THIN LAYER CHROMATOGRAPHY

1. November 8, 2013
[THIN LAYER CHROMATOGRAPHY]
ASSIGNMENT ON THIN LAYER
CHROMATOGRAPHY
SUBMITTED BY:
SUBMITTED TO:
Mr. YUGANTAR KUMAR
Mr. A. N. SAHU
B.PHARM, PART-3RD
ROLL NO. 11121EN003
PHARMACOGNOSY-II
COURSE CODE: PH-3102
DEPARTMENT OF PHARMACEUTICS
IIT (BHU), VARANASI
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CONTENTS:-
TOPIC
PAGE NO.
INTRODUCTION
03
THIN LAYER CHROMATOGRAPHY
04
HISTORY
04
THEORY
07
STEPS OF TLC
11
Rf Value
14
FACTORS AFFECTING THE Rf Value
15
MOLECULAR INTERACTION IN TLC
17
IMPORTANCE & APPLICATION OF TLC
20
SUMMARY
24
REFERENCE
25
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INTRODUCTION:-
Chromatography is the separation of two or more compounds or ions by the distribution
Between two phases, one which is moving and the other which is stationary. These two
phases can be solid-liquid, liquid-liquid or gas-liquid.
Mobile Phase, m
v
A
Stationary Phase, s
Figure shows Schematic presentation of a chromatographic system with partition of analyte
‘A’ between the phases; v mobile phase velocity.
The molecules of the analytes are distributed between the mobile and the stationary phase.
When present in the stationary phase, they are retained, and are not moving through the
system. In contrast, they migrate with the velocity, v, of the mobile phase when being there.
Due to the different distribution of the particular analytes the mean residence time in the
stationary phase differs, too, resulting in a different net migration velocity. This is the
principle of chromatographic separation.
The position of the distribution equilibrium determines the migration velocity. It reflects the
intermolecular interactions of the analyte with the stationary and the mobile phase. However,
during migration of the analytes through the separation capillary broadening of the original
sample zone takes place. This broadening is greater the longer the migration distance is. Two
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effects are therefore counteracting in the chromatographic system: the different
migration is the base of separability by introducing separation selectivity. In contrast, peak
broadening effects lead to a potential overlap of the peaks migrating with different velocity.
They occur in parallel with a dilution of the initial sample zone with the mobile phase.
THIN LAYER CHROMATOGRAPHY:Thin layer chromatography, or TLC, is a method for analyzing mixtures by separating the
compounds in the mixture. TLC can be used to help determine the number of components in
a mixture, the identity of compounds, and the purity of a compound. By observing the
appearance of a product or the disappearance of a reactant, it can also be used to monitor the
progress of a reaction. TLC is a sensitive technique - microgram (0.000001 g) quantities can
be analyzed by TLC and it takes little time for an analysis (about 5-10 minutes).
HISTORY OF TLC:The development of modern thin-layer chromatography has its beginnings in liquid
chromatography. Like most scientific methods, chromatography evolved from initial
phenomenological observations, through early empirical research and the study of the
underlying theoretical principles, and finally, advancements in technique. While the Russian
botanist Tswett is generally credited with the discovery of chromatography for his work in
the separation of plant extracts on a column of sorbent, others observed the phenomenon of
separation before Tswett. For example, in 1844 C. Matteucci observed the rings left by a drop
of chocolate on a piece of paper. In 1850 the German dye chemist F.F. Runge recognized the
possibility of separating inorganic ions when he observed their migration through paper. He
initially described the forces responsible for the separation as the "living forces," but later
attributed the phenomenon to capillary forces (D 'Ascenzo and Nicolini1990). Tswett's
contribution to chromatography was the understanding of adsorption, which led to chemical
separation by liquid column chromatography. Although chromatographic techniques were
little used for the next thirty years, in the 1930’s they were reintroduced by biochemists. In
1941, A.J.P. Martin and R.L.M. Synge introduced partition chromatography. They found that
they could separate amino acids successfully if a water phase was held stationary by
adsorbing it on silica gel, while permitting a mobile chloroform phase to flow over it. Thus
they described the use of a liquid stationary phase with a liquid mobile phase and suggested
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that a gas might also be used as the mobile phase. I n 1952, Martin and Synge were awarded
a Nobel Prize in chemistry for their theoretical development of partition chromatography
(Brooks 1987). The development of TLC can be divided into three eras: the early years
(1938-1951), the classical period (1956-1980), and the modern period (1981 to the present).
