Bele et al., IJPSR, 2011; Vol.

2(2): 256-267
IJPSR (2011), Vol. 2, Issue 2

ISSN: 0975-8232
(Review Article)

Received on 28 July, 2010; received in revised form 16 November, 2010; accepted 18 January, 2010

AN OVERVIEW ON THIN LAYER CHROMATOGRAPHY

Archana A. Bele* and Anubha Khale
H. K. College of Pharmacy, Jogeshwari (W), Mumbai, Maharashtra, India

ABSTRACT
Keywords:
Thin layer chromatography,
capillary action,
Mobile phase,
R value

Correspondence to Author:
Mrs. Archana A. Bele
Lecturer, H.K. College of Pharmacy,
Near MHADA Complex,
Jogeshwari(W), Mumbai,
Maharashtra, India

In the present article attempt has been made to explain the

basic ideas and the significance of Thin layer Chromatography
(TLC) in different analytical methods. As TLC is less time
consuming, low cost, and can be performed with less
complicated technique it has a wide application in
pharmaceutical analysis. If performed precisely 32 amino acids
can be separated by TLC. Also it has a wide application in
identifying impurities in a compound. It can be used as a
preliminary analytical method prior to HPLC. The concept of TLC
is simple and samples usually require only minimal
pretreatment. TLC can be used to monitor the progress of a
reaction, identify compounds present in a given substance. TLC
is also used to separate the identical compounds in a mixture.
Many standard methods in industrial chemistry, environmental
toxicology, food chemistry, water, inorganic and pesticide
analysis, dye purity, cosmetics, plant materials, and herbal
analysis rely upon TLC as the preferred approach.

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INTRODUCTION: Thin layer chromatography (TLC)

is a chromatography technique used to separate
mixtures. Chromatography was discovered by M.
Tswett in 1906.Thin layer chromatography is
performed on a sheet of glass, plastic, or aluminum
foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminum oxide, or
cellulose (blotter paper). This layer of adsorbent is
known as the stationary phase. After the sample
has been applied on the plate, a solvent or solvent
mixture (known as the mobile phase) is drawn up
the plate via capillary action. Because different
analytes ascend the TLC plate at different rates,
separation is achieved.
Thin layer chromatography can be used to:
Monitor the progress of a reaction, identify
compounds present in a given substance,
determine the purity of a substance. Separation of
compounds is based on the competition of the
solute and the mobile phase for binding places on
the stationary phase. For instance, if normal phase
silica gel is used as the stationary phase it can be
considered polar. Given two compounds which
differ in polarity, the more polar compound has a
stronger interaction with the silica and is therefore
more capable to dispel the mobile phase from the
binding places.
Consequently, the less polar compound
moves higher up the plate (resulting in a higher Rf
value). If the mobile phase is changed to a more
polar solvent or mixture of solvents, it is more
capable of dispelling solutes from the silica binding
places and all compounds on the TLC plate will
move higher up the plate. Practically this means
that if you use a mixture of ethyl acetate and
heptane as the mobile phase, adding more ethyl
acetate results in higher Rf values for all
compounds on the TLC plate. Changing the polarity
of the mobile phase will normally not result in
reversed order of running of the compounds on the
TLC plate.

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Principle of TLC 1 : Thin layer chromatography uses

a thin glass plate coated with either aluminum
oxide or silica gel as the solid phase. The mobile
phase is a solvent chosen according to the
properties of the components in the mixture. The
principle of TLC is the distribution of a compound
between a solid fixed phase (the thin layer) applied
to a glass or plastic plate and a liquid mobile phase
(eluting solvent) that is moving over the solid
phase. A small amount of a compound or mixture is
applied to a starting point just above the bottom of
TLC plate.
The plate is then developed in the
developing chamber that has a shallow pool of
solvent just below the level at which the sample
was applied. The solvent is drawn up through the
particles on the plate through the capillary action,
and as the solvent moves over the mixture each
compound will either remain with the solid phase
or dissolve in the solvent and move up the plate.
Whether the compound moves up the plate or
stays behind depend on the physical properties of
that individual compound and thus depend on its
molecular structure, especially functional groups.
The solubility rule Like Dissolves Like is followed.
The more similar the physical properties of the
compound to the mobile phase, the longer it will
stay in the mobile phase. The mobile phase will
carry the most soluble compounds the furthest up
the TLC plate. The compounds that are less soluble
in the mobile phase and have a higher affinity to
the particles on the TLC plate will stay behind 1.
R values : The behavior of an individual
compound in TLC is characterized by a quantity
Known as R and is expressed as a decimal fraction.
The R is calculated by dividing the distance the
compound traveled from the original position by
the distance the solvent travelled from the original
position (the solvent front).

