CHROMATOGRAPHY

Separation Methods: A technique by which a mixture of components is resolved into individual

components is known as separation technique. Separations may be performed by making use of many
different methods and techniques which are based on sound fundamental chemical and physical
properties. The area of separation has rapid growth in recent years. A separation may be used for
purification, qualitative identification and quantitative determination. Many different separation
methods now are available some of which are given below:
S.No.
1
2
3
4
5

Separation Method
Precipitation
Distillation
Sublimation
Crystallization
Floatation

Ultrafiltration

7
8

Extraction
Ion exchange

Chromatography

Basis
Difference in solubilities
Difference in the boiling points
Difference in vapour pressures
Difference in solubilities
Difference in density between substance
and liquid
Difference in particle size of the
substances
Difference in solubility
Difference in exchangeability of ions
with another ion
???

CHROMATOGRAPHY: The term chromatography (Greek: Khrmatos - colour and graphos written)
was first discovered by Mikhail Tswett, a botanist and physical chemist in 1906 based on his
experiments. Of all the available separation methods, chromatography has the unique position of being
applicable to all types of problems in all areas of science.
Mikhail Tswett, in his experiment, a leaf extract sample in petroleum ether was allowed to pass
through a column of CaCO3. Pure ether was continued to flow through the column, as a result of which
various chlorophyll pigments, etc., were separated into a series of differently coloured and easily
distinguished zones. In his first monograph he writes:
If a petroleum ether solution of chlorophyll is filtered through a column of adsorbent, CaCO3,
then the pigments, according to their adsorption sequence, are resolved from top to bottom into various
coloured zones, since the stronger adsorbed pigments displace the weaker adsorbed ones and force them
further downwards. So the different components of the pigments are resolved on the CaCO3 column
according to a law and can be estimated on it qualitatively and also quantitatively. Such a preparation I
term a chromatogram and the method, the chromatographic method.

The chromatographic technique consists of two phases

viz. stationary phase and mobile phase. The stationary phase
may be a solid or liquid or liquid supporting on solid and a
mobile phase may be a liquid or solution or gas. When the
mixture of constituents to be resolved is in contact with both the
phases, the separation of mixture into individual components
may be carried out by means of difference of one of physical or
chemical properties of the constituents of the mixture. For

Concentration of
substance

Chromatography may be regarded as an analytical technique employed for the purification and
separation of organic and inorganic substances.

Time of elution or volume

of eluent

Chromatogram

example, in the adsorption chromatography, the separation of mixtures is carried out due to difference in
the adsorbabilities of constituents of the mixture. When the mixture is allowed to pass through
stationary phase, since constituents adsorb on the stationary phase with different affinities, strongly
adsorbed one will replace the weakly adsorbed one, pushes to downward and adsorb at the top of the
column, while the weakly adsorbed constituent will adsorb below the first one. When a suitable solvent,
which is known as eluent, is allowed to pass through the column, weakly adsorbed constituent comes
first from the column and then the strongly adsorbed constituent. When the concentrations of
constituents in the eluent are plotted against time or volume of eluent, the spectrum obtained is known as
chromatogram.
Classification of chromatography: Chromatography may be classified into following, based on the
principle on which the separation of the components is carried out.
a) Adsorption Chromatography: The technique in which separation of the components will be carried
due to difference in the adsorbilities of components on the surface of the adsorbant is called
adsorption chromatography. In this technique the stationary phase is solid and the mobile phase is
either a gas or a liquid. When a solute is in contact with two phases it distributes between them due
to adsorption. The distribution coefficient, K, is given by
Concentration of solute in stationary phase
K
Concentration of solute in mobile phase

b) Partition Chromatography: When a solute is in contact with a liquid stationary phase and either a
gaseous or liquid mobile phase, it will distribute between the two phases. Now the partition
coefficient may be represented by Nernsts distribution law as
Concentration of solute in stationary phase
K
Concentration of solute in mobile phase
Separation of the mixture of components can be made if there is a difference in the partition
coefficients of the components. Such technique is called partition chromatography.
c) Ion exchange Chromatography: Ion exchange resins consist of high polymeric, cross linked high
molecular weight organic materials containing large number of negative or positive groups and
counter cations or anions. If the counter ion is cation the resin is called as cation exchange resin
while anion is the counter ion then it is called as anion exchange resin. When a solute containing
ions is in contact with the ion exchange resin, the counter ions of the resin can be exchanged by the
ions of solute. If there is difference in the exchangeability of different ions present in the sample
with counter ion of the resin, the sample can be separated. Such technique is called ion exchange
chromatography.
d) Exclusion Chromatography: Chromatographic process in which separation of the sample takes
place according to molecular size is called exclusion chromatography. Gel permeation is widely
using for the separation of sugars, polypeptides, proteins, liquids, polymers.

