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53
Planar Chromatography UNIT 6 PLANAR CHROMATOGRAPHY
Structure 6.1 Introduction
Objectives 6.2 Paper Chromatography
Principle Stationary Support Solvent Systems Development of
Chromatogram Detection Methods Applications
6.3 Thin Layer Chromatography Stationary Phases Mobile Phases
Apparatus and Requirements Detections Methods Plate Concept Applied
to TLC High-Performance Thin Layer Chromatography (HPTLC)
Applications
6.4 Quantitative Aspects of PC and TLC 6.5 Comparison of PC and
TLC 6.6 Summary 6.7 Terminal Questions 6.8 Answers
6.1 INTRODUCTION So far you have studied about the general
principles of chromatography where theoretical principles including
resolution and plate concept were dealt. You now know that
chromatography is now a very powerful separation technique used not
only for the separation of complex mixtures but it is also used for
quantification of each constituent. You have also learnt about
classification of various chromatographic techniques which include
a wide range of ion-exchange chromatography, affinity
chromatography, gel filtration, electro chromatography, zone
electrophoresis, size exclusion chromatography etc.
In this unit, you will learn about planar chromatography, which
includes paper chromatography (PC), thin layer chromatography (TLC)
and electro chromatography (EC). Each of these techniques make use
of a stationary phase in the form of a sheet or flat surface of a
paper or any other material such as metal, glass or plastic plate
coated with suitable adsorbent with the help of a binder. The
mobile phase moves through the stationary phase by capillary
action, assisted by gravity or an electrical potential. Here, we
shall discuss only two techniques i.e. PC and TLC and a comparative
study of their principles, methodology and applications will be
undertaken.
Once upon a time, term planar chromatography included
two-dimensional chromatography though it has now come to signify
the coupling of two chromatographic techniques with different
mechanisms of separation. It is called planar because the
stationary support consists of the plane surface of a paper or
smooth glass plate. It includes paper chromatography (PC) and thin
layer chromatography (TLC) which are the simplest of all other
forms of chromatographic techniques. It also includes
electrophoresis or electro chromatography where the movement of
mobile phase is assisted by electrical potential. However, it will
not be discussed here. The paper chromatography (PC) and thin layer
chromatography (TLC) have the advantage of being simple, fast and
inexpensive. These have been widely used for the
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54
Chromatographic Methods-I
qualitative identification of different constituents in a
mixture though these could be also used for quantitative
determination of the components. However, PC is not used so
commonly these days and as of now, planar chromatography based on
TLC has found widest applications in organic synthesis laboratory,
drug industry, clinical research and for investigating biochemical
processes. In both the cases, sample is spotted with a micropipette
on to a paper or a plate and then the chromatogram is developed
using a suitable organic solvent. Each constituent present in the
sample is identified on the basis of colour development using a
suitable detection system. Different components of a solute travel
with different speeds and the distance travelled by each component
with respect to that of solvent gives a parameter called
retardation factor or Rf.
Even though PC and TLC have many things in common but there are
some basic differences in their operation. Here, we shall discuss
the basic principles, apparatus required, methodology and some
typical applications of both the techniques in the separation of
complex mixtures of organic and inorganic compounds. We shall first
describe paper chromatography, its principle, methodology and
applications. PC is the simplest of all the chromatographic
techniques but it is not so widely used these days. Later, we shall
discuss thin layer chromatography, its methodology and applications
including modern developments of plate concept and high-performance
thin layer chromatography. Remember that though PC and TLC remain
primarily qualitative techniques but these could also be used for
quantitative analysis. A comparative study of the two techniques
will also be presented
Objectives After studying this Unit, you should be able to
explain the meaning of planar chromatography,
describe the principle of paper chromatography (PC), give the
meaning of Rf and factors affecting it,
discuss the type of paper used as support and the types of
solvent mixtures used in paper chromatography,
describe how to run and develop the paper chromatogram,
explain the methodology of separation of inorganic and organic
mixtures,
give the potential applications of paper chromatography,
describe the principle of thin layer chromatography (TLC), list
the types of supports and the types of mobile phases used in
TLC,
explain how to run and develop the thin layer chromatogram,
discuss the advantages of two-dimensional paper and thin layer
chromatography,
apply plate concept to TLC,
describe the basic principle of high-performance TLC (HPTLC),
discuss the potential applications of TLC,
describe the quantitative aspects of PC and TLC; and
compare PC and TLC.
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55
Planar Chromatography 6.2 PAPER CHROMATOGRAPHY
It is one of the oldest and simplest techniques for qualitative
analysis though it can also be used for quantitative determination.
During later part of nineteenth century Runge, Schnbein and
Goppelsroeder separated coloured dyes and other chemicals on paper
or cloth which is considered as old cousin of paper chromatography.
In 1944, R. Consden and A. H. Goron, two coworkers of A. J. P.
Martin, the Nobel Laureate, first reported the separation of a
mixture of amino acids and laid the foundation of paper
chromatography. The technique is based on the movement of solvent
phase in upward or downward direction by gravity and accordingly,
it can be categorized as ascending or descending PC. There is
another version of it, called circular paper chromatography where a
circular paper is taken instead of strip and the solvent phase
moves in circular direction. However, it is not very commonly
used.
6.2.1 Principle The technique of paper chromatography consists
of a sheet of cellulose filter paper which serves as a stationary
phase or separation medium. A small amount (usually a few
micrograms) of solute is placed in a small area near the end of
strip. A solvent is allowed to move from the end of the paper by
capillary action and after equilibration for some fixed period, the
solute migrates from its initial point of application. The
components of mixture are separated completely or partially in
distinct coloured zones or are located by the application of
different reagents or by applying ultra-violet fluorescence.
