analyze environmental samples such as soil and water to
identify pollutants and other contaminants.
 Pharmaceutical Analysis
In the pharmaceutical industry to analyze and identify
different compounds and their impurities. This is
particularly useful in drug development and quality
control.

 Chemical Education
Paper chromatography is a common experiment in high
school and college chemistry courses. It provides students
with hands-on experience in separation techniques and
can help them better understand the principles of
chromatography.
 Thin layer chromatography (TLC)
 A simple, inexpensive chromatographic technique which
is used to separate components from non-volatile
mixtures. eg carbohydrates, fatty acids etc.
 It is a type of adsorption chromatography that uses a
thin layer of adsorbent (silica gel or alumina powder)
held on a glass plate.
 In TLC, adsorbent (silica gel
or alumina powder) is stationary
phase and the mobile phase is liquid.
Hence it is an example of solid–liquid
chromatography.
 Similar to paper chromatography.
Silica gel coated glass plate
 Thin-layer chromatography (TLC) principle
 The sample is applied onto a thin layer of stationary phase.
Stationary phase with loaded sample is then allow to come
in contact with a suitable solvent (mobile phase).
 As the mobile phase (solvent) moves through the stationary
phase along with the components of mixture. Components
of the mixture moves with mobile phase at different speeds.
Hence, they can be separated.
 This separation is based on the different solubility and
adsorbance of components of mixture for stationary phases.
The component that has:
• More affinity towards the S/P – travels slowly (Adsorbed)
• Less affinity towards the S/P – travels fast
 Due to different migration rates of the components, mixture
can be separated and identified based on their Rf values.
 Steps involved in separation of compounds
In TLC is performed in glass slides or aluminum foil, coated
with a thin layer of adsorbent. The adsorbent could be silica
gel or aluminum oxide. The thin adsorbent layer acts as
stationary phase. It is known as TLC plate or chromatoplate.
 Preparation of sample:
The analyte sample is dissolved in appropriate solvent. eg
mixtures of hexane and ethyl acetate.
 Preparation of TLC plate:
A rectangular TLC plate of an
appropriate size (approximate 20* 3.5
cm) is prepared and is coated with
thin layer of silica gel (about 0.2 mm).
Chromato- plate
 Steps involved in ……
 Now, base line is drawn above some distance (about 2.5 cm)
from one end. Sample under the test is spotted on plate with
the help of micropipette followed by dried with hot air.
 Development of chromatogram: The chromatoplate with
sample to be studied is placed in TLC chamber containing
solvent (mobile phase) vertically so that, the spots of plates
remain just above the solvent surface. The chamber is closed
with lid tightly (To prevent from evaporation of solvent).
TLC plate

Chromato- plate TLC chamber

 Steps involved in …….
 Mobile phase travels up plate by capillary action and sample
components migrate varying distances based on their
differential interaction with the stationary and mobile phases.
 When the solvent reaches about 1 cm from the top of the
plate, the plate is removed from the developing chamber and
dried. The separated components appear as spots on the plate
and the retention factor (Rf) of each component is assessed.
Mobile phase
Stationary phase Compounds separated
Sample spotted on TLC plate based on their affinity
 Steps involved in Thin Layer chromatography

Moisture or water affects the development. Hence, chromatoplate is

dried in oven at 100-105O C for half hr (Activation of chromatoplate)
 Steps involved in ……
 Locating the components: The separated components are
located by physical methods such as spraying with fluorescent
reagents.
 UV light, Iodine Staining: is very useful in detecting
carbohydrates since it turns black on contact with Iodine or
Ninhydrin Reagent: often used to detect amino acids and
proteins.
Compounds with more
interaction for stationary phase
move up the plate most slowly
(Lower Rf value) whereas
component with less interaction
towards stationary phase move
up the TLC plate rapidly (Higher
Rf value).
Schematic diagram of TLC
 Applications
 Purity of any sample: used to determine the sample’s purity.
The sample and the standard sample are directly compared;
if any impurities are found, they thus show up as extra spots
and are easily detectable.

 Identification of compounds: Natural products like alkaloids,

glycosides, steroids, etc. can also be purified and identified
using thin layer chromatography.

 Medicine: used to monitor the purity of several drugs,

including sedatives, antihistamines, analgesics etc.
 Applications….
 Biochemical analysis: useful in separation or isolation of
biochemical metabolites from its blood plasma, urine, body
fluids, serum, etc.

 Food industry: used in the food industry, to separate and

identify colors, sweetening agent, and preservative.