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R = Distance of centre of spot from starting point
Distance of solvent front from starting point

The R value is a constant for each component only

under identical experimental condition. It depends
upon number of factors as;
1. Nature of adsorbent: Different adsorbents will
give different R value for same solvent.
Reproducibility is only possible for given
adsorbent of constant particle size and binder.
Plates should be stored over silica gel in
desiccators before use and the sample should
be applied quickly so that the water vapor in
the atmosphere is not adsorbed by the plate.
Because of the difficulties associated with
activation procedures, it is far better to use
plates stored at room temperature and not to
activate them.
2. The mobile phase: The purity of solvents and
quantity of solvent mixed should be strictly
controlled. It should be made freshly for each
run if one of the solvents is very volatile or
hygroscopic. Example- acetone.
3. Temperature: Although precise control of
temperature is not necessary, the tank should
be kept away from sources of heat, direct
sunlight etc. As the temperature is increased,
Volatile solvents evaporate more quickly,
solvents run faster, and R values generally
decrease slightly.
4. Thickness
of
layer:
Standard
plates
approximately 250 micrometer is the
preferable thickness of layer. Below 200, the R
values vary considerably. The layers may be of
higher or lower thickness in individual
compounds.
5. Developing tank: It is important that saturated
conditions are attained for running TLC plates.

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This is best accomplished by using small tanks

with filter paper liners and sufficient solvent,
and by leaving the tank to equilibrate for at
least 30 minutes before running the plates. A
well fitting lid is essential.
6. Mass of sample: Increasing the mass of sample
on the plate will often increase the R of drug,
especially if it normally tails in the system.
However, if a plate is grossly overloaded, this
too will give a tailing spot and will have the
effect of apparently decreasing the R value.
The two situations are normally easy to
distinguish by the intensity of the spot.
7. Chromatographic Technique: Depending upon
the development technique used i.e. ascending,
descending, horizontal etc, the R value change
for the same solvent system.
Plate preparation1: TLC plates are usually
commercially available, with standard particle size
ranges to improve reproducibility. They are
prepared by mixing the adsorbent, such as silica
gel, with a small amount of inert binder like
calcium sulfate (gypsum) and water. This mixture is
spread as thick slurry on an unreactive carrier
sheet, usually glass, thick aluminum foil, or plastic.
The resultant plate is dried and activated by
heating in an oven for thirty minutes at 110 C. The
thickness of the adsorbent layer is typically around
0.1- 0.25 mm for analytical purposes and around
0.5- 2.0 mm for preparative TLC.
Capillary spotters: Place a melting point capillary
and in the dark blue part of the Bunsen burner
flame. Hold it there until it softens and starts to
sag. Quickly remove the capillary from the flame
and pull on both ends to about 2-3 times its
original length. If you pull the capillary inside the
flame, you will have a "piece of art", but not a good
spotter. Allow the capillary to cool down, and then

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break it in the middle. Make sure to break off the

closed end on one of them.
Spotting the plate: The thin end of the spotter is
placed in the dilute solution; the solution will rise
up in the capillary (capillary forces). Touch the
plate briefly at the start line. Allow the solvent to
evaporate and spot at the same place again. This
way you will get a concentrated and small spot. Try
to avoid spotting too much material, because this
will deteriorate the quality of the separation
considerably (tailing). The spots should be far
enough away from the edges and from each other
as well. If possible, you should spot the compound
or mixture together with the starting materials and
possible intermediates on the plate.
Location of spots: The position of various solutes
separated by TLC can be located by various
methods. Colored substances can be seen directly
when viewed against stationary phase, while
colorless substances can be detected only by
making them visible by making use of some
spraying agent, which produces colored areas in
the region which they occupy.
Specifically in TLC following can be used for
spraying the invisible spots:
1. Being purely inorganic in nature, corrosive
agents may also be used for spraying on the
invisible spots.
2. Dilute solution of Potassium dichromate in
concentrated sulfuric acid. In the process,
potassium dichromate (yellow) is reduced to
chromic sulfate (green) by most of the organic
compounds, particularly used for sugars.
3. Vapors of sulfur trioxide, produced on warming
fuming sulfuric acid, chars organic compound
and makes them visible as dark spots.
4. Solution of potassium permanganate.
5. Iodine vapors.