At first, PC was considered as simply a form of liquid-liquid
partition. The hydrophilic fibers of paper can hold (or bind) water
in humid atmosphere such that a large percentage of water, say >
20% by weight, may be held in filter paper. Thus, paper was
considered to be the analog of a column containing a stationary
aqueous phase whence solute molecules get partitioned between this
water and the mobile immiscible organic solvent. Later, this model
was considered to be too simple because separations were also
obtained where mobile phase was miscible with water or in other
cases where it was just aqueous phase. Thus, it cannot be
considered as simply liquid-liquid partition mechanism. Instead,
besides adsorption and hydrogen bonding, interactions between
solutes and the cellulose support are involved. During the pulping
and bleaching operations of paper, carboxylate and other ionizable
groups are introduced into cellulose which makes the paper as ion
exchanger.
Rf value: It is a characteristic parameter called retardation
factor and abbreviated as Rf. It represents the position of an ion
or a substance with respect to solvent phase. Rf of a solute is
defined as the ratio of the rate of movement of the solute to the
rate of movement of the solvent. Rf is most commonly used in paper
and thin layer chromatography and is considered as a characteristic
of nature of the solute sample which may, of course, change with
the solvent phase. It describes relative migration of the solute
with respect to the solvent and may be represented as
m
s
solventthebytraveledDistancesolutethebytraveledDistance
dd
R f == (6.1)
where, ds and dm are linear distances measured from the line of
origin where spots are put as illustrated in Fig. 6.1. By
definition, Rf value cannot exceed 1.0. Ideally, Rf values must be
in the range of 0.1 to 0.9 with a minimum separation of 0.05. In
order to have better separation, two spots must not overlap with
each other and these must be symmetric without any tailing. In
order to avoid tailing, different solvent mixtures, mixed in proper
ratio, must be tried. If the spot of the solute is not symmetric,
then ds is measured from the position of maximum intensity or the
centre of the spot.
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56
Chromatographic Methods-I
Fig. 6.1: Procedure for calculation of Rf value in paper
chromatography
It has been observed that Rf values are influenced by the
impurities in the paper and solvent, temperature and saturation of
the atmosphere. Following factors may be considered; i) Presence of
other ions e.g. presence of chloride is carried out with
nitrate
solutions. ii) Acidity of the original solution-This may be
needed to avoid hydrolysis and its
need in the formation of soluble complex. iii) Development time-
Sometimes it increases with the running time. Therefore
optimum time may be determined. iv) Presence of other cations or
anions as impurities. v) Ambient temperature. Since Rf value
depends on the distribution of complex species of the cations with
different organic solvents, solvents should be carefully
chosen.
The technique of paper chromatography is primarily used for
qualitative identification though it could also be used for
quantitative determination but with a poor precision. Hence, it
could be at best considered as semi-quantitative technique.
SAQ 1 Which of the following phenomenon is responsible for the
rise of solvent in paper chromatography? i) Capillary action ii)
Gravity iii) Ion-exchange iv) Chemical affinity
...
...
...
SAQ 2 What is the ideal range of Rf values?
i) 0.0 to 0.9 ii) 0.5 to 1.0 iii) 0.05 to 0.95 iv) 0.1 to
0.9
...
...
...
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57
Planar Chromatography
6.2.2 Stationary Support As mentioned already, the stationary
support material is a highly purified cellulose filter paper such
as Whatman No. 1, 2, 31 and 3 MM or acetyl acid paper having
hydrophilic affinity for water. The solvent penetrates the fiber
and causes swelling of paper changing its dimensions. Polymeric
cellulose structure contains several thousand anhydroglucose units
linked through oxygen atoms. Alternatively, modified forms of paper
such as impregnated with alumina, silica gel, hydrous zirconium
oxide, ion exchange resin may be used. Sometimes paper is coated
with chelating agent solution such as dimethylglyoxime,
8-hydroxyquinoline etc. However, it should not have any impurities
of Ca2+, Mg2+, Fe3+, Cu2+ etc so as to avoid interference. The
paper is impregnated either neat or dissolved in a volatile
solvent. The solvent should evaporate slowly so that stationary
phase may distribute homogeneously. In this case, coated liquid
phase may interfere with the detection of separated spots. The
paper shows following properties: 1. weak ion-exchange properties
2. adsorptive properties 3. water holding property 4. mild reducing
agent The quality of paper and its porosity play an important role
in paper chromatography as it determines the rate of movement of
the solvent used. Thick paper with increased sample capacity may be
used for preparative studies. The most suitable cardboards are
Schleicher and Schull 2070, SS2171 which can take a load up to 1 g
at each point of application. Generally, a paper strip is cut into
4 cm 30 cm for one dimensional PC. For two dimensional PC, however,
a 30 cm 30 cm square sheet is commonly used.
SAQ 3 Explain why following types of paper can not be used for
paper chromatography? i) Ordinary filter paper
.......................................
.......................................
...
.......................................
ii) Glazed paper ...
.......................................
.......................................
iii) Butter paper ...
.......................................
.......................................
iv) Bond paper ...
.......................................
.......................................
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58
Chromatographic Methods-I
Typical Mobile Phases for P C
Isopropanol,-ammonia -water (9:1:2)
n-Butanol-acetic acid -water (4:1:5)
Water-phenol Formamide-chloroform Formamide-chloroform
-benzene Formamide-benzene Formamide-benzene
-cyclohexane Dimethylformamide
-cyclohexane Kerosene-(7:3)-isopropanol Paraffin
oil-dimethylform
amide -methanol-water
6.2.3 Solvent Systems The nature of solvent plays an important
role in the development of paper chromatogram. The solvent should
be free from impurities and dried before use. Polar phase such as
water is adsorbed by the paper and held stationary whereas the less
polar solvent such as ethanol, acetone, glycol, formamide, acids,
and amines flow through easily. Though pure solvent may be used but
a mixture of solvents is preferred. Many solvent mixtures can be
used provided these are not immiscible with one another. The
following criteria may be adopted for the choice of the
solvent;
The solvent should not react chemically with any of the
components of the sample mixture.
