Chromatography is a laboratory technique used to separate components of a mixture based on how they interact with mobile and stationary phases. It was first developed in 1901 by Russian botanist Mikhail Tswett to separate plant pigments. The components move through the stationary phase at different rates, allowing separation. Chromatography has important analytical and preparative uses and involves terms like chromatograph, eluent, eluate, stationary phase, and mobile phase.

1. 20-Apr-20
Chromatography:
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.
OR/
Chromatography is a laboratory technique that separates components within a
mixture by using the differential affinities of the components for a mobile medium and
for a stationary adsorbing medium through which they pass.
History:
Chromatography has its roots going back to a Russian botanist named Mikhail
Tswett who in 1901 devised a method for separating plant pigments. Tswett
reported this work in 1906 where he described using a petrolium ether/ethanol
mobile phase to separate carotenoids and chlorophylls with calcium carbonate
stationary phase in column chromatography. He named the methodology after the
Greek words chroma (color) and graphein (to write) to form chromatography.
PURPOSE OF CHROMATOGRAPHY
• Analytical
Determine Chemical composition of a sample
• Preparative
Used to purify sufficient quantities of a substance
CHROMATOGRAPHY TERMS
 Chromatograph
Equipment that enables a sophisticated separation
 Eluent
Fluid entering column/ solvent that carries the analyte through the
chromatographic apparatus.
 Eluate
The Mobile phase leaving the column.
 Stationary phase or Immobilized phase
The phase in which the mobile phase is forced through is known as stationary
phase. This phase is always composed of a “solid” phase or “a layer of a liquid
adsorbed on the surface a solid support.”
 Examples: Silica layer - Thin Layer Chromatography
 Mobile phase:
The liquid or gas that flows through a chromatography system, moving the
materials to be separated at different rates over the stationary phase is called
mobile phase.
 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 analyzed in chromatography.
 Chromatogram:
A chromatogram is the visual output of the chromatograph.

2. 20-Apr-20
Classification of chromatography
A) Based on mechanism of separation
I. Adsorption chromatography:
 Adsorption chromatography is one of the oldest types of chromatography.
 Adsorption chromatography is process of separation of components in a mixture
introduced into chromatography system based on the relative difference in
adsorption of components to stationary phase present in chromatography
column.
 Since the adsorption phenomenon is inherent
property of solids, the term adsorption
chromatography is only used where stationary phase
is solid.
Example:
Column chromatography, TLC
II. Partition chromatography:
 Chromatography in which separation is
based mainly on differences between
the solubility of the sample components
in the stationary phase or on differences
b/w the solubility of the components in
the mobile and stationary phases.
 This form of chromatography is based
on a thin film formed on the surface of a
solid support by a liquid stationary
phase. Solute equilibrates between the
mobile phase and the stationary liquid.
Example:
Paper chromatography
III. Ion exchange chromatography:
 In this type of chromatographic technique, the polar molecules and/or ions are
separated based on their affinity to the ion exchanger (that acts as a stationary
phase).
 Ion-exchange chromatography is used for the separation of almost any kind of
charged molecule including large amino acids, small nucleotides, and proteins.
Ion-exchange chromatography is used for the separation of either cations or
anions.
 In conventional methods the stationary phase is an ion exchange resin that
carries charged functional groups that interact with oppositely charged groups
of the compound to retain.

3. 20-Apr-20
IV. Size exclusion chromatography:
 This chromatographic technique is also known as:
 Gel permeation chromatography
 Molecular exclusion chromatography and/or
 Gel filtration chromatography.
 The phenomenon of separation of the components of interest present in the sample
mixture is based on the size and/or molecular weight of individual component present in
the mixture and by the mechanism of filtration through the gel which acts as stationary
phase consisting of heterosporous (pores of different sizes) cross-linked polymeric gels or
beads. Small molecules get trapped into the pores of stationary phase more rapidly as
compared to that of the large molecules that do not entered into the gel easily and cover
the longer distance in stationary phase. The flow of the component of interest through the
gel (stationary phase) is retarded according to their size. The large molecules are eluted
out (cannot enter into the gel or stationary phase due to their large size or molecular
weight). It is applied to separate the large molecular weight macromolecular complexes
such as proteins and industrial polymers for semi-preparative purifications and various
analytical assays.
V. Affinity chromatography:
 Affinity chromatography is a method of separating biochemical mixtures
based on a highly specific interaction such as that between antigen and
antibody, enzyme and substrate, or receptor and ligand.
 When a complex mixture containing specific compound is passed through a
column containing immobilized ligand, the specific compound will bind to the
ligand. Ligand is immobilized to an insoluble solid matrix. A biological
reversible interaction between a compound and a specific ligand occurs as
follows:
 Compound (M) + Ligand (L) ML complex
 This interaction occurs due to Electrostatic or hydrophobic interactions Van
der Waals' forces or
 Hydrogen bonding.
 Compounds not specifically bound are washed away with the buffer.
Compounds specifically bound are recovered from ligand by reversing the
interaction.
1. Based on physical state of mobile phase
a) Liquid chromatography
b) Gas Chromatography:
c) Supercritical fluid Chromatography:
Supercritical fluid chromatography (SFC) is a form of normal
phase chromatography that uses a supercritical fluid such as carbon dioxide as
the mobile phase.