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Other common reagents include saturated solution

of hydrogen sulphide, 0.2N aqueous ammonium
sulphide, 0.1% alcoholic quercetin, 0.2%
methanolic 1-(2-pyridylazo)- 2- napthol, 1%
methanolic oxine , and 0.5% aqueous sodium
rhodizonate. If the adsorbent used for the TLC
plate contains a fluorescing material, the solutes
can be viewed under ultraviolet light.
Development solvents 2: The choice of a suitable
solvent depends upon: Nature of substance, and
adsorbent used on the plate. A development
solvent should be such that, does not react
chemically with the substances in the mixture
under examination. Carcinogenic solvents (benzene
etc) or environmentally dangerous solvents
(dichloromethane etc) should always be avoided.
Solvent systems range from non-polar to polar
solvents. Non-polar solvents are generally used, as
highly polar solvents cause the adsorption of any
component of the solvent mixture. Commonly used
development solvents are petroleum ether, carbon
tetrachloride, pyridine, glycol, glycerol, diethyl
ether, formamide, methanol, ethanol, acetone, and
n-propanol.
Mobile Phase: For silica gel chromatography, the
mobile phase is an organic solvent or mixture of
organic solvents. As the mobile phase moves pass
the surface of the silica gel it transports the analyte
pass the particles of the stationary phase.
However, the analyte molecules are only free to
move with the solvent if they are not bound to the
surface of the silica gel. Thus, the fraction of the
time that the analyte is bound to the surface of the
silica gel relative to the time it spends in solution
determines the retention factor of the analyte. The
ability of an analyte to bind to the surface of the
silica gel in the presence of a particular solvent or
mixture of solvents can be viewed as a the sum of
two competitive interactions. First, polar groups in
the solvent can compete with the analyte for
binding sites on the surface of the silica gel.

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Therefore, if a highly polar solvent is used, it will

interact strongly with the surface of the silica gel
and will leave few sites on the stationary phase
free to bind with the analyte. The analyte will,
therefore, move quickly pass the stationary phase.
Similarly, polar groups in the solvent can interact
strongly with polar functionality in the analyte and
prevent interaction of the analyte with the surface
of the silica gel.
This effect also leads to rapid movement of
the analyte pass the stationary phase. The polarity
of a solvent to be used for chromatography can be
evaluated by examining the dielectric constant ()
and dipole moment () of the solvent. The larger
these two numbers, the more polar is the solvent.
In addition, the hydrogen bonding ability of the
solvent must also be considered. For example
methanol is a strong hydrogen bond donor and will
severely inhibit the ability of all but the most polar
analytes to bind the surface of the silica gel.
Developing a Plate 2: A TLC plate can be developed
in a beaker or closed jar. Place a small amount of
solvent ( mobile phase) in the container. A small
spot of solution containing the sample is applied to
a plate, about one centimeter from the base. The
plate is then dipped in to a suitable solvent, such as
hexane or ethyl acetate, and placed in a sealed
container. The solvent moves up the plate by
capillary action and meets the sample mixture,
which is dissolved and is carried up the plate by the
solvent.
Different compounds in the sample mixture
travel at different rates due to the differences in
their attraction to the stationary phase, and
because of differences in solubility in the solvent.
By changing the solvent, or perhaps using a
mixture, the separation of components (measured
by the Rf value) can be adjusted. The solvent level
has to be below the starting line of the TLC,
otherwise the spots will dissolve away. The lower