The composition of solvent mixture should not change with time.
It means that none of its components should be volatile.
The solvent should not interfere with the detection of
spots.
The distribution ratio should be independent of solute
concentration.
The minimum difference between the Rf values of any two
components should be 0.05 or 0.1 so that they may be separated
easily.
Some of the solvents commonly employed for the separation of
cations are mentioned below; i) n-Butanol saturated with 3M
hydrochloric acid: equal volumes of alcohol and
acid are shaken together and the upper layer is used. ii)
Acetylacetone saturated with water: 7.5 mL acetylacetone is mixed
with 0.05
mL hydrochloric acid and 2.5 mL dried acetone. iii) Acetone
containing 5% (v/v) water and 8% (v/v) hydrochloric acid iv)
Glacial acetic acid containing 25% (v/v) dried methanol v) Methanol
vi) Methyl ethyl ketone containing 30% (v/v) water and 1% (w/v)
potassium
thiocyanate vii) Methyl acetate containing 3% (v/v) methanol and
10% (v/v) water viii) Pyridine containing 10% (v/v) water ix) Dried
n-butanol containing 40% (v/v) dry methanol The solvents must be
refluxed over suitable drying agent such as potassium hydroxide for
acetone and ethyl methyl ketone, or anhydrous calcium sulphate for
n-butanol or as prescribed in literature.
6.2.4 Development of Chromatogram Paper strips are cut in
appropriate size (usually 4-5 cm 35-40 cm for a single spot but
breadth may be changed for multiple spots) and stored under
controlled conditions of humidity. A thin pencil line is drawn
across the paper 2-3 cm from the edge and a circle is put at its
centre. The sample is dissolved in a volatile solvent and it is
spotted in the centre of line using a lambda (or micro) pipette of
usually 5-10 micro liters or even with a capillary in case of
qualitative analysis. The spot must be as small as possible without
any spread for better separation and symmetric spots offer
separation. It is best done by placing the sample drop wise, drying
it with hot air blower (or hair drier) to evaporate the solvent as
illustrated in Fig. 6.2. Sample spot may also be dried using
infrafil lamp in a chamber. After drying, another drop may be put
and dried. Several spots of different samples or a sample and the
standard can be made across the line with a minimum separation of
1- 2 cm. The paper is now ready for development.
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59
Planar Chromatography
Fig. 6.2: Illustration of (a) spotting of solute sample on paper
using a capillary tube and (b) drying process using hair dryer
Important equipment used in paper chromatography is a
development chamber where chromatogram is developed in a controlled
environment. Some commercially available development chambers used
in ascending and descending PC are shown in Fig. 6.3. In ascending
chromatography, the paper is supported by means of a clip as shown
in Fig. 6.3 (a).
Fig. 6.3: Some typical Developing chambers for paper
chromatography: (a) Ascending and (b) Descending
In descending PC, the top edge of the paper is held down by a
glass rod or strip as shown in Fig. 6.3 (b). The development
chamber is presaturated with the solvent
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60
Chromatographic Methods-I
system and then paper is hung with its end dipping in the
solvent system as shown in the figure. The solvent starts rising
slowly and then it stops after some time. It may take an hour or
even longer.
The development time will depend on the complexity of the
mixture of solutes being separated, solvent system and the quality
of paper and the ambient temperature. The diffusion of the solvent
and the resulting separation into spots is termed as development of
the chromatogram. It is essential that paper should be equilibrated
with the solvent vapours in the chamber. For good resolution,
reasonable Rf values must be in the range of 0.4 to 0.8 with
typical separation time being 1 to 2 hours. Following are the main
sources of error:
Lateral diffusion of the solutes.
Variation in the structure of paper.
These become important especially for the concentrated
solutes.
Variation in the geometry of the assembly for the standard and
unknown samples. However, this error can be easily eliminated by
using the same tank for the two samples where similar experimental
conditions are maintained.
6.2.5 Detection Methods After development of chromatogram, the
solvent front is marked and the solvent is dried. The spots of the
separated compounds are then detected in a variety of ways by using
any one of the following methods. i) Reactions with colouring
reagents such as dimethylglyoxime for Ni and
hydrogen sulphide gas for any of Gr II elements ii) Fluorescence
producing reagents iii) Inherent visible colours of the components
such as sulphides and oxides iv) Radioactivity measurement of
radiotracers v) Electrochemical methods such as potentiometry,
conductance measurements and
polarography have been successfully used for the detection and
quantitative analysis in paper chromatography. However, these
methods are of limited importance and not commonly used.
The reagents employed include diphenylthiocarbazone (dithizone),
rubeanic acid, diphenyl dithiocarbazide, alizarin, salicylaldoxime,
morin, potassium ferrocyanide, potassium chromate, ammonium
sulphide and hydrogen sulphide gas. In many cases, two or more of
these reagents are advantageous.
The spots are characterized by their characteristic Rf values.
Identical Rf values for a known and an unknown compound using
several different solvent systems provide a good evidence that two
are same especially if they run side by side along the same strip
of paper.
Example i) In a paper chromatographic separation of cations-
Ag+, Pb+ and Hg+, solvent
front rises to 18.4 cm while cationic spots were observed at
15.8, 12.1 and 5.9 cm, respectively. Calculate Rf values of the
metal ions.
ii) Emission gases of a two wheeler were tested for pollutant
metals by paper chromatography. A spot corresponding to Rf value of
0.65 was observed. What is the possible pollutant metal ion in the
emission gases?