4. 20-Apr-20
Based on the Polarity of Mobile Phase:
a) Normal Phase Chromatography:
In normal-phase chromatography, the stationary phase is polar and the mobile
phase is nonpolar.
b) Reversed-phase chromatography:
It is a technique using alkyl chains covalently bonded to the stationary phase particles
in order to create a hydrophobic stationary phase, which has a stronger affinity for
hydrophobic or less polar compounds.
In Reverse-phase chromatography, the stationary phase is non-polar and the
mobile phase is polar.
3. Based on shape/Geometry of chromatographic bed
a. Planner chromatography (Two dimensional)
Here stationary phase is on a flat plate and the mobile phase moves through
stationary phase due to capillary action.
i. Paper chromatography
ii. Thin layer chromatography
iii. HPTLC
b. Column chromatography (Three dimensional)
Here stationary phase is packed in a tubular column usually made up of glass
or steel.
i. Packed column chromatography
ii. Open tubular column chromatography
COLUMN CHROMATOGRAPHY:
Column chromatography is a form of liquid chromatography in which column is used
to hold the stationary phase. The stationary phase is either a solid or a thin, liquid
film coating on a solid particulate packing material.
Principle
Column is packed with appropriate stationary phase. A mixture of compounds that
needs to be separated is introduced from the top of the column. Components present
in the mixture then move at different rates depending upon their relative affinities
towards the stationary phase. The components having lower adsorption rate and less
affinity with stationary phase will move faster as compared to those components
having more adsorption and more affinity with stationary phase. The components
moving faster are eluted out first, whereas those moving slowly are removed last.
From the distance travelled by solute, a retardation factor is calculated:

5. 20-Apr-20
Types of Column Chromatography
Following are the most common types of column chromatography.
1. Absorption chromatography 2. Partition chromatography
3. Gel chromatography 4. Ion exchange chromatography
Components of Column Chromatography
Following are the main components of column chromatography:
1. Stationary Phase
Stationary phase is solid in column chromatography which should have good
adsorption property. Stationary phase should be selected properly to achieve the
success of column chromatography.
A stationary phase should have the following properties:
1. Should have uniform shape and size of particles (60-200 micron)
2. Mechanically stable 3. Chemically inert
4. Allow free flow of the solvent 5. Freely available
6. Inexpensive 7. Should be colorless so as to observe color bands
Adsorbents (stationary phase)
 Stationary phases used in column adsorption chromatography are also known as
adsorbents. A good number of solid compounds belonging to either ‘organic’ or
‘inorganic’ category are being extensively employed as adsorbents in column
chromatography. Organic substances: Carbon, Starch , Cellulose
 Inorganic substances: Alumina, Silica gel, Kiesulgur, calcium phosphate, calcium
carbonate, and magnesia (MgO)
are the most commonly used adsorbents in column chromatography.
 For less polar compounds alumina is preferred.
 Silica gel also gives good results for compounds having polar functional groups.
Adsorbents should have the following properties:
1. Particles should be uniform in size and have spherical shapes.
2. High mechanical stability.
3. Chemically inert.
4. Useful for the separation of many compounds.
5. Inexpensive and freely available.
B) Mobile Phase
Mobile phase is liquid in case of column chromatography which dissolves the
mixture and transfer it through the column. It acts as a:
1. Solvent: To introduce the sample mixture into the column.
2. Developing agent: To separate the components of interest present in mixture in
the form of bands.
3. Eluting agent: To remove the separated components out of the column.
 The solvent is chosen on bases of the solubility properties of the mixture. Low
boiling point and polarity of the solvents are the important factors in the
selection of a solvent in column chromatography.
 The mostly used solvents are carbon tetrachloride, petroleum ether, ether,
esters, cyclohexane, acetone, toluene, benzene, and water.
C) Column
 It is used to hold the adsorbent or stationary phase. It is made up of neutral glass
which should be of good quality so that it cannot adversely affect the solvent.
Usually, a burette is used as a column having length and diameter ratio of 10:1,
30:1, or 100:1.
 Selection of the column’s dimensions depends upon the number of components in
the sample, type of stationary phase, sample quantity under analysis &
components affinity towards the stationary phase. A narrow column is preferred
over the short and thick column to achieve better separation.

6. 20-Apr-20
Step involved in Column Chromatography (methodology):
1. Preparation of Column
Column is first washed properly with purified water and then with acetone. Then,
it is dried properly. Then column is fixed along a stand with the help of clamp in
such a way that its outlet should be downward. Column should be packed from the
bottom with a cotton wool, glass wool, etc so that adsorbent will not fall out. Then
a layer of purified sand is added.
2. Packing of Column
Two techniques are used for the packing of column. One is dry packing or dry
filling and second one is wet packing or wet filling.
a) Dry Packing of Column
Column is firstly filled with the adsorbent in dry form in this method and then the
solvent is flushed through the column until equilibrium is reached. Air bubbles
may be entrapped between the mobile phase and stationary phase due to which
cracks or void space may appear in the adsorbent layer. To address these
problems tapping is done while packing of the column. A schematic
representation of dry packing of column has been represented in Fig below.
b) Wet Packing of Column
The adsorbent is suspended in the mobile phase and stirred very well to remove
all air bubbles. The resulted slurry is then poured in to the column. Stationary
phase (adsorbent) settles uniformly and no cracks are formed in the column. The
solid settle down while the solvent remains upward. The extra solvent above the
silica jell bed is removed until it reaches about 1 cm above its level. Again a small
layer of purified sand is added over the silica jell and now the column is ready to
be loaded with the sample.
3. Sample introduction in column chromatography (Loading):
a) Wet loading:
Dissolve the sample in the initial
mobile phase and apply by pipette to
the top of the column. This is very
good method but in most of cases the
samples are not soluble in the initial
mobile phase so we have to used dry
loading method.
b) Dry loading:
Dissolve the sample in any volatile solvent. The sample solution
is then adsorbed on small amount of adsorbent (stationary
phase) and the solvent is allowed to evaporate. The dry
adsorbent loaded with the sample is then applied to the column.