S.No.
1

Chromatographic method
Adsorption chromatography

Partition chromatography

3
4

Ion exchange chromatography

Exclusion chromatography

Basis
Difference in the adsorbability of substances on
the surface of an adsorbent
Difference in the solubility of substance between
two liquids
Difference in the exchangeability of ions
Difference in the molecular weights and sizes

Chromatography may also be classified, based on the stationary and mobile phases.
S.No.
1
2
3
4
5
6
7
8
9

10

Chromatographic
Method
Liquid Chromatography

Stationary
Phase
Solid or liquid

Mobile
Phase
Liquid

Liquid Solid
Chromatography (LSC)
Liquid liquid
chromatography (LLC)
Liquid exclusion
chromatography (LEC)
Ion exchange
chromatography
High performance liquid
chromatography (HPLC)
Gas Chromatography
(GC)
Gas liquid
chromatography (GLC)
Paper chromatography

Solid

Liquid

Liquid

Liquid

Liquid

Liquid

Thin layer
chromatography (TLC)

Solid (Ion
exchange resion
Solid

Basis
Adsorption, Partition or
Ion exchange
Adsorption or ion
exchange
Partition

Molecular weights and

sizes
Solution Exchange of ions
Liquid

Adsorption or Partition

Solid

Gas

Adsorption

Liquid

Gas

Adsorption

Liquid
supporting on
paper
Solid

Liquid

Adsorption or partition

Liquid

Adsorption

PAPER CHROMATOGRAPHY
Paper chromatography was first introduced by Schonbein in 1961 under the name Capillary
Analysis, but the technique became popular in 1963 through the work of Consden, etc. It may be
regarded as an extension of the application of partition chromatography and it is remarkably simple form
of chromatographic methods.
In paper chromatography, a coarse paper serves instead of packed column and the silica gel as
the solid support for the polar phase, is replaced with filter paper. An organic solvent, partially miscible
with water (ex. butanol) is most suitable in paper chromatography.
Paper chromatography may therefore be defined as the technique in which the analysis of an
unknown substance is carried out mainly by the flow of solvent on specially designed filter paper. The
two solvents used in paper chromatography are immiscible or partially miscible with each other.
Types of paper chromatography
There are two main types of paper chromatography.
Paper partition chromatography: Paper partition chromatography is a technique in which paper is used
as support with one solvent as stationary phase and the other solvent is mobile phase.

Paper adsorption chromatography: In this chromatography, a modified paper impregnated with an

adsorbent, like silica alumina is used as an adsorbent and the solvent is allowed to flow over the
unknown components.
Principle: Paper chromatography may be regarded as a type of partition chromatography in which the
stationary phase is water absorbed on the hydrophilic surface of the paper. The organic solvent then acts
as a mobile phase. The general principles developed for column chromatography are also applicable as
well to paper chromatography. Thus paper may be regarded as consisting of a series of theoretical
plates.
A drop of the solution containing sample is introduced at some point on the filter paper, and
when the edge of the paper is in contact with the suitable solvent which is known as developer, the
solvent migrates due to the capillary action. This process is called development. When the movement
of mobile phase is in upward direction, the development is known as ascending development, and when
the movement of mobile phase is in downward direction, the development is known as descending
development. In this process the constituents of the sample also migrate along with the mobile phase.
Since the constituents of the sample are now in contact with both mobile phase and stationary phase,
they distribute between both the phases. Paper may be regarded as consisting of a series of theoretical
plates, at each plate, equilibrium occurs according to the relation
K

C
Conc of constituent in the stationary phase
s
Conc of constituent in the mobile phase
Cm

where K is partition coefficient.