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edge of the plate is then dipped in a solvent. The

solvent (eluent) travels up the matrix by capillarity,
moving the components of the samples at various
rates because of their different degrees of
interaction with the matrix (stationary phase) and
solubility in the developing solvent. Non-polar
solvents will force non-polar compounds to the top
of the plate, because the compounds dissolve well
and do not interact with the polar stationary phase.
Allow the solvent to travel up the plate until ~1 cm
from the top. Take the plate out and mark the
solvent front immediately. Do not allow the solvent
to run over the edge of the plate. Next, let the
solvent evaporate completely.
Precautions during sample application;
1. Sample should be dissolved in a nonpolar
solvent as polar solvent has a tendency to
spread out the starting spot.
2. Solvent used for dissolving sample should be
volatile.
3. While applying sample, the surface of the
adsorbent should not be disturbed as this
distorts the shapes of the spots on subsequent
developed chromatogram, hindering the
accuracy of quantitative measurements.
4. The sample spot should be within 2-5 mm in
diameter.
The TLC Experiment:

LC CHAMBER FOR DEVELOPMENT

WITH A LID OR A CLOSED JAR

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After ~5 Min

After ~10 Min

After Drying

Visualization: When the solvent front has moved

to within about 1 cm of the top end of the
adsorbent (after 15 to 45 minutes), the plate
should be removed from the developing chamber,
the position of the solvent front marked, and the
solvent allowed to evaporate. If the components of
the sample are colored, they can be observed
directly. If not, they can sometimes be visualized by
shining ultraviolet light on the plate or by allowing
the plate to stand for a few minutes in a closed
container in which the atmosphere is saturated
with iodine vapor. Sometimes the spots can be
visualized by spraying the plate with a reagent that
will react with one or more of the components of
the sample.
Analysis: The components, visible as separated
spots, are identified by comparing the distances
they have traveled with those of the known
reference materials. Measure the distance of the
start line to the solvent front. Then measure the
distance of center of the spot to the start line.
Divide the distance the solvent moved by the
distance the individual spot moved. The resulting
ratio is called Rf-value. As the chemicals being
separated may be colorless, several methods exist
to visualize the spots. Often a small amount of a
fluorescent compound, usually manganeseactivated zinc silicate, is added to the adsorbent
that allows the visualization of spots under a

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blacklight (UV254). The adsorbent layer will thus

fluoresce light green by itself, but spots of analyte
quench this fluorescence, Iodine vapors are a
general unspecific color reagent, Specific color
reagents exist into which the TLC plate is dipped or
which are sprayed onto the plate. Once visible, the
Rf value, or retention factor, of each spot can be
determined by dividing the distance traveled by the
product by the total distance traveled by the
solvent (the solvent front). These values depend on
the solvent used, and the type of TLC plate, and are
not physical constants.
Preparative TLC8: TLC can also be used on a small
semi-preparative scale to separate mixtures of up
to a few hundred milligrams. The mixture is not
"spotted" on the TLC plate as dots, but rather is
applied to the plate as a thin even layer
horizontally to and just above the solvent level.
When developed with solvent the compounds
separate in horizontal bands rather than
horizontally separated spots. Each band (or a
desired band) is scraped off the backing material.
The backing material is then extracted with
a suitable solvent (e.g. DCM) and filtered to give
the isolated material upon removal of the solvent.
For small-scale reactions with easily separated
products, preparative TLC can be a far more
efficient in terms of time and cost than doing
chromatography. Obviously, the whole plate
cannot be chemically developed or the product will
be chemically destroyed. Thus this technique is
best used with compounds that are colored, or
visible under UV light. Alternatively, a small section
of the plate can be chemically developed e.g.
cutting a section out and chemically developing it,
or masking most of the plate and exposing a small
section to a chemical developer like iodine.
Applications 1-6, 9, 10: Thin layer chromatography
has been a useful tool in numerous applications of
pharmaceutical importance.

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1. TLC of amino acids: TLC of amino acids is more

difficult than TLC of inks, because amino acids
are colorless. Therefore, one cannot see the
spots with the naked eye once the plate is fully
developed and dried. To see the spots, it is
necessary to use either the ninhydrin or the
black-light visualization techniques.
E.g., Amino acids, proteins and peptides 8: A
mixture of 34 amino acids, proteins and
peptides has been successfully separated and
isolated from urine using silica gel plates. All
these substances were found to be ninhydrin
positive. The development were carried out
first with chloroform-methanol-20%ammonium
hydroxide (2:2:1) and then with phenol-water.
2. Pharmaceuticals and drugs: TLC is used in the
identification, purity testing and determination
of the concentration of active ingredients,
auxiliary substances and preservatives in drugs
and drug preparations, process control in
synthetic manufacturing processes. Various
pharmacopoeias have accepted TLC technique
for the detection of impurity in a drug or
chemical
E.g., Antibiotics: Penicillins have been
separated on silica gel G by using the two
solvents, acetone- methanol (1:1) and isopropanol-methanol (3:7). As the detecting
agent, the iodine-azide reaction was employed
by spraying the dried plates with a 0.1 %
iodine solution containing 3.5% of sodium
azide.
3. Separation of multicomponent pharmaceutical
formulations: It is also used in separation of
multicomponent pharmaceutical formulations.
4. Qualitative analysis of alkaloids: It is used in
qualitative analysis of alkaloids in control phase
of both pharmaceutical formulations and