7. 20-Apr-20
B) Mobile Phase
Mobile phase is liquid in case of column chromatography which dissolves the
mixture and transfer it through the column. It acts as a:
1. Solvent: To introduce the sample mixture into the column.
2. Developing agent: To separate the components of interest present in mixture in
the form of bands.
3. Eluting agent: To remove the separated components out of the column.
 The solvent is chosen on bases of the solubility properties of the mixture. Low
boiling point and polarity of the solvents are the important factors in the
selection of a solvent in column chromatography.
 The mostly used solvents are carbon tetrachloride, petroleum ether, ether,
esters, cyclohexane, acetone, toluene, benzene, and water.
C) Column
 It is used to hold the adsorbent or stationary phase. It is made up of neutral glass
which should be of good quality so that it cannot adversely affect the solvent.
Usually, a burette is used as a column having length and diameter ratio of 10:1,
30:1, or 100:1.
 Selection of the column’s dimensions depends upon the number of components in
the sample, type of stationary phase, sample quantity under analysis &
components affinity towards the stationary phase. A narrow column is preferred
over the short and thick column to achieve better separation.
Step involved in Column Chromatography (methodology):
1. Preparation of Column
Column is first washed properly with purified water and then with acetone. Then,
it is dried properly. Then column is fixed along a stand with the help of clamp in
such a way that its outlet should be downward. Column should be packed from the
bottom with a cotton wool, glass wool, etc so that adsorbent will not fall out. Then
a layer of purified sand is added.
2. Packing of Column
Two techniques are used for the packing of column. One is dry packing or dry
filling and second one is wet packing or wet filling.
a) Dry Packing of Column
Column is firstly filled with the adsorbent in dry form in this method and then the
solvent is flushed through the column until equilibrium is reached. Air bubbles
may be entrapped between the mobile phase and stationary phase due to which
cracks or void space may appear in the adsorbent layer. To address these
problems tapping is done while packing of the column. A schematic
representation of dry packing of column has been represented in Fig below.

8. 20-Apr-20
b) Wet Packing of Column
The adsorbent is suspended in the mobile phase and stirred very well to remove
all air bubbles. The resulted slurry is then poured in to the column. Stationary
phase (adsorbent) settles uniformly and no cracks are formed in the column. The
solid settle down while the solvent remains upward. The extra solvent above the
silica jell bed is removed until it reaches about 1 cm above its level. Again a small
layer of purified sand is added over the silica jell and now the column is ready to
be loaded with the sample.
3. Sample introduction in column chromatography (Loading):
a) Wet loading:
Dissolve the sample in the initial
mobile phase and apply by pipette to
the top of the column. This is very
good method but in most of cases the
samples are not soluble in the initial
mobile phase so we have to used dry
loading method.
b) Dry loading:
Dissolve the sample in any volatile solvent. The sample solution
is then adsorbed on small amount of adsorbent (stationary
phase) and the solvent is allowed to evaporate. The dry
adsorbent loaded with the sample is then applied to the column.
4. Development of the column:
The process by which solutes are carried through the stationary phase by the
mobile phase is called development or chromatographic development.
Development Procedures in chromatography
Basically three methods are used for chromatographic development.
 Frontal analysis
 Displacement analysis
 Elution analysis
Of these methods elution is by far the most widely used. In fact frontal analysis is
never used as a practical analytical method. Displacement techniques are
occasionally used.
a) Frontal Analysis
Here the sample is fed continuously onto the column as a dilute
solution in the mobile phase in contrast to displacement & elution
development, where discrete samples are placed on the system and the
separation subsequently processed. Frontal analysis can only separate
part of the first compound in a relatively pure state, each subsequent
component being mixed with those previously eluted.
Explanation:
Consider a three component mixture, containing solutes (A), (B) and
(C) as a dilute solution in the mobile phase that is fed continuously
onto a column. Suppose C is most strongly held by the stationary phase
and A is least strongly held. The first component to elute will be that
solute held least strongly (A) in the stationary phase. Then the second
solute, (B), will elute but it will be mixed with the first solute, since the