Figure shows a time line for the history of TLC.
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Figure shows one of the first TLC plates developed by Stahl in 1956, showing a
valerian oil separation. The plate was sprayed with SbCl3 solution in CHCl3
and subsequently heated
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THEORY:In thin layer chromatography, a solid phase, the adsorbent, is coated onto a solid support as a
thin layer (about 0.25 mm thick). In many cases, a small amount of a binder such as plaster of
Paris is mixed with the absorbent to facilitate the coating. Many different solid supports are
employed, including thin sheets of glass, plastic, and aluminium. The mixture (A plus B) to
be separated is dissolved in a solvent and the resulting solution is spotted onto the thin layer
plate near the bottom. A solvent, or mixture of solvents, called the eluant, is allowed to flow
up the plate by capillary action. At all times, the solid will adsorb a certain fraction of each
component of the mixture and the remainder will be in solution. Any one molecule will spend
part of the time sitting still on the adsorbent with the remainder moving up the plate with the
solvent. A substance that is strongly adsorbed (say, A) will have a greater fraction of its
molecules adsorbed at any one time, and thus any one molecule of A will spend more time
sitting still and less time moving. In contrast, a weakly adsorbed substance (B) will have a
smaller fraction of its molecules adsorbed at any one time, and hence any one molecule of B
will spend less time sitting and more time moving. Thus, the more weakly a substance is
adsorbed, the farther up the plate it will move. The more strongly a substance is adsorbed, the
closer it will stay near the origin. Several factors determine the efficiency of a
chromatographic separation. The adsorbent should show a maximum of selectivity toward the
substances being separated so that the differences in rate of elution will be large. For the
separation of any given mixture, some adsorbents may be too strongly adsorbing or too
weakly adsorbing. Table 1 lists a number of adsorbents in order of adsorptive power.
Table 1. Chromatographic adsorbents. The order in the table is approximate,
since it depends upon the substance being adsorbed, and the solvent used for
elution.
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The eluting solvent should also show a maximum of selectivity in its ability to dissolve or
desorbs the substances being separated. The fact that one substance is relatively soluble in a
solvent can result in its being eluted faster than another substance. However, a more
important property of the solvent is its ability to be itself adsorbed on the adsorbent. If the
solvent is more strongly adsorbed than the substances being separated, it can take their place
on the adsorbent and all the substances will flow together. If the solvent is less strongly
adsorbed than any of the components of the mixture, its contribution to different rates of
elution will be only through its difference in solvent power toward them. If, however, it is
more, strongly adsorbed than some components of the mixture and less strongly than others,
it will greatly speed the elution of those substances that it can replace on the absorbent,
without speeding the elution of the others. Table 2 lists a number of common solvents in
approximate order of increasing adsorbability, and hence in order of increasing eluting
power. The order is only approximate since it depends upon the nature of the adsorbent.
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Mixtures of solvents can be used, and, since increasing eluting power results mostly from
preferential adsorption of the solvent, addition of only a little (0.5-2%, by volume) of a more
strongly adsorbed solvent will result in a large increase in the eluting power. Because water is
among the most strongly adsorbed solvents, the presence of a little water in a solvent can
greatly increase its eluting power. For this reason, solvents to be used in chromatography
should be quite dry. The particular combination of adsorbent and eluting solvent that will
result in the acceptable separation of a particular mixture can be determined only by trial.
Table 2. Eluting solvents for chromatography
If the substances in the mixture differ greatly in adsorbability, it will be much easier to
separate them. Often, when this is so, a succession of solvents of increasing eluting power is
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used. One substance may be eluted easily while the other stays at the top of the column, and
then the other can be eluted with a solvent of greater eluting power. Table 3 indicates an
approximate order of adsorbability by functional group.