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vegetable drugs. TLC has been used for the

isolation and determination of alkaloids in
toxicology where the 30-60 minute runs give a
great advantage in comparison to the 12-24
hours required for paper chromatography.
Purine alkaloids have been separated by TLC on
silicic acid, silica gel and aluminum oxide. The
spots are visualized by spraying first with an
alcoholic iodine-potassium iodine solution
followed by 25% HCl- 96% ethanol (1:1).
5. Clinical chemistry and Biochemistry: For the
determination of active substances and their
metabolites in biological matrices, diagnosis of
metabolic disorders such as phenylketonuria,
cystinuria and maple syrup disease in babies. It
serves as an useful tool in analysis of urinary
constituent derived from lipids in analysis of
many urinary constituents such as steroids,
amino acids, porphyrins and bile acids. Urinary
analysis by TLC is most effective when done in
conjunction with other chromatographic
processes, so that minor metabolites can be
detected and resolved completely free of other
components.
6. Cosmetology: In the identification of dye raw
materials and end products, preservatives,
surfactants, fatty acids, constituents of
perfumes.
7. Food Analysis: For the determination of
pesticides and fungicides in drinking water,
residues in vegetables, salads and meat,
vitamins in soft drinks, banned additives in
Germany (e.g. sandalwood extract in fish and
meat products), compliance with limit values
(e.g. polycyclic compounds in drinking water,
aflatoxins in milk and milk products). A typical
separation of dyes in spinach looks like this:

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NaCl and NaBr in micellar solutions on the

mobility and separation efficiency of amines is
also assessed.

10. Applications related to Organic Chemistry 1-6:

8. Analysis of Heavy Petroleum Product8: Thinlayer chromatography (TLC), which is

commonly used in the analysis of complex
mixtures, is seldom used in the investigation of
petroleum products, maybe the most complex
objects. In particular, with respect to heavy
petroleum products, no such information has
been found in the literature. At the same time,
the simplicity, economy, and efficiency of this
technique in comparison with column
chromatography are advantages that are
widely known. TLC technique used (in the
preparataive variant) for a rapid determination
of the group composition of heavy petroleum
products (asphalts, pitches, resids), and in
connection
with
spectroscopic studies of the chemical
composition of the fractions obtained.
9. Separation of aromatic amines: Cationic and
non-ionic surfactant-mediated systems have
been used as mobile phases in thin-layer
chromatographic separation of aromatic
amines on silica gel layers. The effect of
surfactant concentration below and above its
critical micellar concentration on mobility of
amines was examined. The influence of organic
and inorganic additives such as alcohols, urea,

It has been widely used for checking

number of other separation processes. TLC
has also been applied successfully in various
purification
processes,
checking
of distillation fractions and for checking the
progress of purification by molecular
distillation.

TLC has been used as an analytical tool in

organic chemistry due to its high speed of
separation and its applicability in a large
number of chemical compounds. Its
important use is in the separation and
isolation of individual components of a
mixture, but in organic chemistry it has also
been used for: Checking the purity of
samples, as purification process, for
identification of organic compounds, for
studying various organic reactions, in
characterizing and isolating a number of
compounds such as acids, alcohols, glycols,
amides, alkaloids, vitamins, amino acids,
antibiotics, food stuffs and examination of
reaction. The reaction mixture is examined
by TLC to assess whether the reaction is
complete or otherwise.The method is also
used in checking other separational
processes and purification processes like
distillation, molecular distillation etc.