 Pharmaceutical industry: has also been used in separating

multi-component pharmaceutical formulations.
 Difference Between Paper and Thin Layer
Chromatography (with Comparison Chart) -
Biology Reader

Paper chromatography | PPT (slideshare.net)

Madhusudan Bachute

Thin layer chromatography: Principle, Advantages, Disadvantages

(scienceinfo.com)
Thin Layer Chromatography Applications
The qualitative testing of Various medicines such as sedatives, local
anaesthetics, anticonvulsant tranquilisers, analgesics, antihistamines,
steroids, hypnotics is done by TLC.
TLC is extremely useful in Biochemical analysis such as separation or
isolation of biochemical metabolites from its blood plasma, urine, body
fluids, serum, etc.
Thin layer chromatography can be used to identify natural products like
essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc.
It is widely used in separating multicomponent pharmaceutical
formulations.
It is used for the purification of samples and direct comparison is done
between the sample and the authentic sample.
It is used in the food industry, to separate and identify colours, sweetening
agent, and preservatives
It is used in the cosmetic industry.

31
the separation of the analyte occurs by the process of partition
between the water molecules (present in the surface of the
cellulose of which the filter paper serving as liquid stationary
phase and any solvent used as mobile phase.

STATIONARY PHASE AND PAPERS USED Whatman filter

papers of different grades like No.1, No.2, No.3, No.4,
No.20, No.40, No.42 etc are used. In general this paper
contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose
TLC system components consists of:
TLC plates, preferably ready made with a stationary phase:
These are stable and chemically inert plates, where a thin
layer of stationary phase is applied on its whole surface layer.
The stationary phase on the plates is of uniform thickness and
is in a fine particle size.
TLC chamber- This is used for the development of TLC plate.
The chamber maintains a uniform environment inside for
proper development of spots. It also prevents the evaporation
of solvents, and keeps the process dust free.
Mobile phase- This comprises of a solvent or solvent mixture
The mobile phase used should be particulate-free and of the
highest purity for proper development of TLC spots. The
solvents recommended are chemically inert with the sample,
a stationary phase.
A filter paper- This is moistened in the mobile phase, to be
placed inside the chamber. This helps develop a uniform rise
in a mobile phase over the length of the stationary phase.
 The Rf value for a particular substance is always the same if
the same solvent, same thickness of stationary phase, same
amount of spotted samples are used at same temperature.

Component with less affinity

towards stationary phase move
up the TLC plate.
Non-polar compounds move up
the plate most rapidly (higher Rf
value), whereas polar
substances travel up the TLC
plate slowly or not at all (lower
Rf value).
 Paper chromatography Thin layer chromatography

(Statio
nary)

(Mobile)

Separation of the components of Separation of analyte is

mixture occurs by the partition
based on the adsorption
between the stationary phase
process (Interaction of
(water molecules present in the
surface of the cellulose of filter the adsorbate with the
paper), and mobile phase (solvent) adsorbent).
 Paper Chromatography
 A technique in which analysis of unknown substance is carried
out mainly by the flow of solvent on specially designed filter
paper. The movement of the substances is due to the
differences in their solubility in a given solvent.
Principle:
 Type of liquid-liquid partition chromatography because the
substances are partitioned or distributed between two liquid
phases. Cellulose layers in filter paper contains moisture
which acts as stationary phase & organic solvents/buffers are
used as mobile phase. i.e. paper is inert support
A small spot of the mixture to be separated is placed on the
filter paper, and the paper is then placed in a solvent.
The solvent moves up the paper by capillary action, and as it
moves, it carries the different components of the mixture
along with it.
 Principle:
 When a mobile phase (solvent) moves along the
stationary phase (Paper), the components of the mixture
distributed or partitioned themselves between two
phases (stationary and mobile)
 The mobile phase (solvent) moves up the paper by
capillary action, and as it moves, it carries the different
components of the mixture along with it.
 The movement of the different components is based on
their solubility in the solvent. The component, more
soluble in stationary phase moves slowly while the
component which is less soluble in stationary phase
move fast up the paper.
 As a result of this differential movement, the
components from the mixture is separated.
Differential coefficient = Solute in stationary phase
Solute in mobile phase
 Principle:
 When a mobile phase (solvent) moves along the
stationary phase (Paper), the components of the mixture
distributed or partitioned themselves between two
phases (stationary and mobile).
 The mobile phase (solvent) moves up the paper by
capillary action, and as it moves, it carries the different
components of the mixture along with it.
 The movement of the different components is based on
their solubility in the solvent. The component, more
soluble in stationary phase moves slowly while the
component which is less soluble in stationary phase
move fast up the paper.
 As a result of this differential movement, the
components from the mixture is separated.
Differential coefficient = Solute in stationary phase
Solute in mobile phase
 Chromatography………
Basic Principle:
 The sample is applied onto a stationary phase and sample
loaded stationary phase is allow to come in contact with
mobile phase. “The mobile phase (solvent) moves through
the stationary phase, leading to separation based on
various properties”.
 Components of the mixture moves with mobile phase
through the stationary phase at different speeds. Hence
they are separated from one another. eg.​ In Paper
Chromatography:​ Paper strip acts as "stationary phase"
while solvent act as "mobile phase“.
 Chromatogram: A visible record (such as a graph) showing
the separation of the components of a mixture
by chromatography.
 Chromatograph: Instrument used.