Since there is difference in the distribution of constituents in the mobile and stationary phases,
the constituents move with different velocities and retain on the paper as spots at different positions.
The positions of the spots can be determined either by physical means or by chemical means and they
can be expressed by their retention factor (RF), as

RF

Dis tan ce travelled by the component from the origin

Dis tan ce travelled by the solvent from the same place

The RF values of the components of a mixture can be compared

Location of spots and measurement of RF value
The drop of solution of the mixture to be chromatographed is applied at about one centimeter
from one end of the paper, by means of a capillary tube and the paper is allowed to dry. The position of
the sample on the paper at which it is applied is marked with the help of pencil. The paper is then placed
in a container containing a suitable solvent, called developer. The two ends of the paper should not be
bent, so that it is hanged with the help of clips. Now the container is covered with lid to avoid loss of
solvent due to evaporation. After some time the solvent moves up by capillary action and carrying the
constituents of the sample along with, with different speeds, according to their partition coefficients.
After the solvent traversed sufficient length, the paper is taken out of the container, the position of the
solvent front upto which it is moved, is marked with pencil and the paper is again allowed to dry.
The locations of the constituents of the sample are identified either by means of physical methods
or by means of chemical methods. If the substances are coloured, their positions can be identified easily
because they form coloured spots or strains in the paper. If the substances exhibit fluorescence when

held under UV light, their positions can be determined under UV light. When the physical methods can
not be used, the positions of the constituents are identified by using chemical methods. In chemical
methods, the colourless substances form coloured spots with some reagents, which are known as
visualizing or locating reagents. In chemical methods, suitable locating reagent (for example, ninhydrine
for identifying the amino acids) is sprayed on the paper, which develops coloured spots with constituents
and they can be identified.
The position of the components can be expressed by their retention factor (RF), which may be
defined as:
RF

Dis tan ce travelled by the component from the origin line

Dis tan ce travelled by the solvent from the origin line

Suppose a mixture contains three different components A, B and C which travel through a
distance of d1, d 2 and d3 respectively and solvent travels distance of d as shown in the fig. So the RF
values of the components are
d
d
d
R F ( A) 1 ;
RF ( B) 2 ;
and
R F (C ) 3
d
d
d
The RF value depends on various factors:
a)
b)
c)
d)
e)

The nature of the solvent (mobile phase)

The medium used for separation or the quality of filter paper used
The nature of mixture to be separated
The temperature
The size of the container in which the experiment is performed

The RF values of the components of a mixture can be compared with known RF values by
keeping the above factors as nearly constant as possible. It should be noted that the similar RF values do
not indicate that the components are identical, because different components may have identical RF
values in a given solvent system. For example, RF values of amino acids alanine and tyrosine in phenol
water are same (0.55). It is therefore necessary to use different solvent systems for identifying the
compounds.
Technique of paper chromatography:
a) Selection and preparation of paper: The basic material of the paper used in paper
chromatography is the mixture of 98.99% -cellulose and the remaining is -cellulose. The
choice of paper depends upon the thickness, flow rate, purity and net strengths. For example,
Whatmann No. 4 is used when speed of development is to be taken into consideration, while No.
20 is suitable when a good resolution is required. If the paper does not have requisite properties,
it can be modified to achieve the desired properties. The papers can be modified by several
ways, for example, they can be impregnated with diato-maceous earth, silica gel and ion
exchange resins etc. After selecting the required quality of paper, then it is prepared in the
required size and shape, because paper chromatography depends on the size and shape of the
paper, especially when descending development technique is to be used.
b) Preparation of sample: Normally paper chromatography is applied for liquid samples. If the
sample is solid, it may be dissolved in suitable volatile solvent and a minimum amount of this
solution is then applied on the paper carefully by suitable means.

c) Choice of solvents: Paper chromatography is essentially a partition chromatography, there are

wide verity of combinations of stationary and mobile phases. The selection of stationary and
mobile phases purely depends on the nature of sample to be analyzed.
Stationary phase: The stationary phases that are used can be classified as follows:
i) Aqueous stationary phase: Water is best example for this which is rapidly held by paper.
ii) Hydrophylic stationary phase: Methanol, formamide, glycol, glycerol etc are the common
solvents.
iii) Hydrophobic stationary phase: Kerosene, aromatic and aliphatic hydrocarbons etc.
The paper is equilibrated with stationary phase by suspending the paper in a closed chamber,
whose atmosphere is saturated with vapours of stationary phase
Mobile phase: Numerous combinations are possible for mobile phase. Individual solvents,
mixtures of two or more solvents of different compositions, solution of salts, buffer solutions are
generally used. For example, isopropanol ammonia water (9 : 1 : 2), n-butanol acetic acid
water (4 : 1 : 5) are suitable for separation of hydrophilic substances and kerosene 70%
isopropanol etc are suitable for separation of hydrophobic substances.
In general, one phase systems are much used in paper chromatography than two or three
phase systems.