High sensitivity of TLC is used to check

purity of sample, because high sensitivity
enables impurities to be observed in so
called pure samples. With the help of TLC it
is possible to know whether a reaction is

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complete and had followed the expected

course. The nature of byproducts can also
be ascertained by using TLC. If the reaction
does not proceed as desired or expected,
then an examination of the behaviour of
the spots with standard reagents may
sometimes give information for the rapid
identification of the products.
Problems in TLC:
Over-large Spots: Sample spots made using TLC
capillaries should be no larger than 1-2 mm in
diameter, because component spots in the
developed plate will be no smaller than, and will
usually be larger than, the size of the initial spot. If
the initial spot is larger than 2 mm in diameter,
then components with similar Rf values may not be
resolved because their spots will be so large that
they will overlap considerably and may appear to
be one large spot. Small initial spots, on the other
hand, maximize the potential of complete
separation of components.
Uneven Advance of Solvent Front: A common
problem in TLC is uneven advance of solvent along
the plate. Instead of a straight line, the solvent
front may appear to bow either up or down in the
center. Uneven advance of solvent leads to uneven
advance of substance spots, and inaccurate Rf
values result. A frequent cause of uneven solvent
advance is the use of a developing chamber that
does not have a flat bottom. Glass bottles usually
have bottoms that curve upward from the edges to
the center. If the bottom of the TLC plate is placed
on this curved surface, the shape of the solvent
advance line may mirror the shape of the container
bottom. It is therefore important to use flatbottomed developing tanks in TLC. A bowed
solvent front may also result if too little developing
solvent is placed in the chamber; if the plate is cut
improperly, so that the sides are not exactly
perpendicular to the bottom edge; and if the slide

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is excessively tilted in the chamber. Care in

choosing and using a developing chamber is the
best defense against curved solvent fronts. Water
is seldom used as a developing solvent because it
has a tendency to produce a dramatically curved
front. This may be due to its unusually high surface
tension.
Streaking: Sometimes a substance will move along
a TLC plate as a long streak, rather than as a single
discrete spot. This is the result of spotting the plate
with too much substance, more than the moving
solvent can handle. The solvent moves as much
substance as it can, but a substantial amount of
substance is left behind. The substance is dragged
along by the solvent leaving a trail of substance
that may sometimes span the entire distance
between the starting line and the solvent front.
Streaking can be eliminated by systematically
diluting the spotting solution until development
and visualization show the substances moving as
single spots, rather than elongated streaks.
Specific TLC Procedures:
1. Separartion for alanine, glycine, threonine,
and proline: TLC of amino acids is more difficult
one cannot see the spots with the naked eye
once the plate is fully developed and dried. To
see the spots, it is necessary to use either the
ninhydrin or the black-light visualization
techniques. Observe the spots, and decide
whether or not a chosen solvent system has
been effective in moving an amino acid or in
separating a mixture. Therefore the process of
finding an effective solvent system can be long
and painstaking. As points of general
information, amino acids are quite polar and
tend to move on silica gel plates with polar
solvents. They have Rf values close to 1 when
water or concentrated ammonia is used as the
developing solvent, probably because of their
high solubility in water. Diluting a polar solvent

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with a less polar one results in smaller Rf

values, roughly in proportion to the amount of
less polar solvent used, Thus, alanine, glycine,
threonine, and proline all have Rf values of
around 0.60 when developed with a 50/50
mixture of water and n-propanol, and around
0.40 when developed with a 30/70 mixture of
concentrated NH3 and n-propanol. The
following procedure assumes the use of 50/50
water/n-propanol as the developing solvent,
but one can try other polar/non-polar
combinations.
Experimental Procedure In the hood, prepare 10
mL of a mixture consisting of 50% 1-propanol and
50% water by volume, and pour about half of this
into a clean developing tank. Make sure that the
level of liquid in the tank is no higher than 5 mm,
and close the lid. Prepare a solution of about 0.001
g of amino acid in 0.2 mL of water. Dissolve the
acid, and then draw some solution up in a spotting
capillary and double-spot a properly marked and
activated TLC plate. Allow the plate to dry for 5
minutes, and then lower the plate into the
developing tank so that its bottom is submerged in
the developing solvent. Close the lid, and allow the
plate to develop until solvent has risen to the
pencil line at the top of the plate.
Remove the plate from the tank and place it
in an oven at 50 oC to dry. When the plate is dry,
visualize it using ninhydrin spray or iodination.
Circle the amino acid spots with pencil, and
calculate Rf values. Compare the measured Rf
values with the values for the amino acids. On this
basis, identity of amino acid is done. In
combination with other data , obtain, information
which will help unambiguously to identify amino
acid. Suppose that amino acid has Rf value similar
to that of alanine, one should then prepare a small
amount of alanine solution and spot it alongside
amino acid on a new TLC plate. Develop, dry, and
visualize the plate to confirm that amino acid

