Separation of Polar and Hydrophilic Compounds Using a Zwitterionic Stationary Phase in Hydrophilic Interaction Liquid Chromatography
Application And Importance Of Thin Layer Chromatography In Analysis And Research Work
Pramod S. Agrawal
Thin layer chromatography (TLC)
is very useful to chemists as an analytical technique to separate and identify
the compounds in a mixture. Only very small samples of the analyte are needed
much less than a milligram.
The technique is non-destructive, that is, the molecules in the mixtures are
separated physically without being chemically altered. Analytical
TLC is used in drug analysis, consumer product monitoring, environmental pollution
studies, forensics and many other applications.
Thin layer chromatography is so widely used that it has becomes an essential
technique for analyst and research workers. Thin layer chromatography is the
almost universal analytical technique in chemical analysis for organic and inorganic
The identification of unknown compounds is an important task of the organic
chemist. The identity of a particular compound is usually determined by measuring
more than one of its physical properties, for instance, by measuring its melting
or boiling point and its spectroscopic characteristics. Chromatography can also
be used to support a compound’s identification if its chromatographic properties
are compared with those of known compounds.
Thin-layer chromatography (TLC) is a very commonly used technique in synthetic
chemistry for identifying compounds, determining their purity and following
the progress of a reaction. It also permits the optimization of the solvent
system for a given separation problem. In comparison with column chromatography,
it only requires small quantities of the compound (~ng) and is much faster as
Thin-layer chromatography (TLC) is a chromatographic
technique that is useful for separating
organic compounds. Because of the simplicity and rapidity of TLC, it is often
used to monitor the progress of organic reactions and to check the purity of
TLC is a simple, quick, and inexpensive procedure that gives the chemist a
quick answer as to how many components are in a mixture. TLC is also used to
support the identity of a compound in a mixture when the Rf of a
compound is compared with the Rf of a known compound (preferably
both run on the same TLC plate). Chromatography works on the principle that
different compounds will have different solubilities and adsorption to the two
phases between which they are to be partitioned 9.
As the solvent rises by capillary action up through the adsorbent, differential
partitioning occurs between the components of the mixture dissolved in the solvent
the stationary adsorbent phase. The more strongly a given component of a mixture
is adsorbed onto the stationary phase, the less time it will spend in the mobile
phase and the more slowly it will migrate up the plate 9.
The following are some common uses of Thin-Layer Chromatography
- To determine the number of components in a mixture.
- To determine the identity of two substances.
- To monitor the progress of a reaction.
- To determine the effectiveness of a purification.
- To determine the appropriate conditions for a column chromatographic separation.
- To monitor column chromatography.
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 the TLC plate. The plate is then placed in a 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 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 a compound moves up the plate or stays behind depends on the physical
properties of that individual compound and thus depends on its molecular structure,
especially functional groups. The solubility rule "like dissolves like"
is followed. The more similar the physical properties of the compound is to
the mobile phase, the longer it will stay dissolved 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.
The Rf Values
The behavior of an individual compound in TLC is characterized by a quantity
known as Rf and is expressed as a decimal fraction. The Rf is calculated
by dividing the distance the compound traveled from the original position by
the distance the solvent traveled from the original position (the solvent front).
Rf value = Distance the compound traveled from
the original position
Distance the solvent traveled from the original position
The distance a compound travels indicates that compound's physical characteristics.
The greater the similarity to the mobile phase, the further it will be pulled
up through the stationary particles on the TLC plate. Therefore, the more a
sample's components are like the eluting solvent the closer to a value of one
the Rf will be for that component. Hence, known Rf values can be compared to
those of unknown substances to aid in their identifications. Rf value is a
constant for each component only under identical experimental condition. It
depends upon number of factors as4:
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 desiccator 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
The Mobile phase
The purity of solvents and quantity of solvent mixed should be
strictly controlled. It should made freshly for each run if one of the solvents
is very volatile or hygroscopic, for example acetone.
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 solvents evaporate more quickly, solvents run faster,
and Rf values generally decrease slightly.
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.
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.
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.
Depending upon the development technique used i.e. ascending, descending, horizontal
etc. the Rf value changes for the same solvent system.
Eluting Solvents for Chromatography
Least Eluting Power
Hexane or Pentane
The non-polar solvents at the top of the list are often used as a base and
a few percent of a stronger eluting, more polar solvent is added. As the eluting
power of the added solvent increases, the amount that is generally added decreases.
“Medium polar” solvents like diethyl ether and ethyl acetate may be used 1%-50%
combination with hexane making these mixtures very tunable and common. Stronger
eluting solvents like methanol cannot be used as more than 10% of the solution
or the silica gel will dissolve in them causing problems with the separation.