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61
Planar Chromatography
Solution: Here, Rf value for Ag+ = 15.8/18.4 = 0.86 Rf value for
Pb+ = 12.1/18.4 = 0.66 Rf value for Hg+ = 5.9 /18.4 = 0.32 As Rf
value of 0.65 is comparable to 0.66 corresponding to Pb+ , it can
be concluded that the pollutant in the emission gases is likely to
be lead.
6.2.6 Applications Paper chromatography has been widely used for
separation and identification of cations in inorganic mixtures,
organic functional groups, proteins and enzymes in biochemical
work. It is particularly useful in the separation of closely
related compounds such as isomers, homologues, isotopes and species
having different valency or oxidation states. It has been found
especially useful in the following:
identification of trace metals in ores,
checking purity of pharmaceuticals and fermentation,
ripening of fruits and fermentation products,
detection of adulterants and contaminants in foods and drinks by
comparing chromatogram of pure compound with that of the
adulterant.
Sometimes, it happens that all the components of a mixture can
not be separated using a single solvent system; some components
separate better in one solvent and some in another. In such cases,
two-dimensional paper chromatography may be employed. In this
technique, a square paper of 30 cm x 30 cm is taken and the sample
is spotted at one corner of the sheet. Chromatogram is developed
using one solvent whence solute migrates parallel to one edge of
the paper. Next time, the paper is turned at 90o and developed in a
second solvent system which carries the solutes into the unused
portion of the paper. A typical chromatogram of a protein
hydrolysate using ninhydrin-stained spots is shown in Fig. 6.4.
Fig. 6.4: Illustration of two-dimensional paper chromatogram of
a protein hydrolysate where solute is placed in corner at H and it
is first run towards the right with an acetic acid-butanol solvent
and then at perpendicular using phenol + resol/water solvent
Some typical applications are described below:
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62
Chromatographic Methods-I
i) Separation and Identification of Mn, Ni, Co and Zn A
combination of Mn, Ni, Co and Zn may be separated by paper
chromatography using Whatman No. 1 filter paper strip by developing
with a mixture of acetone, water-hydrochloric acid system. The
spots are located by the colouring reagents. These elements are
identified by comparing with their characteristic Rf values of
known with those in unknown mixture. The characteristic Rf values
are as follows:
Ni - 0.09 Mn - 0.21 Co - 0.43 Cu - 0.61
ii) Separation of a mixture of pesticides A combination of
pesticides such as aldrin, endrin, lindane, heptachlor, DDT, BHC
etc. may be separated on Whatman No. 1 filter paper treated with
refined mineral oil in diethyl ether (5% v/v) and washed with 75%
aqueous acetone. The mobile phase used is 3:1 methanol-water (v/v).
The Rf values for aldrin, heptachlor, DDT, endrin and lindane were
found to be 0.37, 0.48, 0.60, 0.62 and 0.89, respectively.
iii) Speciation of Different Anions of Sulphur In its unique
application, paper chromatography has been successfully used for
speciation studies of sulphur such as S2-, SO32-, SO42-,S2O32-,
S4O62- using radiotracer 35S. Whatman No. 1 filter paper was used
along with solvent mixtures of dioxane, n-butyl alcohol-1N ammonia
(1:1:1) and acetone-iso-propanol-liquor ammonia (1:1:1). Different
spots corresponding to various anions can be identified by
radioactivity measurements using a G. M. counter. The Rf values for
SO42-, S2O32-, S4O62- and S2- were found to be 0.12, 0.43, 0.54 and
0.77, respectively. Similarly, Rf values for S2O32- and SO32- were
found to be 0.14 and 0.61, respectively.
Paper treated with silicone or paraffin oil permits reversed
phase paper chromatography where mobile phase is a highly polar
solvent. This technique is referred to as reverse-phase PC.
Commercially available papers coated with adsorbent or ion-exchange
resin offer additional applications of adsorption and ion-exchange
paper chromatography.
iv) Autoradiography using Radiotracers In this technique, a
radiotracer or a radiolabelled solute sample is used. After
development of chromatogram, it is kept in contact with a
photographic film of the same size. After some time when the film
is developed then dark spots are observed in place of spots
corresponding to the movement of solute components. This is called
autoradiography. It has been found to be especially useful for the
study of distribution and metabolism of compounds administered to
the plants or animals. In a unique experiment, the Nobel Laureate
Calvin and coworkers identified intermediate steps involved in the
photosynthesis of carbohydrates from atmospheric CO2 in the
presence of light and chlorophyll by using 14C, 32P and 3H. Plants
were placed in the atmosphere containing 14C labeled CO2 and
irradiated with light as shown in Fig. 6.5.
The plants were removed after different irradiation periods of
light and the molecular components were separated using paper
chromatography. The presence of radioactive atoms in a compound by
following autoradiography was taken as a proof of the involvement
of that compound in the photosynthesis.
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63
Planar Chromatography
Fig. 6.5: Illustration of Autoradiography method used for the
determination of compounds involved in photosynthesis
6.3 THIN LAYER CHROMATOGRAPHY Thin layer chromatography is
different from paper chromatography in that a flat
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64
Chromatographic Methods-I
surface of glass, metal or plastic coated with adsorbent such as
silica gel, alumina or any other material, is used. The technique
discovered by Ismailoff and Scraiber in 1938 is faster, more
sensitive and has better resolution than paper chromatography. TLC
is often used to develop optimal conditions for separation by
liquid column chromatography. The technique has become a workhorse
of the drug industry for purification of products. It has also
found widespread use in clinical, industrial and environmental
laboratories. Recent developments have elevated it from the level
of semi-quantitative analytical procedure to one in which highly
reliable quantitative separations can be performed.