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indeed has exactly the same value as alanine, and

that the spot is the same shape and color. Finally, it
is very important to be observant of detail in doing
TLC. In addition to the Rf value for a substance, the
shape of the spot produced by a particular
developing solvent and the shade of color
produced by iodine or ninhydrin can be
characteristic of the substance. For example, when
alanine, glycine, threonine, and proline are spotted
side-by-side on a plate and developed with 70% npropanol/30% conc NH3 following observations can
be made:
Amino
Acid

Solvent

Spot Color
after
Iodination

Spot Color
with
Ninhydrin

Rf
Value

Spot
Shape

alanine

50/50
water/npropanol

white on
brown
bkgrnd

purple

0.65

circle

glycine

30/70
conc
NH3/npropanol

white on
brown
bkgrnd

pink

0.25

elongated
oval

glycine

50/50
water/npropanol

white on
brown
bkgrnd

pink

0.55

circle

threonine

50/50
water/npropanol

white on
brown
bkgrnd

purple

0.57

circle

proline

50/50
water/npropanol

white on
brown
bkgrnd

yellow
with pink
border

0.65

circle

Rf values of amino acids

2. Separation of dyes in spinach: On a balance

weigh out 0.5 grams of fresh spinach and
combine with 0.5 grams of anhydrous
magnesium sulfate and 1.0 grams of sand.
Transfer these materials to a mortar and using
a pestle grind the mixture until a fine dry
powder is obtained (grind the mixture really
well). The anhydrous magnesium sulfate will
remove the water from the leaves. Transfer the
powder (2.0 grams total) to a small test tube
and combine with 2.0 mL of acetone. Stopper

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International Journal of Pharmaceutical Sciences and Research

the test tube and shake vigorously for

approximately one minute. You need to make
sure that the solid and solvent are well mixed.
Allow this mixture to stand for 10 minutes,
and then using a pipette carefully transfer the
solvent above the solid into a small micro
centrifuge tube. Use care not to transfer any of
the solid material. The solvent extract should
be green. Cap the micro centrifuge tube to
minimize solvent evaporation. Obtain a TLC
chamber (a 400 mL glass beaker covered with
parafilm or aluminum foil) and add developing
solvent (a mixture of petroleum ether, acetone,
cyclohexane, ethyl acetate and methanol). The
solvent should completely cover the bottom of
the chamber to a depth of approximately 0.5
cm. Keep the chamber covered so that
evaporation doesnt change the composition of
the solvent. Allow the TLC plate to develop
(separation of pigments) for approximately 10
minutes. As the solvent moves up the TLC plate
you should see the different colored pigments
separating.
Remove the TLC plate from the chamber
when the solvent front is approximately 1.0cm
from the top of the TLC plate. With a pencil,
mark the level of the solvent front (highest
level the solvent moves up the TLC plate) as
soon as you remove the strip from the chamber
(the solvent evaporates and disappears
quickly). Also measure the pigment distances
quickly as some pigments (especially the betacarotene) may fade over time.
Beta-carotene is the most non-polar
pigment (highest Rf) and its band will be
yellow. Chlorophyll a has a larger Rf than does
chlorophyll b. For the following calculations
mark the center of the initial pigment dot; this
will be the starting point for all the following
measurements. Also mark the middle point of