More than 1% of pyridine or acetic acid isn’t often necessary, a drop or two
is more common, while these two additives are next to each other on the list,
they can have very different effects on a separation depending on the functional
groups in the molecules being separated. Water is very strongly eluting and
its presence as an impurity in your solvent can be problematic.
The functional groups of the molecules in your mixture effect how strongly
they are adsorbed by the stationary phase. Very “greasy” non-polar substructures,
usually made entirely of carbon and hydrogen, are hardly adsorbed by silica
gel at all. Polar groups, with oxygen and especially nitrogen are more strongly
adsorbed. The ability to hydrogen bond with the silica gel creates a strong
adsorbing interaction in alcohols, carboxylic acids and amines.
Adsorbability of Organic Compounds by Functional Group
Least Strongly Adsorbed
Saturated hydrocarbons; alkyl halides
Unsaturated hydrocarbons; alkenyl halides
Aromatic hydrocarbons; aryl halides
Aldehydes and ketones
Most Strongly Adsorbed
Acids and bases (amines)
Method of Thin Layer Chromatography
Thin-layer chromatography consists of a stationary phase immobilized on a glass
or plastic plate, and an organic solvent. The sample, either liquid or dissolved
in a volatile solvent, is deposited as a spot on the stationary phase. The constituents
of a sample can be identified by simultaneously running standards with the unknown.
The bottom edge of the plate is placed in a solvent reservoir, and the solvent
moves up the plate by capillary action. When the solvent front reaches the other
edge of the stationary phase, the plate is removed from the solvent reservoir.
The separated spots are visualized with ultraviolet light or by placing the
plate in iodine vapor. The different components in the mixture move up the plate
at different rates due to differences in their partitioning behavior between
the mobile liquid phase and the stationary phase 10.
Applications and Importance of Thin Layer Chromatography
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.
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.
Dye raw materials and end products, preservatives, surfactants, fatty acids,
constituents of perfumes.
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).
Groundwater analysis, determination of pollutants from abandoned armaments
in soils and surface waters, decomposition products from azo dyes used in textiles.
Analysis of Inorganic Substances
Determination of inorganic ions (metals).
Electrolytic technology (meta-nitrobenzoic acid in nickel plating baths).
Applications Related To Organic Chemistry1-5
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
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
· 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.
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
Application of TLC For Separation of Inorganic Ions3
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.
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.
TLC has been used for the isolation and deterimination 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 iodine-potassium iodine solution followed
by 25% HCl – 96% ethanol (1:1).
Penicillines have been separated on silica gel ‘G’ by using the two solvents,
acetone-methanol (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 Product8
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 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.
Thin-layer chromatographic separation of aromatic amines7
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, NaCl and NaBr in micellar
solutions on the mobility and separation efficiency of amines was also assessed.
Software For TLC6
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. A variety of computational techniques exist, employing
either quantum mechanics (ab initio methods), hybrid theoretical and experimental
parameters (semiempirical models), or basic mechanical analysis (molecular mechanics).
1.D. A. Skoog,
F.J.Holler and T.A. Nieman, “Principles of instrumental analysis, Saunders college
publishing, 5th edition, 761-766.
Instrumental methods of chemical analysis, Goel publishing house, Meerut, 6th
S.K Anand, Instrumental methods of chemical analysis, Himalaya publishing house,
5th edition, 2.599-2.616.
K.R Mahadik, S.G Wadodkar, H.N. More, A textbook of pharmaceutical analysis,
Instrumental methods, Nirali Prakashan, vol.2, 9th edition, 18-30.
5. A.H Beckett,
J.B. Stenlake, Practical pharmaceutical chemistry, CBS publishers, 4th edition,
J.Amburgey-Peters, Experiment 1: SPARTAN’02 exercise on lewis structures, polarity,
and thin layer chromatography, Chemistry 211, Fall 2006.
7.M. Ali and V.Agrawal,
Thin-layer chromatography of aromatic amines, Separation Science and Technology,
37(2002), 363 - 377.
Application of thin-layer chromatography for analysis of heavy petroleum products,
Plenum Publishing Corporation, 1985, 462-464.
(Adapted from Mohrig, 1st ed., 151-162.
C. S. Magdum, Bansode Shubhnagi, Deshmukh Swapnil, Ali Asif.
Department of Pharmaceutical Chemistry, Appasaheb Birnale College of Pharmacy,
Pramod S. Agrawal
Appasaheb Birnale College of Pharmacy,South Shivaji Nagar, Sangli-Miraj Road,
Mob.No. 09881510272, E.mail. email@example.com
Separation of Polar and Hydrophilic Compounds Using a Zwitterionic Stationary Phase in Hydrophilic I
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