9. 20-Apr-20
sample is introduced continuously. Finally,
the third solute (C), will elute in conjunction
with (A) and (B). It is clear that only solute
(A) is eluted in a pure form and, thus, frontal
analysis would be quite inappropriate for
most practical analytical applications. This
development technique has been completely
superseded by elution development and so it
is rarely used and probably of academic
interest only.
b) Displacement analysis
The principle involved in this method is that small volume of
mixture of components (sample) is introduced into the column
and the usual ‘elution’ is performed by means of a solvent
consisting of a solute (also called displacer) that possesses high
degree of adsorptivity for the adsorbent packed in the column.
Then the adsorbed components present in the ‘sample mixture’
are displaced by the ‘added solute’ (displacer) from the eluting
mobile phase. Each displaced component present in the sample
mixture now helps to displace another solute (sample
component) that is less strongly adsorbed. In this way, the least
strongly adsorbed component is flushed out of the column first.
Explanation:
Suppose we have a mixture that has the same three compounds
i.e. A, B and C. Again lets suppose that C is the most strongly
bound component whereas, A is the least
strongly bound solute in the sample. When this
sample is loaded to the column and a mobile
phase containing a displacing agent D having
higher affinity for the stationary phase than all
the components of the sample is added, the
displacer will displace all the components from
the stationary phase and each displaced
component will then displace the next less
strongly bound component from the stationary
phase and separation will occur in the form of
discrete zones as shown in fig.
 Displacement development is only effective with a solid stationary phase where the
solutes are adsorbed on its surface.
 It is primarily used for purification purpose rather than for analytical purpose.
c) Elution analysis
 Elution analysis refers to the specific removal of chemical entities
from a chromatographic support (stationary phase) by the aid of
solvent.
 Elution analysis is carried out by introducing the sample in as small
volume as possible onto the head of the column. The mobile phase
is then allowed to flow through the system. With the passage of
time the ‘mobile phase’ moves down the column and the mixture of
‘analytes’ undergo resolution into various ‘distinct zones’ by the
fact that the analytes in the mixture get adsorbed to various degree.
Compounds can be isolated in a relatively pure state.
 Explanation:
Consider a mixture of solutes A, B and C, placed initially at one end
of a bed of stationary phase. suppose that C is more strongly
retained than B which in turn is more strongly retained than A.

10. 20-Apr-20
If a mobile phase, less strongly retained
than either A or B, is caused to pass
through the bed, then it will wash A, B
and C through at different speeds
according to the degree of retention of the
two solutes. If the difference in migration
rates is sufficiently great, then A, B and C,
which are initially superimposed, will
gradually separate to form three distinct
zones with pure eluent separating them.
 Importance:
 It is a common method used in column chromatography.
 Elution development is the only development technique employed in both GC & LC.
 Each component of Sample mixture is eluted out as separated components.
Types of elution techniques
i) Isocratic Elution Technique
Solvents (mixture) having the same composition or solvent of same polarity are
used in this technique throughout the process of separation. One of the most
commonly used solvents for isocratic elution technique is chloroform.
ii) Gradient Elution Technique
Solvents having gradually increasing polarity or increasing strength of elution are
used in this technique during the process of separation. For example, initially
benzene, then chloroform, then ethyl acetate then methanol.
5. Eluting the sample:
Once the sample solution is added onto the stationary phase, solvent (mobile
phase) is added to the top of the column at a rate sufficient to ensure a “Head” of
liquid on the top layer of sand at any point of chromatogram development. The
composition of the solvent can be changed as the column progresses. The
constituents of the sample will pass down the column at varying rates.
6. Detection and recovery of the compounds
By visual examination of the column:
 After developing the chromatogram by running the solvent, different bands of
components are formed. If the bands are colored then we can detect the
components visually.
 The colorless components may also be detected visually if they fluorescence.
Ex : Quinine & Ergotamine.
 Recovery of the components after detection on the column requires ‘extrusion’ of
the column of adsorbent and isolation of each zone for extraction with solvents.
 In case of plastic tubing the zones are isolated by cutting tubing into sections.
Eluting the various components with solvents:
 For colorless compounds the eluate is collected as a large number of fractions,
each of small volume.
 Each fraction is examined appropriately for the presence of a compound by:
o By simple spot tests by paper or TLC
o By passing UV light, compounds can be detected visually.
o By reagents such as Ninhydrin, ceric sulfate or I2 vapors can be used for
visualization of colorless compounds.
o By spectrophotometry

11. 20-Apr-20
Factors Affecting Column Chromatography
1. Dimension of the column:
Length of the column should be more than its width. Normally 10:1, 30:1, or 100:1
ratios of length and diameter are used.
2. Particle size of column packing:
separation to be improved by decreasing the particle size of the adsorbent.
3. Activation:
Proper activation of the adsorbent is needed.
4. Column’s temperature:
Temperature should be managed properly as high temperature can enhance the
process of elusion however, it does not improve separation.
5. Column packing:
Column should be properly packed with the adsorbent and bottom should be also
filled with cotton wool or anything else used for this purpose.
6. Solvents:
proper selection of solvents is important. Less viscous solvents give better results.
Advantages
1. It is simple and easy technique.
2. Mixture of any type or quantity can be easily separated using this technique.
3. Many types of solvent/mobile phase can be used in this technique.
4. Automation is possible when using this technique.
5. It is an inexpensive technique.
6. This technique can be used both on small and on large scale.
Disadvantages
1. It is a time consuming process, especially when components show colorless bands.
2. In this technique, a large amount of the solvent is required for the proper elusion.
3. It is a simple technique but if automated then it becomes more complex and hence
more expensive.
Applications of Column Chromatography:
1. It is used to separate a mixture of compounds into its components of interest.
2. It is used for removal of impurities.
3. The active constituents of many drugs can be isolated by using this technique.
4. It is used for estimation of drugs in drug formulations.
5. It is also used to isolate many metabolites from the biological fluids like blood or
serum.
6. Secondary metabolites like glycosides in digitalis leaf can be isolated by using this
technique.
7. It can be used to separate natural complex mixtures like alkaloids, glycosides.
8. It is used for Separation of amino acids, proteins, phospholipids and triglycerides.
Thin Layer Chromatography:
It is a type of planar chromatography in which compounds are separated on a thin
layer of adsorbent material, typically a coating of silica gel on a glass plate or plastic
or Aluminium sheet.
Principle
1. Separation of individual component present in sample mixture is dependent on the
relative affinity of individual component with stationary phase and the mobile phase.
2. If the affinity of individual component present in a sample mixture is high with
mobile phase, then it travels over the surface of the stationary phase eluted out.
Whereas, if the affinity of individual component present in sample mixture is higher
with stationary phase, then it travels slowly and retained in the stationary phase.
Thus, the separation of components in the mixture is achieved.
3. Once the separation occurs, individual components of interest are visualized as
spots appeared at different points on TLC plate.