Samples

Solvent flow

Solvent flow

d) Development: The method of

paper chromatography may be one
dimensional or two dimensional,
depending upon the nature of the
sample to be analyzed. A typical
arrangement of one dimensional
paper chromatography is shown in
the fig.

Developer

Developer

A strip of selective filter

Ascending
Descending
development
development
paper usually 15 to 30 cm in
length and few cm in width is laid
flat. A line was drawn with pencil at about one cm from the one end. A minute drop of sample
solution is placed on the line and the position of the sample is marked with pencil. The original
solvent in which the sample is dissolved is allowed to evaporate. The portion of the paper
nearest to the sample is then brought in contact with a suitable solvent, called developer in a
suitable container. Now the container is closed with air tight lid to prevent loss of solvent due to
evaporation. After some time the solvent moves either upward against the gravity (ascending
development) or downward (descending development) by capillary action and carrying the
constituents of the sample along with it, at different speeds, according to their partition
coefficients. After the solvent has traversed to sufficient length, the paper is removed from the
solvent, the position of the solvent upto which it moves is marked with pencil and then it is dried
by keeping the paper in an oven for few minutes. The finished dried paper is called
chromatogram.
e) Identification of position of constituents: The locations of the constituents of the sample are
identified either by means of physical methods or by means of chemical methods. If the
substances are coloured, their positions can be identified easily because they form coloured spots
or strains on the paper. If the substances exhibit fluorescence when held under UV light, their

positions can be determined under UV light. If the sample is radio active the positions of the
constituents are identified with the help of radiation counters. When the physical methods can
not be used, the positions of the constituents are identified by using chemical methods. In
chemical methods, the colourless substances form coloured spots with some reagents, which are
known as visualizing or locating reagents. In chemical methods, suitable locating reagent (for
example, ninhydrine for identifying the amino acids) is sprayed on the paper, which develops
coloured spots with constituents and they can be identified. Physical methods for identification
of the locations of sample are always advisable than chemical methods.

The position of the components can be expressed by their retention factor (RF), which may be
defined as:

RF

Dis tan ce travelled by the component from the origin line

Dis tan ce travelled by the solvent from the origin line

The RF values of the components of a mixture can be compared with known RF values by
keeping the above factors as nearly constant as possible. It should be noted that the similar RF values do
not indicate that the components are identical, because different components may have identical RF
values in a given solvent system. For example, RF values of amino acids alanine and tyrosine in phenol
water are same (0.55). It is therefore necessary to use different solvent systems for identifying the
compounds.
Precautions in Paper Chromatography
The following are some important precautions to be taken in paper chromatography.
1. Precautions must be taken in establishing the vapour solvent equilibrium.
2. In order to get reproductibility, the stability of the solvent mixture is first ensured
3. Heavy metals and complexing agents should not be used as they convert the solute into a complex
with different properties resulting multiple spots.
4. Natural salts should not be used as they disturb the cellulose water complex, which will result in
separation of liquid water on paper.
Applications of paper chromatography
Applications of paper chromatography are numerous and appear to be endless. It has been
widely used for qualitative and quantitative analysis for various substances. The following are some
important applications of paper chromatography.
a) In qualitative analysis: The sample and standards are chromatographed in similar conditions and RF
values of the components of the sample and standards are determined in several solvents and they are
compared and then the nature of the components of the sample are identified.
b) In quantitative analysis: Paper chromatography has been widely used for quantitative analysis of
inorganic, organic and biochemical compounds, but inorganic separations are quite possible. The
chromatogram of the sample is obtained by developing the sample in a suitable solvent. The amount
material in each spot can be measured either by direct methods or by prior elution methods. The
method by which the compound is to be analysed depends on the properties (both physical and
chemical) of the compound, composition, capacity of the system and the degree of the accuracy
required.