Table 3. Adsorbability of organic compounds by functional group
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STEPS OF THIN LAYER CHROMATOGRAPHY:
Thin Layer Chromatography is a three step process which involves spotting of TLC plate,
development of that plate and visualization under UV rays. So, steps of TLC are as follows:-
SPOTTING THE TLC PLATE:-
One advantage TLC has over other separation methods is that it is truly a micro scale
technique. Only a few micrograms of material in solution is necessary to observe the solute
on a TLC plate. Dissolve a few milligrams of material in a volatile solvent creating a dilute
solution. Choose a volatile solvent that completely dissolves the sample. However, if it is
partially soluble, since such only low concentrations are needed, normally you will be able to
observe the compound. Once the sample is prepared, a spotting capillary must be used to add
the sample to the plate. The spotting capillaries must be extremely small. In fact, the opening
at the end of a regular Pasteur pipette is too big for spotting a TLC plate. CG capillary
columns donated by Restek, a chromatography company located in Bellefonte, are used to
spot the plates. These columns are very small bore and can be cut into three inch sections to
provide very good TLC spotting tubes. The solution can be drawn up the tube by capillary
action (hence the name) and spotted on the plate at the hash mark labelled in pencil. This is
known as the origin and is shown in Figure. Since a TLC plate can run three, if not four
mixtures at one time, it is very important to properly label the plate. Notice that pencil is
always used to mark a TLC plate since the graphite carbon is inert. If organic ink is used to
mark the plate, it will chromatograph just as any other organic compound and give incorrect
results.
To spot the plate, simply touch the end of the capillary tube to the coated side of the plate.
The solvent should evaporate quickly leaving your mixture behind on the plate. You may
have to spot the plate a couple of times to ensure the material is present, but do not spot too
much sample. If too much solute is added to the plate, a poor separation will result. Smearing,
smudging and spots that overlap will result making identification of separated components
difficult.
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DEVELOPMENT:Once the dilute solution of the mixture has been spotted on the plate, the next step is the
development. Just like paper chromatography, the solvent must be in contact with the
stationary phase. Figure shows a wide-mouth bottle commonly used to develop TLC plates.
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The bottle is filled with a small amount of the mobile phase and capped with a cork. In
addition, a piece of filter paper is put in the bottle to help create an atmosphere saturated with
solvent. Use your tweezers to place the plate in the development chamber; oils from your
fingers can sometimes smear or ruin a TLC plate. Also make sure the origin spots are not
below the solvent level in the chamber. If the spots are submerged in the solvent, they are
washed off the plate and lost. Once the solvent has run within a centimetre of the top of the
plate, remove it with tweezers. Using a pencil, immediately draw a line across the plate where
the solvent front can be seen. The proper location of this solvent front line will be important
for later calculations.
VISUALIZATION:Some organic compounds are coloured. If you are fortunate enough to be separating organic
molecules that are coloured such as dyes, inks or indicators, then visualizing the separated
spots is easy. However, since most organic compounds are colourless, this first method does
not always work. In most cases observing the separated spots by UV light works well. TLC
plates normally contain a fluorescent indicator which makes the TLC plate glow green under
UV light of wavelength 254 nm. Compounds that absorb UV light will quench the green
fluorescence yielding dark purple or bluish spots on the plate. Simply put the plate under a
UV lamp, and the compounds become visible to the naked eye. Lightly circle the spots, so
that you will have a permanent record of their location for later calculations.
Figure shows two ways of using UV light to visualize the spots
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Another useful visualizing technique is an iodine (I2) chamber. Iodine sublimes and will
absorb to organic molecules in the vapour phase. The organic spots on the plate will turn
brown and can be easily identified. Also circle these observed spots, since the colour stain
will eventually fade from the plate. Sometimes, a combination of both a UV lamp and iodine
is needed to observe all the spots. Some compounds are not “UV active”, that is, they do not
absorb light at the wavelength of 254 nm. Using both methods will ensure correct
identification of all the spots on the TLC plate.