6.3.1 Stationary Phases The stationary phase used in TLC can be
an adsorbent, an ion exchanger, a molecular sieve or it can serve
as the support for a liquid film. It consists of a finely divided
powder of particle size 5 to 50 m. A variety of stationary supports
are available for coating the smooth surface of glass, metal or
plastic. Plastic sheets have the advantage that these can be easily
cut to any shape or size as required l. Silica gel G (60-120 mesh)
remains the most frequently used coating materia. It contains
hydroxyl groups on the surface which form hydrogen bonds with polar
molecules. The adsorbed water prevents other polar molecules from
reaching the surface. Hence, the gel is activated by heating to
remove the adsorbed water. In modified silica gel, H and OH groups
can be replaced by other functional groups similar to bonded phase.
Alumina containing hydroxyl groups or oxygen atoms is another
commonly used adsorbent. Other adsorbents widely used are powdered
cellulose, magnesium silicate, calcium silicate, activated
charcoal, natural diatomaceous earth, ion exchange resins such as
Dowex-50W-strong acid cation exchanger in sodium or hydrogen form
and Dowex-1-strong base anion exchanger in chloride form.
Kieselghur is often used for the separation of sugars. In fact, any
adsorbent material that can be used in adsorption or column
chromatography can be used in TLC.
Here, an aqueous slurry of the powder is prepared by mixing the
adsorbent with a binder such as plaster of Paris (CaSO4) and
polyvinyl alcohol to help it adhere to the backing material. It is
uniformly spread over the plate manually or by using one of the
commercial forms of spreader as shown in Fig. 6.6. The thickness of
the layer is usually in the range 0.1 to 0.3 mm but for preparative
work much thicker layers are preferred. The solvent is evaporated
off and adsorbents are activated by placing in an oven at 110o C
for a few hours. Precoated ready-to-use plates and sheets are also
commercially available.
Fig. 6.6: Apparatus used for preparing TLC plates where the
applicator is filled with the slurry of adsorbent and binder in a
solvent.
Thin layers of Sephadex superfine gel can be prepared for size
exclusion. Gel is soaked in water for a few days and then spread on
the plate. These are not dried but stored wet. Capillary action
through these molecular sieves is much slower, typically of the
order of 1 to 2 cm/hour. Hence, it takes longer period to develop
these plates.
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65
Planar Chromatography
The following precautions must be followed while handling TLC
plates: 1. The surface of the TLC plates should not be touched. The
plates should be
handled carefully by holding at the edges so as to avoid any
contamination due to sweat.
2. The plates should be cleaned thoroughly so as to remove any
extraneous material that might contaminate the adsorbent.
SAQ 4 Explain why the flat surfaces of the following materials
cannot be used as an inert support for the coating of the adsorbent
in TLC? i) Plywood ...
...
...
ii) Asbestos sheet ...
...
...
iii) Card board ...
...
...
iv) Glass with granulated surface ...
...
6.3.2 Mobile Phases The choice of the mobile phase is largely
empirical but some general guidelines can be formulated. A mixture
of organic solvents and water with the addition of acid, base, or
complexing agent to optimize the solubility of the components of a
mixture can be used. It may be emphasized that a large degree of
trial and error is involved in the selection of the mobile phase.
The polar solvents can themselves become strongly adsorbed; thus,
producing an undesirable situation of partition system. The
following criteria may be adopted. i) The developing solvent must
be of the highest purity because even trace
amounts of impurities may yield irreproducible results. ii) Good
separation of polar or ionic solutes can be achieved with a mixture
of
water and n-butanol. The criteria used for the selection of
developing solvent are the same as for column chromatography.
iii) The eluting power of solvents increases in the order of
their polarities e.g. from hexane to acetone to alcohol to water.
An eluotropic series, given in Table 6.1, can be used for the
selection of the best solvent or the solvent mixture for a
sample.
iv) If the stationary phase is hydrophobic, mixtures of benzene,
cyclohexane, and chloroform in different ratios provide
satisfactory mobile phase.
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66
Chromatographic Methods-I
Table 6.1: Eluotropic series of mobile phase
Solvent Solvent strength
o
n- Pentane 0.00 Cyclohexane 0.04 Carbon tetrachloride 0.18
Toluene 0.29 Chloroform 0.40 Methylene chloride 0.42
Tetrahydrofuran 0.45 Acetone Methyl acetate Acetonitrile
0.56 0.60 0.65
n- and iso-propanol 0.82 Ethanol 0.88 Methanol Water Ethylene
glycol
0.95 1.00 1.11
6.3.3 Apparatus and Requirements Commonly, the plates in sizes
of 2.510, 2.515, 2.520, 520, 1020, and 2020 in centimeters are
available. These are of two types: conventional and
high-performance. The former have thicker layers of about 0.25 mm
with particle size of < 20 m. On the other hand, the high
performance plates usually have thickness of 0.1 mm and particle
size of
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67
Planar Chromatography
The plate is immersed in the developing solvent avoiding its
direct contact. The developing solvent travels up the plate and
after passing the point of sample application; it dissolves the
sample and carries it up the plate. Thus, the sample distributes
itself between the moving solvents and the stationary phase.
After the developer has traveled about two-third of the length
of the plate, it is removed from the container and dried. The
positions of the components are then determined by any of the
methods described in the next section.
6.3.4 Detection Methods The detection of the spots in TLC is
easier than in PC as silica and alumina used as support are inert
than paper and hence, strongly reactive reagents can be used to
locate the compounds. A universal technique involves the use of
iodine vapours for colourless or non-fluorescent spots. It consists
of exposing the developed plate to iodine vapours which interact
with the sample components, either chemically or by solubility to
produce colour. Alternatively some other colour forming reagents
called chromogenic reagents as given in Table 6.2 may be used.
Table 6.2: Some Chromogenic Reagents Used for Identification in
Thin Layer Chormatography
Reagent Application
Ninhydrin or isotin Amino acids 2,4-dinitophenylhydrazine
Ketones and aldehydes H2S water, diphenylcarbazide Metals Rubeanic
acid Metals Aniline phthalate Sugars Chloroplatinic acids Alkaloids
Bromothymol blue Lipids Antimony trichloride Steroids, essential
oils
Now-a-days, the thin layer plates and sheets incorporating
fluorescent dyes in powdered adsorbent, are commercially available.