c) Purity of the substance: Paper chromatography can also be used in checking purity of the substance
and in checking other separation methods. For example, a small amount of purified substance is
tested and developed by paper chromatography. If a single spot is observed on the paper, then it is
conformed that the compound is pure, while if two or more spots are observed, the compound
contains impurities.
d) In process: Since paper chromatography is simple, it is ideally suited for rapid analysis of reaction
mixture of process. Whether the process is completed or not can be tested by analyzing the reaction
mixture collected from the process.
e) As a separation technique: Paper chromatography can be used successfully for the separation of
complex mixture of drugs, metal ions, dyes, amino acids, sugars etc. One of the classical example is
the separation of amino acids. The sample and the standard amino acid solutions are spotted on
Whatmann filter paper No. 1 (8 x 10 inch) at 0.5 inch from the bottom. Now the paper is developed
in a mixture of 95% of ethyl alcohol and 5% water. The chromatogram is dried and then 0.2% of
ninhydrin solution in water is sprayed. The different amino acids appear as blue like spots.
Qualitative identification may be carried out by comparing the RF values of each amino acid.
Quantitative analysis may also be made by comparing the intensity of the colour with standards.
THIN LAYER CHROMATOGRAPHY
Thin layer chromatography (TLC) was first discovered by Izmailev and Shraiber in 1938, but it
was further developed by Meinhard and Hall et all and popularized by E. Stahl. TLC is a simple,
versatile, inexpensive separation technique. In TLC, separation of mixture is carried out on the
stationary phase of thin layer coated on a glass, metal or plastic plate by adsorption, partition, exclusion
or ion exchange process, but the first two are the most important. The TLC has several advantages over
paper chromatography, some of them are mentioned below:
1. TLC requires less time, (15 45 min) so it can be employed for checking the industrial process
control.
2. It requires less amount of substance than paper chromatography.
3. It can be performed on an analytical as well as a preparative scale.
4. The sharpness of TLC is very high.
5. Compounds which are encountered in trace amounts (e.g. narcotics, air pollutants, pesticides etc)
can be readily detected due to its high sensitivity.
6. Strong acids can also be used for identification of position of constituents, where as it not
possible in paper chromatography.
7. The substances can be easily recovered by removing the spots of the TLC.
8. TLC plates may be heated for several hours in an oven, without causing any damage to the
plates.
9. In TLC even corrosive reagents may be coated on glass plates.
Principle of Thin Layer Chromatography
In general, TLC is carried out experimentally like a sheet method but the sheet exhibits the
properties of a column. So the TLC is working on the principle of column chromatography. The
separation of the mixture by TLC is normally carried out on the thin layer of stationary phase coated on
glass, metal or plastic plate. All chromatographic principles such as adsorption, partition, exclusion and
ion exchange chromatography are applicable to TLC. Adsorption and partition chromatography have
widely used, but the choice of the chromatographic principle is determined by the chemical nature of the
compounds to be resolved.

The thin layer of stationary phase of TLC may be regarded as consisting of a series of theoretical
plates. The resolution of the sample is now carried out at each plate due to difference in the adsorption,
or solubility etc.
As like paper chromatography, a drop of the solution containing sample is introduced at some
point on the thin layer, and when the edge of the layer is in contact with the suitable solvent which is
known as developer, the solvent migrates due to the capillary action. This process is called
development. In the development process the constituents of the sample also migrate along with the
mobile phase. Since the constituents of the sample are now in contact with both mobile phase and
stationary phase, they distribute between both the phases. The thin layer of the stationary phase may be
regarded as consisting of a series of theoretical plates, at each plate, equilibrium occurs according to the
relation
K

C
Conc of constituent in the stationary phase
s
Conc of constituent in the mobile phase
Cm

where K is partition coefficient.