Rf Value:In addition to qualitative results, TLC can also provide a chromatographic measurement
known as an Rf value. The Rf value is the “retardation factor” or the “ratio-to-front” value
expressed as a decimal fraction. The Rf value can be calculated as:
This number can be calculated for each spot observed on a TLC plate. Essentially it describes
the distance travelled by the individual components. If two spots travel the same distance or
have the same Rf value then it might be concluded that the two components are the same
molecule. For Rf value comparisons to be valid; however, TLC plates must be run under the
same exact conditions. These conditions include the stationary phase, mobile phase, and
temperature. Just as many organic molecules have the same melting point and colour, many
can have the same Rf value, so identical Rf values doesn’t necessarily mean identical
compounds. Additional information must be obtained before this conclusion can be made. It
is important to restate that this number is only significant when the same chromatographic
conditions are used. Figure shows a diagram of a typical TLC plate and how the distances are
measured to calculate the Rf value.
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Factors Affecting the Rf value:1. Nature of Adsorbent
Different adsorbents will give different Rf valve for same solvent. Reproducibility is only
possible for given adsorbent of constant particle size and binder. Plates should be the stored
over silica gel in a desiccators before use and the sample should be applied quickly so that the
water vapour 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 make freshly for each run if one of the solvents is very
volatile or hygroscopic, for example acetone.
3. Temperature
Although precise control of temperature is not necessary, the tank should be kept away from
draughts, sources of heat, direct sunlight, etc. As the temperature is increased, volatile
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solvents evaporate more quickly, solvents run faster, and Rf values generally decrease
slightly.
4. Thickness of Layer
Standard plates approximately 250 µm is preferable thickness of layer. Below 200 µm the Rf
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. 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 Rf of a 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 Rf
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 Rf value changes for the same solvent system.
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MOLECULAR INTERACTIONS IN TLC:There are three components in TLC: The TLC plate (stationary phase), the development
solvent (mobile phase), and the sample to be analyzed (solute). In our experiment the TLC
plate consists of a thin plastic sheet covered with a thin layer of silica gel, a portion of the
structure of which is shown here.
Silica gel consists of a three-dimensional network of thousands of alternating silicon and
oxygen bonds, with O-H groups on the outside surface. Silica gel is simply very finely
ground very pure sand. It should be noted that silica gel is highly polar and is capable of
hydrogen bonding. Consider the side-on view of the development of a TLC plate below. As
the solvent travels up the plate, over the spot, equilibrium is set up, as development solvent
competes with the TLC plate for the solute. The silica gel binds to the solute and the
development solvent tries to dissolve it away, carrying the solute(s) along as the solvent
travels up the plate.
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A balance of intermolecular forces determines the position of equilibrium and thus the ability
of the solvent to move the solute up the plate. In other words, would the spot prefer to be
stuck on the plate or would it prefer to move along with solvent.
The balance depends upon (1) the polarity of the TLC plate (constant and high), (2) the
polarity of the development solvent (can be varied by using different solvents), and (3) the
polarity of the compounds in the spot (this varies depending upon what compounds are in the
spot). For example, if a sample consists of two components, one more polar than the other,
the more polar will tend to stick more tightly to the plate and the less polar will tend to move
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along more freely with the solvent. Using a more polar development solvent would cause
both to move along further. If the approximate structures of the solutes are known, it is
possible to make an educated guess as to what solvent or mixture of solvents to use. In
practice though, for a given mixture of compounds to be analyzed, a solvent or mixture of
solvents is chosen by trial and error to give the best separation. (A caveat: the polarity
argument is helpful in understanding the principles of TLC. Because most compounds have
some polarity the argument works well. For compounds having very low polarity however, a
lower-polarity solvent may be more effective in moving the solute up the plate.)
This interaction can be understood more easily using the following example illustrated here
below:-
The polarity of molecules, solutes and solvents alike, is ordered as follows, from least to most
polar: Alkanes (least polar), alkyl halides, alkenes, aromatic hydrocarbons, ethers, esters,
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ketones, aldehydes, amines, alcohols, and carboxylic acids (most polar). Note however that
many molecules contain multiple functional groups and that the overall polarity would be
determined by all of the groups.
Applications and Importance of Thin Layer Chromatography:1. Pharmaceuticals and Drugs
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.