12. 20-Apr-20
Types of Thin Layer Chromatography
 Based on Nature of Phases
 Based on the nature of phases used in TLC, it can be classified into the following
two types:
1. Normal Phase TLC
Normal phase is the terminology used when the stationary phase is polar ; for
example silica gel, and the mobile phase is an organic solvent or a mixture of
organic solvents which is less polar than the stationary phase.
2. Reversed Phase TLC
Reversed phase is the terminology used when the stationary phase is rendered less
polar and the mobile phase is a mixture of water and organic solvent which is more
polar than the stationary phase.
Based on Purpose of Use
TLC can also be classified into two types based on the purpose of Use.
Analytical TLC
The main purpose of analytical chromatography is qualitative/ quantitative
separation of the components of a sample.
Preparative TLC
The main purpose of preparative chromatography is to isolate and purify a
reasonable quantity of a specific substance from a sample so it is basically
performed for purification purposes.
Components of Thin Layer Chromatography
Following are the components of TLC:
a) Developing Chamber
This is used for the development of TLC plate. It create and maintain the
environment for TLC, prevents the evaporation of solvents, and keeps the process
dust free.
b) Solid Support
 It is used to support the stationary phase (adsorbent) as a thin film coat. The
sorbents may be coated on glass, plastic, or aluminum supports.
 Glass plates having specific dimensions like 20 cm X 20 cm, 20 X 10 cm, 20 cm X 5
cm can be used. These dimensions are used since the width of the commercially
available TLC spreader is 20 cm.
 Glass plates of different dimensions can also be used when the TLC plates are
prepared without the use of TLC spreader.
 Microscopic slides can also be used for some applications like monitoring the
progress of a chemical reaction. The development time is much shorter like 5 min.
 Glass has the advantage that it can be easily cleaned, repeatedly used, and
withstands the usual solvents and revealing reagents, including most corrosive
substances.
 Borosilicate glass can be used if the developed chromatogram is to be subjected to
high temperatures (> 150 °C), e.g., in using charring techniques to reveal the
chromatogram.
 In general, the glass plates should be of good quality and should withstand
temperatures used for drying the plates.
 After use, plates are cleaned by washing under running water, immersed for 24 hr
in a tank of distilled water containing 1% of an aqueous detergent, and finally
washed in distilled water and left to dry.
 Plates with a plastic support have an additional distinguishing feature relative to
glass-backed plates: They can be cut with scissors. This is advantageous, for
example, when small plates are desired but only 20x20 cm plates are on hand.
 With plastic and Aluminum-backed plates it is also easy to elute separated
substances from the developed layer by cutting out an appropriate region and
immersing the cut piece in a suitable extraction solvent.

13. 20-Apr-20
c) Stationary Phase (Adsorbents)
 It is used to provide facility for the adsorption of analyte on TLC film.
 The common adsorbents used in TLC are silica gel, alumina, kieselguhr & powdered
cellulose.
 Silica gel:
It is the most widely used adsorbent and is slightly acidic. A binding agent, usually
gypsum (calcium sulphate) is often incorporated to hold the adsorbent firmly on the
plate. Two ultra violet indicators are incorporated, either singly or together, in several
varieties of gel. Preparations without binding agent should be used if these additives
react with compounds to be separated.
 Layers 0.25 mm thick can be prepared by spreading an aqueous slurry of adsorbent with
a commercial applicator on glass plates. The slurry is prepared by thoroughly mixing 25
gms of silica gel G (Merck) with 50 mL of distilled water in a mortar. Thick layers (1-2
mm) of silica gel can be prepared by slurring silica gel G with water in the ratios 25 : 40.
The layers are air dried for about 10 minutes and then activated by heating in an oven at
about 110 ° C for 2 hours.
Aluminium oxide (Al2O3):
It is slightly basic. It is also available commercially with and without binder.
Kieselguhr (Diatomaceous Earth):
It is available both with and without a binder. Its capacity of resolving constituents is
less than either silica gel or alumina.
Cellulose and derivatives:
It is used only as support for the stationary liquid phase in partition TLC, in the same
way as sheet of cellulose paper acts as a support in paper partition chromatography.
These adsorbents are commercially available in various forms e.g., particle size, degree
of acetylation, with or without binders like starch or Plaster of Paris.
Since TLC plates are prepared by spreading an aqueous slurry of the finely ground
adsorbent over the surface of a glass plate or a microscope slide. The type of mechanism
involved in separation depends upon the composition of the layer and the manner in
which it has been prepared.
For example. if a silica gel film is deposited from aqueous solution and allowed to
set up at room temperature, the particles remain coated with a thin film of water. If
the sample is then developed with an organic solvent. Separation will most likely
be the result of a liquid-liquid partitioning. On the other hand, if the silica film is
dried by heating, partition may involve solid - liquid adsorption phenomena.
S.no Name of the Adsorbents Composition Adsorbent : Water
1 Silica gel H Silica with out binder 1 : 1.5
2 Silica gel G Silica with Calcium sulphate 1 : 2
3 Silica gel GF254 Silica + binder & Fluorescent indicator 1 : 2
5 Cellulose powder Cellulose powder with binder 1 : 6
6 Keiselguhr G Diatomaceous earth + binder 1 : 2
7 Polyamide powder Polyamide 1 : 9
8 Alumina G Al 2O3 + Binder 1 : 2
9
Alumina
Neutral
Acidic
Basic
Al 2O3 without Binder
1 : 1.1
d) Mobile phase
The solvent or the mobile phase used depends upon various factors as mentioned
in column chromatography. Some of the factors are:
1. Nature of the substances to be separated
2. Nature of the stationary phase used
3. Mode of chromatography (Normal phase or reverse phase)
4. Separation to be achieved – Analytical or preparative
The developing solvent must be of high purity. The presence of small amounts of
water or other impurities can produce irreproducible chromatograms.
The following gives a list of solvents (of increasing polarity)
 Petroleum ether, Carbon tetrachloride, Cyclohexane, Carbon disulfide, Ether,
Acetone, Benzene, Toluene, Ethyl acetate, Dichloromethane, Chloroform,
Alcohols, Water, Pyridine, organic acids, inorganic acid.