Since there is difference in the distribution of constituents in the mobile and stationary phases,
the constituents move with different velocities and retain on the layer as spots at different positions. The
positions of the spots can be determined either by physical means or by chemical means and they can be
expressed by their retention factor (RF), as

RF

Dis tan ce travelled by the component from the origin

Dis tan ce travelled by the solvent from the same place

The RF values of the components of a mixture can be compared with RF values of known
substances obtained in the similar conditions.
Experimental Techniques: The experimental technique can be divided into six basic operations. These
are
a) Selection of adsorbent: In general, TLC is carried out experimentally like a sheet method but the
sheet exhibits the properties of a column technique. Therefore, any material that used in column
chromatography as stationary phase, can be used in TLC as stationary phase, provided the stationary
phase is available in small enough particle size and held in some way in sheet form. The thin layer
should be consistent throughout.
The common adsorbents used in TLC are silica gel alumina, kieselguhr and powdered cellulose.
Silica gel is the most widely used adsorbent is slightly acidic in nature. A binding agent, usually
plaster of Paris is often incorporated to hold the adsorbent firmly on the plate. While choosing the
adsorbents, one must consider the characteristics of the compounds to be separated.
b) Preparation of Thin layer plates
i) Application of adsorbent on the plates: The mixture of binding and adsorbent is made into slurry
with some solvent and smeared on the glass, metal or plastic plate. To apply uniform thick layer,
the plates must be flat and clean. Various methods are available to apply the slurry onto plates are
as follows:

a) Pouring: In this method, a measured amount of the slurry is put on a given size plate which is
kept on level surface. The plate is then tipped back and forth to spread the slurry uniformly
over the surface of the plate. Though this method is simple, yet is not used because it is
difficult to obtain uniform layers.
b) Dipping: In this method, plates are prepared by dipping the two plates jointly at a time, in
chloroform or chloroform-methanol slurries of the adsorbent. This method is also not of much
use.
c) Spraying: In this method, the slurry is sprayed with small point sprayers for distribution of the
slurry on the plate. This technique is also not in use because it is difficult to obtain uniform
thick layers.
d) Spreading:
In
this
method, the slurry is
placed in an applicator
(Fig) which contains a
narrow slit at the bottom.
The slurry is applied on
the plate in the form of
uniform thick layer by
Spreading Applicators
either
moving
the
applicator over the stationary plate or the applicator is held stationary and the plate is moved
through. The thickness of the layer is controlled by the number of tape layers that are used.
The thickness of the thin layer can be made from 0.1 2 mm by using this method.
ii) Activation of adsorbent: After making thin layers on the plates, they are dried for 30 minutes in
air and then in an oven at 1100C for another 30 minutes to remove solvent associated with the
layer. This drying process makes the adsorbent layers active. In order to obtain very active layers,
silica gel and alumina plates can be heated to 1500C for about 4 hours.
iii) Purification of silica gel layers: Silica gel G contains iron as impurity which interferes in the
resolution of the constituents. Therefore it becomes essential to purify the adsorbent. Iron can be
removed by giving the coated and air-dried plates a preliminary development with methanol-conc.
HCl (9 : 1 v/v). The iron gets migrated with solvent front to the upper edge of the plate. The
plates are again dried and activated at 1100C.
c) Sample application: The methods of application of sample on the layers are similar to those used in
paper chromatography. Normally TLC is applied for liquid samples. If the sample is solid, it may
be dissolved in suitable volatile solvent and a minimum amount of this solution is then applied on the
layer carefully with the help of capillaries, micropipettes etc. In analytical TLC, 0.1% solutions of
the sample are normally applied.
d) Choice of developers (Mobile solvents): The choice of solvent depends on the (i) nature of the
substance to be separated and (ii) nature of adsorbent. Almost all solvents which have been used in
the paper chromatography, can also be used in TLC. More polar solvents produce the greater
migration and a better separation. It has been observed that a combination of two solvents give
better results than single solvent. If one does not know about the nature of the compounds of the
mixture to be separated, the best elute (solvent) is found by trial and error method. If one knows the
chemical nature of substance to be separated, it is possible to know suitable solvent by using Stahls
triangle (Fig) which is inter-relating adsorbent activity, nature of solvent and nature of sample. If the