When these are held under ultraviolet radiation, dark spots glow
where sample spots occur due to fluorescent substances.
Alternatively, an immobile fluorescent substance may be added while
preparing the slurry so that the separated compounds appear as dark
spots against fluorescing background when the plate is viewed under
the ultraviolet light.
Another common technique used for the identification of organic
compounds is by spraying the plate with concentrated sulphuric acid
solution and then heating it in an oven. Charring of the organic
compounds make them appear as black spots.
The Rf values in TLC are difficult to reproduce because of
experimental variables. Following factors may be considered.
i) Nature of the adsorbent-its chemical nature, particle size,
surface area and binder. Also its activity, thickness and
uniformity.
ii) Nature of mobile phase-its purity, moisture content,
precision of mixing in case of mixture and volatility.
iii) Amount of sample used iv) Vapour-pressure equilibrium
between the plate and the atmosphere of the
development chamber v) Ambient temperature
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68
Chromatographic Methods-I
A typical thin layer chromatogram of a mixture is shown in Fig.
6.8.
Fig. 6.8: Thin layer chromatogram of a mixture
The chromatograms can be stored for future reference or their
photographs can be made.
Solved example Methanolic extract of a medicinal herb Terminalia
Arjuna bark powder was passed through a silica gel column and
eluted by various solvents. Petroleum ether-ethyl acetate (10:1)
eluant spotted on TLC where three components A, B and C
corresponding to Rf values of 0.63, 0.72 and 0.79, respectively
were observed. One of these compounds was a carboxylic acid which
was identified as tartaric acid. In order to further confirm, a
known standard tartaric acid was run on TLC where solvent front ran
up to 8.6 cm and its spot was found at 6.3 cm. Which one of the
spots corresponds to tartaric acid?
The Rf value for standard tartaric acid = 6.3/8.6 = 0.73
Thus, the standard value of 0.73 is comparable with 0.72 for B
compound. Hence, B is tartaric acid.
6.3.5 Plate Concept Applied to TLC Plate concept and its terms
as developed for column chromatography can be applied to thin layer
chromatography after slight modifications. As described in sub-Sec.
6.2.1 and Eq. 6.1, the retardation factor Rf is given by the
equation Rf = ds/ dm. In order to adapt, thin layer chromatography
with the plate concept, a chromatogram may be visualized as a plot,
shown in Fig 6.9, where the spot diameter may be considered as
width in Eq. 6.6. Further, ds and dm may be defined in terms of
retention time of solute (tr) and retention time of mobile phase
(tm), respectively.
Fig. 6.9: Schematic representation of thin layer chromatogram
with plot
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69
Planar Chromatography
For the mobile phase, tm may be defined as the distance divided
by its linear velocity, u tm = ds /u (6.2)
The solvent does not reach the same point until the mobile phase
has travelled the distance dm. Therefore,
tr = dm /u (6.3) Substituting the values of tr and tm in
equation tr = tm (1 + k), we get
k = (dm ds)/ ds (6.4) The capacity factor k can also be
expressed in terms of the retardation factor Rf as
k = (1 ds/dm) / (ds/dm) = (1 Rf)/ Rf (6.5) This approach can be
used for the method development in column chromatography. It is not
easier to calculate capacity factor by thin layer chromatography
but it is more rapid than obtaining it from column chromatography
experiment. Also, this can be used for calculating the plate height
by first determining the number of plates as follows:
N = 16 (ds / w)2 (6.6) where w is the spot width as shown in
Fig. 6.9. Thus, the plate height H is represented by
H = dm /N (6.7)
6.3.6 High-Performance Thin Layer Chromatography (HPTLC) In
order to have better resolution and sensitivity in TLC, it can be
coupled with high-performance liquid chromatography (HPLC), mass
spectrometry (MS), Fourier-transform infrared (FTIR) etc. As
already mentioned in Sec. 6.3, TLC can be compared with
liquid-liquid chromatography where solute is detected in the
presence of the mobile phase which can be so chosen that it may not
interfere with the detection wavelength. Reverse phase version of
TLC has been proposed where better separation can be obtained than
that with normal TLC. Thus, the performance of TLC can be improved
by improving the quality of adsorbent and sample application
procedure done by choosing smaller particle size, reducing the
plate length and using smaller quantity of sample as described
below.
Quality of Adsorbent: Specially purified silica gel with average
particle diameter of 3-5 m is used and it may also be modified with
chemically bonded layers. Such an adsorbent may have 5000
theoretical plates providing a much improved performance over the
conventional TLC. Thus, a better separation may be achieved in much
shorter time.
Methodology of Sample Application: The amount of sample applied
in HPTLC is much smaller, typical volumes being 100-200 nL with a
compact diameter of 1.0-1.5 mm. Number of samples may be increased
because of its compact size. After developing the plate up to a
distance of 3 to 6 cm, compact separated spots are obtained which
may have 10 times better detection limits than the conventional
TLC.
Application of the sample on the plate is a critical process in
HPTLC. A convenient spotting device used is platinum-iridium
capillary of 100-200 nL whose tip is polished to provide a smooth,
planar surface of area 0.05 mm2. It is used with a mechanical
applicator which minimizes damage to the plate surface.
Quantitative analysis requires scrapping of the spot followed by
dissolution or direct scanning. In order to shorten the procedure,
solid phase extraction column is used to concentrate the sample,
followed by automatic sampling device so that multiple samples may
be processed in a short period and highly reproducible results are
obtained.