14. 20-Apr-20
 For silica gel TLC, the four solvents used most often are hexane as the diluent, with
dichloromethane, chloroform and methyl tert-butyl ether (MTBE) or diethyl ether.
 In case RP-TLC Two-solvent mixtures composed of water plus an alcohol, acetonitrile,
acetone, dioxane, or an ether are widely used, with methanol-water (8:2) a convenient
first-try solvent for C8 or C18 layers.
e) Reagents:
Since many compounds separated by thin layer chromatography are colorless, their
positions are thus located or detected with the help of some reagents, known as locating or
detecting reagents. Iodine vapor and sulphuric acid (mixed with aromatic aldehydes or
oxidizing agents like HNO3, chromic acid etc.) are common locating reagents. Other
reagents include Ferric chloride, 3,5-Dinitro benzoic acid, Ninhydrin, ceric sulfate and
Dragendroff’s reagent.
e) Pipette
Very thin glass pipette is used to load the samples on the stationary phase of TLC film.
f) Forceps
They are used to handle the TLC plates during and after experiment.
Working on Thin Layer Chromatography (protocol)
The following steps are involved while working on TLC.
1. Preparation of TLC Plates
At first, the suspension or slurry of coating material is prepared with water in a
stoppered conical flask by vigorous shaking for 3 minutes. Usually 40 g of silica is
mixed with a small amount of inert binder like gypsum and 80 ml of water to make 5
plates of 0.5mm thickness. Then the slurry is applied on the solid surface that acts as
stationary phase by one of the following methods as described below.
a). Pouring
In this technique, TLC plate is kept on a level
surface and the measured amount of slurry of
adsorbent is poured on plate. Then the plate is
tilted back and forth to spread slurry of adsorbent
and finally dried. The disadvantage is that
uniformity in thickness can not be ensured.
b). Dipping
In this technique, two TLC plates at a time, back to back are
dipped in slurry of adsorbent in chloroform or other
volatile liquid. It is a convenient process for making
several plates for rapid qualitative separation. After
dipping plates are allowed to dry for 5-10 min and if made
with aqueous slurry, it is further dried and activated at 100
°C for 30 min.
Disadvantage is thickness of the layer is not known as well
as unevenness in the layer.
c). Spraying
In this technique, sprayer is used to distribute slurry of adsorbent on TLC plate.
Nowadays this technique is not used.
Uneven coverage by spray and lack of reproducibility is the problem here.
d). Spreading
In this technique, the slurry of adsorbent is
placed on applicator (reservoir) and the
applicator is either moved on the stationary
phase or it is kept static and TLC plate is
pushed or pulled through the applicator. The
thickness of the adsorbent layer is adjusted
by using a knob in the spreader.
Advantage: It gives quite uniform thin layer on the glass plates.
2. Activation of Adsorbent
Once the TLC plate is prepared, it is dried by keeping it in air for 30 min and then,
it is kept in oven for another 30 min at 110 °C for the complete evaporation of
solvent. This drying technique activates the adsorbent layer on TLC plate. The
most commonly used active layers on TLC plates are silica gel and alumina. These
adsorbents on TLC plate can be activated by heating at 150 °C for 4 h.