triangle is rotated so that the corner M points to the type of mixture to be separated, the corners S and
E specify the necessary activity of the adsorbent and optimum polarity of the eluent respectively.
e) Development of chromatogram: The development tank used in paper chromatography can also be
used in TLC. Either ascending or descending chromatography can be used in the TLC, however
ascending chromatography is the most common technique in TLC.
A minute drop of sample is applied on the thin layer of normally
20 cm length, at about one cm from one end and the position of the
sample is marked with pencil. The original solvent in which the
sample is dissolved is allowed to evaporate. The plate is placed in the
development chamber at an angle of 45 0C as shown in the fig. The
bottom of the chamber is covered up to nearly 1mm by a solvent. It is
important in TLC that the chamber must be perfectly saturated with
solvent vapour, so as to avoid unequal solvent evaporation from the
developing plate. For this three sides of the chamber are lined with
solvent impregnated paper while top is covered tightly with the lid.
Now the solvent rises upward by capillary action and carrying the
constituents of the sample along with it, at different speeds, according
to their partition coefficients. After the solvent has traversed to
sufficient length, the plate is removed from the solvent, then the
position of the solvent front is marked with pencil and finally it is
dried by keeping the plate in an oven for few minutes.

Solvent front

Thin layer

Solvent

Ascending development
technique

f) Identification of position of constituents: The locations of the constituents of the sample are
identified either by means of physical methods or by means of chemical methods. Most of the
methods used for detecting the positions of the substances on paper chromatography, are also
applicable to TLC. If the substances are coloured, their positions can be identified easily because
they form coloured spots or strains on the paper. If the substances exhibit fluorescence when held
under UV light, their positions can be determined under UV light. If the sample is radio active the
positions of the constituents are identified with the help of radiation counters. When the physical
methods can not be used, the positions of the constituents are identified by using chemical methods.
In chemical methods, the colourless substances form coloured spots with some reagents, which are
known as visualizing or locating reagents. In these chemical methods, suitable locating reagent is
sprayed on the layer, which develops coloured spots with constituents and then they can be
identified. The corrosive reagents like chromic acid and conc. sulphuric acid can be used in TLC as
visualizing agent, but they can not used in paper chromatography. Some important visualizing
reagents are given below. Physical methods for identification of the locations of sample are always
advisable than chemical methods.
S.No.
1
2
3
4
5
6
7

Visualization Reagent
Aniline phthalate
SbCl3 in CHCl3
Bromocresol purple
Bromocresol green
2,4- dinitrophenyl hydrazine
Ninhydrin
Ferric chloride

Type of compounds separated

Reducing sugars
Steoids, glycosides, Vitamin A etc
Halogen ions except F- dicarboxylic acids
Carboxylic acid
Aldehydes and ketones
Aminoacids
Phenols

g) Evaluation of the chromatogram: After detecting the separated constituents on the plate and
marking their positions, the chromatogram is evaluated, and the evaluation may be qualitative or
quantitative.

a) Qualitative analysis: This is same as with paper chromatography. The position of the
components can be expressed by their retention factor (RF), which may be defined as:

RF

Dis tan ce travelled by the component from the origin line

Dis tan ce travelled by the solvent from the origin line

The RF values of the components of a mixture can be compared with known RF values. It
should be noted that the similar RF values do not indicate that the components are identical,
because different components may have identical RF values in a given solvent system.
b) Quantitative analysis: Quantitative results are usually obtained by either two methods.
i) First method: In one method, the constituent which forms spot on the layer is separated from
the sheet and diluted to known volume. Subsequently the concentration of constituent is
measured by using any one of the quantitative method like gravimetric, volumetric, colourimetric
method that depends on the nature of the substance.
ii) Second method: This method is performed directly on the layer. This method involves the
measurement of spot area. The spot areas are computed by a planimeter or by transferring the
zone area to a piece of graph. The amount of substance is measured from area of the spot, since
the square root of the area is directly proportional to the logarithm of the amount of the
substance.
The concentration of the substance can also be measured directly by the technique known as
densitometry.
Applications of Thin Layer Chromatography: The thin layer chromatography has been used for both
qualitative and quantitative analysis in many fields.
1. TLC can be used in the identification of compounds in drugs, biochemical preparations, natural
products, environment and organic chemistry.
2. A botanist can identify easily the flavanoids obtained from plants.
3. A technician can detect trace pesticides in water
4. TLC can be used to identify the presence of poison, illicit drugs etc in the specimens.
5. TLC has been used for checking the purity of the substances
6. It can be helpful in the checking of other separation methods
7. The TLC can be used in the process control as it requires less time.
8. The technique can be useful in the isolation of amino acids. A mixture of 34 amino acids, proteins
and peptides has been successfully separated and isolated from urine using silica gel plates.
9. A large number of vitamins, antibiotics and food products have also been separated by using TLC