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70
Chromatographic Methods-I
Detection: In situ quantitative analysis is carried out by
direct photometric measurements. For this purpose, single or double
beam spectrophotometers are available. Scanning densitometers based
on reflectance and absorbance of light are often used to measure
the individual spots. The chromatogram is scanned with a moving
beam of light and the intensity of reflected light from the plate
surface is measured. The difference in light intensity between the
thin layer plate without sample and with spot is displayed as peaks
in the scan. The areas of peaks correspond to the amount of solute
component in the sample. An alternate procedure consists in
measuring the light transmitted through the plate.
Photodensitometers measure the transmitted or reflected light to
produce a photograph revealing dark and light zones for the areas
of constituents in the sample. The standard deviation for
quantitative determinations by densitometry is better than 5%.
CAMAG, Switzerland have developed a HPTLC Vario system with key
features of development with six different solvents side by side,
sandwich as well as tank configuration making directly comparable
results, and six different conditions of equilibration including
relative humidity.
6.3.7 Applications Thin layer chromatography is widely used for
qualitative analysis; almost any mixture can be partially resolved.
Inorganic separation of metals in alloys, soils and geological
samples and so also organic compounds formed during synthesis work
or the analysis of natural products can be easily achieved by TLC.
It is ideally suited for following the course of complex reactions,
quality control, purity checks, plant extracts, biochemical
preparations, clinical diagnosis, forensic tests etc. It is
primarily used for qualitative identification by comparison of the
Rf values with those of standards run under identical conditions or
by removing the material from the chromatogram and subjecting it to
further tests by other techniques. The versatility of this
technique has resulted in a rapid spread of its use in all the
fields. Particularly sharp separations have been obtained for
vitamins, fatty acids, lipids in serum, amino acid s, dyes and
glycerides, pesticides, sugars, etc.
In a typical case, presence of gallic acid in trifala (a mixture
of Harad, Baheda and Amla) was identified by thin layer
chromatography in ethyl acetate-methanol (7:3) solvent system. The
standard sample of gallic acid was spotted along side of unknown
and compared as shown in Fig. 6.10. It is observed that the two
samples give spots with Rf = 0.86. Presence of gallic acid was
further confirmed by elemental analysis, infrared, NMR and GC-MS
methods.
Fig. 6. 10: Identification of gallic acid in trifala using ethyl
acetate methanol (7:3) solvent system where comparable Rf values
are observed
Similar to the two-dimensional paper chromatography described in
sub-Sec. 6.2.5 and Fig. 6.4, the two-dimensional thin layer
chromatography has been found to be very
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71
Planar Chromatography
effective for the separation of complex mixtures. The sample is
spotted in one corner and developed with the first solvent. After
thorough drying, the plate is turned through 90o and developed with
the second solvent. The whole process of the development of
chromatogram is shown in Fig. 6.11. It can be compared with a
standard map of known mixtures.
Fig. 6.11: Schematic representation of two-dimensional thin
layer chromatography; (a) sample is spotted in one corner, (b)
results obtained after first separation, (c) rotation of plate by
90 and (d) final chromatogram
6.4 QUANTITATIVE ASPECTS OF PC AND TLC As already emphasized, PC
and TLC are primarily qualitative techniques used for
identification of inorganic and organic compounds. However, both
the techniques can also be used for quantitative analysis after
taking proper care and suitable calibration so that reproducible
and accurate results are obtained. Here, the most important aspect
is that all the chromatographic conditions must be well
standardized. The following precautions must be taken. i) Standards
and samples must be applied to the paper or plate in spots of
similar
size and at similar concentration using propipette. ii) All the
solvents must be of high purity. While using mixtures, these should
be
prepared carefully. iii) Development chamber must be brought in
equilibrium in the same manner. After the development of
chromatogram, the spot area must be removed carefully by cutting
the paper or scratching out from the plate and measured. Relative
precision of these measurements is usually of the order of 5-10%
but can be 1-2%. The main difficulties in quantitative measurements
lie in defining the boundaries of spots and controlling chromogenic
reactions in a reproducible manner. Following are some of the
physical and chemical methods employed. 1. Visual comparison of
spots: The standards containing known amounts and
samples must be run on the same sheet or plate, and their
relative intensity or area may be compared visually.
2. Physical measurement of coloured spots: The colour intensity
may be measured by transmission or reflectance using
spectrophotometer. Scanning photo densitometers which measure spot
intensity are also available. For organic fluorescent substances,
the fluorescence intensity may be measured under illumination with
ultraviolet radiation. Full spectrum recording and multiple
wavelength scanning capabilities are available commercially.
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72
Chromatographic Methods-I
3. Spot area measurement: The spot area determined by using
transparent graph paper and counting the squares within a spot, is
proportional to the logarithm of the amount of substance. A
standard is run under identical and controlled conditions and a
calibration plot is made between area vs log of concentration.
4. Radioactivity measurements: In this method, radiotracers or
radiolabelled substances are used for spotting. The paper strip or
plate after development can be scanned for activity. A comparison
of the activity for the sample with that of standard may give
quantitative results. Alternatively, before spotting pencil lines
are drawn on the paper strip at an interval of one half cm and
after development these strips are cut and then counted for
radioactivity using GM counter or scintillation gamma ray
spectrometer. Also, an X-ray film may be kept in contact with the
developed paper or thin layer plate so that black spots will appear
at the location of the separated constituents.
5. Removal of spots: The spot is cut from the paper or removed
from the plate by scrapping of the adsorbent. The substance is
eluted or extracted from the spot paper or adsorbent (using dil
hydrochloric acid and slight warming for inorganic substance and
suitable organic solvent for organic substance). The solution is
made up to a standard volume and then measurements are made by
spectrophotometry or any other technique.