15. 20-Apr-20
3. Preparation of Mobile Phase
 Proper solvent selection is perhaps the most important aspect of TLC, and
determining the best solvent may require a degree of trial and error.
 If a development solvent of too high a polarity is used, all components in the mixture
will move along with the solvent and no separation will be observed (Rf’s will be too
large). If the solvent is of too low a polarity like pentane & hexane, the components
will not move enough, and again separation will not occur (Rf’s will be too small).
 Typically an effective solvent is one that moves all components of your mixture off
the baseline, but does not put anything on the solvent front. The best developing
solvent should give an Rf value of 0.3 to 0.7 for the desired compound and a ∆Rf of at
least 0.1 between the desired compound and any impurities (For column
chromatography the correct solvent system should give an Rf b/w 0.2 & 0.3).
 In practice, different solvents or mixtures of solvents are tried until a good
separation is observed.
 A common starting solvent is 1:1 hexane : ethyl acetate. Varying the ratio can have
a pronounced effect of Rf .
4. Marking on TLC Plate
A line about 1 cm is marked below the
top of TLC plate and another line about
2 cm from the bottom (base line).
Vertical marks are made on the base
line to represent the actual points of
samples application. Care must be taken
during the marking on TLC plate so that
the adsorbent material should not be removed from the sides, top, or bottom of
TLC plate. If more than one sample is applied on the plate, then there should be at
least 2 cm distance b/w the two spots.
5. Preparation of Sample and Standard Solutions
Samples and standards are applied in the form of 0.1-1 % solutions in a nonpolar,
volatile solvent. The solvent should be nonpolar to minimize the spreading at the
point of application. It should also be volatile, so that it quickly evaporates.
6. Spotting on TLC Plate
For spotting the sample and standard solution on TLC plate, capillary tube,
micropipette, calibrated glass tube or Hamilton-type micro syringe is used. 2-5µl
of sample solution is spotted. The diameters of the spots should be between 2
and 4 mm. Care must be taken not to disturb the adsorbent surface when
applying the solution.
Application of a large sample, in case of
preparative TLC, by repetitive spotting is both
tedious and unsatisfactory. A convenient
method is to use a commercial band applicator.
7. Development
Development in TLC refers to the process by which the
developing solvent moves through the sorbent layer.
Developing chambers
It can be a specially designed chamber, a jar with a
lid, or a beaker with a watch glass on the top.
The specialized chambers can be cylindrical in
shape, rectangular and may be single or double
troughed or of some other shape. The actual design
of chamber depends on whether ascending or
descending or some other type of chromatography
is to be used.
The solvent is poured into the chamber to a depth
of just less than 0.5 cm. To aid in the saturation of

16. 20-Apr-20
the TLC chamber with solvent vapors, you can line part of the inside of the chamber
with filter paper. This will act as a wick and thus saturates the chamber with solvent
vapors. Then the beaker is covered with a watch glass and allowed to stand for a
while. After this, TLC plate is placed in it for development.
Chromatography in saturated atmosphere have the following advantages.
 It yields straight solvent fronts
 Developing time is reduced to one third.
 Rf values are much less than in unsaturated ranks.
Development technique
Different development techniques are used for
efficient separations. They are:
i) Ascending technique
In this technique the sample is spotted at one end of the plate
and then placed vertically in a container saturated with
developer vapor. The solvent percolates through the sorbent
material by capillary action (Against gravitational force) moving
the components to differing extents, determined by their
distribution coefficient, in the direction of flow of the eluant. This
is the simplest technique and remains the most popular.
ii) Descending technique
In descending technique, the top of the plate, where the spots
are located, has solvent from a trough fed onto it via a wick;
some solvent of the same composition is placed in the bottom
of the tank but the plate is supported above the solvent level.
Since the apparatus is more cumbersome, this method is
rarely used.
In order to improve the resolution for particularly difficult separations a number
of modifications to the above technique have been developed.
iii) Continuous development
When there are small differences in RF values, the development distance is
increased in order to achieve complete separation. This is done by continuous
development, in which a solvent is forced to run over the edges of the
chromatographic plates, where it can be collected instead of being left to
evaporate. This method is often conducted by descending techniques, but it can
also be applied to ascending technique. In fact continuous development combines
some of the features of column chromatography with those of thin layer
chromatography. Separated compounds can be isolated by collecting the eluate
from the end of the plate. Modifying descending development to continuous one
has the advantage of quicker solvent flow, due to the action of capillary and
gravity forces on the eluate.
iv) Horizontal development
The chromatographic plate
with samples applied at one
or both ends is placed facing
downward on the edge of
solvent troughs. Only a
small part of the plate is in
contact with the mobile
phase through small
capillary slits in the spacer.
Capillary forces move the mobile phase toward the middle of the plate. The
development is finished as the mobile phase (from the left and the right) has
almost reached the middle of the plate. The main advantage is that the plate can be
used for many more samples.