ISSN: 0975-8232

each pigment band and the solvent front. The

literature gives Rf values of 0.61 and 0.49 for
pheophytin a and pheophytin b. Use these
values to help identify which spots are due to
those compounds. Use the pure extract of carotene to identify the location of that band.
3. Identification
of
naphthodianthrones:
Hypericum perforatum extracts containing
naphthodianthrones have been identified by
TLC using silica gel plates with fluorescence
indicator and ethyl acetate-formic acidwater(30:2:3 v/v/v) or toluene- ethyl acetateformic acid-water(50:40:5:5 v/v/v/v) as the
mobile phase. TLC can also be used for the
identification of indolic alkaloids isolated from
various Rauwolfia Species by using silica gel
plates as stationary species; acetone-light
petroleum-diethyl amine(2:7:1 v/v/v) and 1%
solution of ammonium cerium (IV) sulfate for
the visualization of spots.
4. Identification
of
drugs:
Aspirin,
acetaminophen, ibuprofen, and caffeine: Draw
a light pencil line about 1 cm from the end of a
chromatographic plate, and on this line spot
aspirin, acetaminophen, ibuprofen, and
caffeine, which are available as reference
standards. Use a separate capillary for each
standard. Make each spot as small as possible,
preferably less than 0.5 mm in diameter. Use
the blower to facilitate the evaporation of the
solvent between applications. Examine the
plate under the ultraviolet (UV) light to see that
enough of each compound has been applied; if
not, add more. On a separate plate run three of
the unknowns and one of the aspirin standard.
The unknown sample is prepared by crushing a
part of a tablet, adding this powder to a test
tube or small vial along with an appropriate
amount of ethanol, and then mixing the
suspension. Not all of the tablet will dissolve,
but enough will go into solution to spot the

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International Journal of Pharmaceutical Sciences and Research

plate. The binder starch or silica will not

dissolve. The 1% solutions should be prepared
and ready to use. After the solvent has risen to
about 2/3 of the length of the plate, remove
the plate from the developing chamber. Quickly
mark the solvent front with a pencil and allow
the solvent to dry. Examine the plate under UV
light to see the components as dark spots
against a bright green-blue background. Outline
the spots with a pencil. The spots can also be
visualized by putting the plate in an iodine
chamber made by placing a few crystals of
iodine in the bottom of a capped jar. Calculate
the Rf values for the spots.
Substances:
Rf Values:

Aspirin
0.45

Caffeine
0.08

Acetaminophen
0.24

Ibuprofen
0.60

5. Separation of Inorganic Ions: TLC has been

used for separating cationic, anionic, purely
covalent species and also organic derivatives of
the metals. In order to carry out TLC of groups
of cations, silica gel is first washed with acid
and water to remove impurities of sodium,
magnesium, calcium and iron. But this
treatment removes the calcium sulphate
binder. Therefore, calcium sulphate must be
replaced by starch or some other suitable
binder. After washing and drying of TLC plate,
the spots of cations or anions to be separated

ISSN: 0975-8232

are applied on this plate. The plate is then kept

in a close chamber and the lower part of the
plate is then dipped into a solvent. It is than
removed from chamber and dried, visualized
for spots by suitable visualizing reagents.
REFERENCES:
1.

Singhal S., Singhal N., Agarwal S., Pharmaceutical Analysis

II, Thin layer chromatography, Pragati prakashan, First
edition, 2009, 98-111.
2. Kasture A.V., Mahadik K.R, Wadodkar S.G, More H.N., A
textbook of pharmaceutical analysis, Instrumental
methods, Nirali Prakashan, 9th edition, 2005, vol II, 18-30.
3. Quach, H. T, Steeper, R L, Griffin, G. W, Separation of plant
pigments by thin layer chromatography. Journal of
chemical education. 2004, 81, 385-7.
4. Skoog D. A., Holler F.J. and Nieman T.A., Principles of
instrumental analysis, Saunders college publishing, 5th
edition, 2006 ,761-766.
5. Chatwal G. R., Anand S.K, Instrumental methods of
chemical analysis, Himalaya publishing house,5th
edition,2008, 2.599-2.616.
6. Beckett A.H, Stenlake J.B., Practical pharmaceutical
chemistry, Thin layer chromatography, CBS publishers, 4th
edition, 2005, 115-128.
7. Ali M. and Agrawal V., Thin-layer chromatography of
aromatic amines, Separation Science and Technology,
2002, 37, 363 - 377.
8. Sharma B.K., Instrumental methods of chemical analysis,
Goel publishing house, Meerut, 5th edition, 2007, 241-264.
9. Vidya Sagar, Instrumental methods of drug analysis,
Pharma Med Press, First edition 2009,263.
10. Justus G. Kirchner, Thin-layer chromatographic
quantitative analysis, Journal of Chromatography A ,1
August 1973, Volume 82, Issue 1, 101-115.

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