2. Clinical Chemistry, Forensic Chemistry and Biochemistry
Determination of active substances and their metabolites in biological matrices, diagnosis of
metabolic disorders such as PKU (phenylketonuria), cystinuria and maple syrup disease in
babies.
3. Cosmetology
Dye raw materials and end products, preservatives, surfactants, fatty acids, constituents of
perfumes.
4. Food Analysis
Determination of pesticides and fungicides in drinking water, residues in vegetables, salads
and meat, vitamins in soft drinks and margarine, 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).
5. Environmental Analysis
Groundwater analysis, determination of pollutants from abandoned armaments in soils and
surface waters, decomposition products from azo dyes used in textiles.
6. Analysis of Inorganic Substances
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Thin layer chromatography technique is also used in the determination of inorganic ions
(metals).
Applications Related To Organic Chemistry:1) It has 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.
2) 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 etc.
· Examination of reaction.
3) 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 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.
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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 separation processes and purification
processes like distillation, molecular distillation etc.
Application of TLC for Separation of Inorganic Ions:Nowadays, 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 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. After that
it is removed from chamber and dried visualized for spots by suitable visualizing reagents.
Examples
1. Amino acids, proteins and peptides
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. Alkaloids
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
aluminium oxide. The spots are visualized by spraying first with an alcoholic iodinepotassium iodine solution followed by 25% HCl – 96% ethanol (1:1).
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3) Antibiotics
Penicillines have been separated on silica gel ‘G’ by using the two solvents, acetonemethanol (1:1) and iso-propanol-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.
Applications of Thin Layer Chromatography in Analysis of Heavy Petroleum Product
Thin-layer 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 preparative 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.
Thin-layer chromatographic separation of aromatic amines
Cationic and non-ionic surfactant-mediated systems have been used as mobile phases in thinlayer 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.
Software for TLC
Thin layer chromatography (TLC) relies upon polarity and the strength of intermolecular
interactions between the stationary phase (silica gel), mobile phase (solvent), and chemical
sample to separate the components of a mixture. The rational selection of a proper solvent is
one of the most important factors in achieving good resolution and unambiguous results.
Thus, by investing some time in understanding the structure and properties of organic
solvents more fully, you will be better equipped to carry out TLC experiments. Rather than
examining solvents in the “wet” laboratory, by using Spartan ’02, a software package that
calculates the structure and properties of compounds.
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SUMMARY:Thin layer chromatography involves the use of thin layer of some adsorbent as stationary
phase coated on some inert support (such a glass or plastic sheet), while the solvent, the
mobile phase percolates through the layer. Mixture to be analysed is deposited near the
bottom of plate. The solvent is made to move up the plate.
As it passes over the deposit various components of the mixture are carried up and deposited
as separate spots on the plate. The adsorbent commonly used are silica gel, alumina,
Kieselguhr, Cellulose powder etc. while the choice of solvent depends on the solubility's of
constituents of the mixture. The Rf values of various constituents may be calculated from the
distances traversed by the spot and the solvent from the place of origin or the point where the
mixture is deposited.
Thin layer chromatography, is therefore, similar to paper chromatography. Instead of filter
paper, a thin layer of some adsorbent is used as the stationary phase. Thin layer
chromatography has many advantages over paper chromatography. The technique is less time
consuming and convenient. There is a greater sharpness of separation and a higher sensitivity.
The spots can be scrapped off with the help of a knife dissolved and analysed quantitatively
by using suitable analytical techniques. Thin layer chromatography has proved to be very
useful in the analyses of adulterated food stuffs, determination of organomercurials in fishes,
phenolics and aromatic alcohols in beer, organ chlorine and carbamate pesticides in fruits and
vegetables.
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REFERENCE:
A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, and P.W.G. Smith. Vogel's
Textbook of Practical Organic Chemistry (5th Ed.).

Scott, R. P. W. Techniques and Practices of Chromatography; 2nd Ed.; Marcel
Dekker, 1995.

Joseph Sherma, Bernard Fried (1991): Handbook of Thin-Layer Chromatography
Marcel Dekker, New York.

Plant drug analysis: A Thin Layer Chromatography Atlas by Hildebert Wagner, 2nd
Edition.
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