17. 20-Apr-20
v) Multiple Development
In this technique the plate is developed a number of times in
solvent, the plate being dried between each development. This
significantly improves resolution for substances with Rf values
below 0.5 (poorly resolved solutes). This procedure may
be repeated as many times as necessary to achieve a
satisfactory separation. Multiple development are performed
over same separation distances, using the same solvents.
Advantages:
 Enhanced efficiency.
 Separation of wider polarity range of analytes.
vi) Stepwise development
This technique is used to separate a mixture containing solutes of widely different
polarities. For example, to separate polar additives in a lubricating oil, the plate is first
developed with a nonpolar mobile phase such as toluene for the full length of the
plate. This moves the hydrocarbon base oil to the top part of the plate (15-18 cm),
leaving the polar additives undisturbed on the start point. The plate is then removed
from the developing chamber, the mobile phase evaporated, and the plate redeveloped
the normal distance of 10 cm in a polar mobile phase to chromatograph the polar
additive mixture.
vii) Gradient development
During gradient elution, the plate is vertically developed with a certain mobile phase.
Capillary forces cause mobile phase flow. After the elution is completed, the plate is
removed from the chamber and the mobile phase evaporates. After this, the plate is
placed in a new chamber with a mobile phase having a different elution strength.
These steps can be repeated several times Gradient elution is typically used in those
cases where the range of polarity between the analytes is large.
viii) Two dimensional technique
Complex samples that are not completely
separated with the conventional vertical
development can be subjected to two-
dimensional TLC. In this case, only one
sample is applied at one of the lower
corners of the rectangular plate and the
plate is developed and dried. Following
this, the plate is rotated 90° and
developed with a second (different)
mobile phase.
Advantages:
 More complex samples can be separated using this mode of TLC,
Disadvantage:
 It is not possible to run more than one sample at a time.
ix) Circular Development
Mobile phase is slowly applied to the center of a circular
sample spot in the middle of a horizontal TLC plate.
Sample components move and separate outward in the
form of concentric rings.
 Once the development of TLC plate is done
through ascending, descending or any
other method, TLC plate is taken out and
solvent front is quickly marked on TLC
plate. Then TLC plate is allowed to be air
dried.

18. 20-Apr-20
8. Detection
If the analyte is colored, then it can be visually detected easily but for the
detection of colorless analytes, two types of techniques are used:
Destructive Techniques
In these techniques, specific reagent, e.g., ninhydrin and stannus chloride is used
in the form of spray on TLC slide. Ninhydrin and stannus chloride react with
analyte and produce specific color depending on the nature of analyte. 1%
vanillin reagent in sulfuric acid is also used to visualize most of the organic
compounds except some alkaloids for which Dragendorff’s reagent is used.
Non-destructive Technique
In this technique, different methods are used. For example, for the detection of
radioactive materials, Geiger Muller counter is used, whereas for the detection of
fluorescent compounds, UV chamber is used. Iodine chamber is also used for the
detection of analyte on TLC plate.
Retardation Factor (Rf Value):
o In TLC, the movement of a compound is described using the retardation factor Rf.
o The Rf of a compound is defined as the ratio of the distance moved by the
compound to the distance moved by the mobile phase (solvent front) from the
origin line. Mathematically;
where dR is the distance that the analyte has
traveled (migrated) from the origin spot and dM is the
distance that the solvent front has traveled. It is a unit
less quantity.
The larger the value for Rf, the lower the interaction
between the compound and the stationary phase.
Rf values range from 0 (no elution) to 1 (no affinity for stationary phase) and the value
for Rf is specific for a compound when the chromatographic conditions are kept
constant (same mobile phase, same plate).
When measuring distances for Rf value, linear measurement are drawn from the most
intense (concentrated) portion of the spot, which is usually the estimated middle of
the spot. If the spot is irregular in shape an attempt should be made to locate a center
of mass for measurement.
The relationships between the Rf value and the retention factor (capacity factor) (k)
and between the Rf value and the efficiency (N) are given by:
Where W is diameter of the chromate-
graphic spot after elution.
Advantages
1. TLC is a cheaper chromatographic technique.
2. The purity standards of the given sample can be easily assessed.
3. The separation process is faster and Quick recovery of separated constituents.
4. TLC helps to isolate most of the components.
5. TLC helps with the visualization of separated components spots easily.
6. TLC is a simple process with short development time.
7. TLC helps to identify the individual components easily.
8. TLC mostly requires very simple equipments, such as: micro-slides; specimen jars
with lid; strips of glass sheet; small chromatank etc
9. TLC may be used for adsorption, partition (including reversed phase) or ion-
exchange chromatography
10. In conjunction with densitometric detection, it can be used as a quantitative
technique.

19. 20-Apr-20
Disadvantages
1. It cannot differentiate between the enantiomers and some isomers.
2. Results produced by TLC are very difficult to be reproduced.
3. Only soluble components of interest present in mixture can be separated.
4. As TLC operates in open system, environmental factors like temperature and
humidity may interfere with the overall results.
5. Sensitivity is often limited
6. Not suitable for volatile compounds
7. Requires more operator skill for optimal use than HPLC.
Applications
1. Used in pharmaceutical and natural product analysis.
2. Used for the separation of active pharmaceutical ingredients (API) from the
mixture and sample solutions.
3. Used to determine impurities in pharmaceutical raw materials and formulated
products.
4. Identification of API from the mixture during the synthesis of API, namely:
morphine in apomorphine .
5. Used in pharmacokinetic studies of pharmaceutical products.
6. Determination of essential oils in herbal drugs and assay of herbal medicines.
7. Used for the isolation and identification of carotenoid pigments.
8. Used for the detection of proteins and peptides.
9. Potentially useful in cleaning validation, which is part of the manufacture of
pharmaceuticals.
10. Used for detection of Foreign alkaloids present in alkaloidal drugs, for instance:
atropine sulphate ; codeine;
11. Used for detection of foreign steroids present in steroidal drugs, for example:
betamethasone valerate;
12. Analysis of environmental samples for the detection of various pollutants.
13. Used for the identification of pesticides and toxins present in food items.
14. Used for the screening and detection of drug abuse in urine samples.
15. In clinical laboratories, TLC is widely used for separation of carbohydrates in
biological samples.
16. TLC is the most widely used for the routine analysis of porphyrins.
17. Used for detection of homocysteine, lipids, and protein antigens.