CAMAG, Switzerland, pioneers in modern equipments of TLC, have
come up with a Planar Chromatography Manager winCATS which offers a
new approach to thin layer chromatography. It is a 32bit Windows
software designed to control, monitor and document all steps of TLC
analysis. CAMAG instruments like the automatic TLC sampler, scanner
etc. can be linked to winCATS by means of a software interface and
offer unique features.
6.5 COMPARISON OF PC AND TLC Even though PC and TLC both are
considered as planar chromatographic techniques where a plain or
flat surface is used and the main parameter is Rf . But, both these
are widely different in terms of stationary support, developing
chamber, detection methods and even in applications.
The PC has limited applications but the TLC has wide range of
applications especially in drug industry and biochemical processes.
For this reason, the PC technique has been superseded by TLC in
analytical laboratories though it is still used for demonstrating
the general principles of chromatographic separations. A comparison
of TLC and PC separation of nucleotides is shown in Fig. 6.12
(a) (b) Fig. 6.12: A comparison of separation of nucleotides by
TLC and PC under identical
conditions: Solvent; saturated ammonium sulphate-1M sodium
acetate-iso-propanol (80:18:2) (a) Thin layer chromatogram using
cellulose as adsorbent, Run time, 90 min(b) Paper chromatogram
using Scheicher and Schull 2034 paper, Run time, 135 min
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73
Planar Chromatography
In the above figure one-dimensional development using identical
conditions shows the superiority of cellulose layer TLC over PC for
separating various mixtures. It may be observed that TLC shows
reduced degree of spot diffusion or better resolution especially
for samples 1 to 4.
The following points of differences are of special significance:
i) In case of PC, cellulose paper acts as the stationary support
that is flexible
whereas in TLC, suitable adsorbent material is coated on to a
glass, metal or plastic sheet that is rigid.
ii) In PC, the solvent rises by capillary action but in TLC
solvent moves similar to that in liquid column chromatography.
iii) The development of chromatogram in PC may take several
hours but TLC is much faster as it takes only half an hour or
so.
iv) PC may be classified as ascending, descending or circular
types though it is generally carried out in ascending mode. On the
other hand, TLC is primarily ascending though it has a horizontal
version as well.
v) Developing chambers used in PC and TLC are somewhat different
as the paper needs to be hung with a rod whereas the plate touches
the bottom of the chamber in later case.
vi) For quantitative analysis, the paper strip with solute spot
needs to be cut with scissors whereas in TLC, the spot portion
needs to scrapped. In both the cases, the solute is dissolved
though paper is removed whereas adsorbent in TLC is filtered
off.
vii) PC takes more time and is less reproducible whereas TLC is
much faster and more reproducible.
viii) PC and TLC both have been carried out using coated liquid
phases and reversed phase versions have been developed.
ix) The plate concept has been adapted to TLC and high
performance TLC has been developed whereas this is not the case
with PC.
x) The choice of paper in PC is limited whereas in TLC, a
variety of stationary phase surfaces are available.
xi) Not many automated instruments have been developed in PC
contrary to TLC where automated sampler, capillary dispenser,
developing chamber and documentation system with camera or software
have been developed.
6.6 SUMMARY In this unit, you have learnt about planar or
two-dimensional chromatography which includes paper chromatography
and thin layer chromatography. The main parameter in both the cases
is retardation factor, Rf , which is defined as the relative
movement of the solute components to that of the solvent phase. In
both the cases, the details of stationary phase, mobile phase,
detection methods and some typical applications are discussed. The
plate concept has been adapted to TLC so that the separation
process can be better understood. Modern developments in TLC
include development of high performance TLC. A comparison of paper
chromatography and thin layer chromatography is presented. Though
both the techniques are primarily used for qualitative
identification of inorganic and organic compounds but TLC is now
being widely used for quantitative analysis. Use of radiotracers in
both the techniques is also discussed.
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74
Chromatographic Methods-I 6.7 TERMINAL QUESTIONS
1. Spots 3 and 4 in Fig. 6.12 correspond to specific nucleotides
3-GMP and 2-GMP, respectively. Determine the Rf values in PC and
TLC, compare and comment on them.
2. A mixture of U, Mg and Al was separated by paper
chromatography and then quantitatively determined by
spectrophotometry. 100 ppm standards of each of these were prepared
and reacted with oxine for U, magneson for Mg, aluminon for Al
giving absorbance values 0.85, 0. 80, 0.65, respectively. Spot
areas of paper chromatogram were cut, dissolved and their
absorbances were found to be 0.39 for U, 0.42 for Mg and 0.53 for
Al. Calculate concentrations of each of three elements in the
mixture.
3. i) Explain the basic principle of Reverse Phase
Chromatography and its applicability to PC and TLC.
ii) Explain reverse phase PC and TLC. In what respect are these
different from normal PC and TLC?
4. Enumerate reasons for lack of modern developments in paper
chromatography whereas in case of TLC, plate concept has been
adapted and it has been further developed as HPTLC.
5. i) Explain the characteristics of paper used in PC. ii) Can
van Deemter equation be applied to TLC? iii) Similar to iodine used
for detection in TLC, can one use Br2 for the
detection of organic compounds. iv) Explain how Rf value is
different from retention time, tr. How do you
justify the use of retention time in TLC? 6. A six component
mixture of compounds obtained by extracting plant leaves with
an organic solvent was subjected to two-dimensional thin layer
chromatography by placing a portion of the extract in one corner.
First, it was developed with solvent A and after drying the plate
and turning it to right angle, a different solvent was allowed to
flow perpendicular to the direction of the first solvent. The black
dot represents the start of original sample. Finally, the plate was
sprayed with a reagent and the blue coloured spots were observed as
shown in Figure.
7.
The Rf values of six components as reported for known compounds
are listed in Table. Identify the numbered compounds in figure on
the basis of their Rf
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75
Planar Chromatography
values.
Compound Rf (solvent ) Rf (solvent B) 1 0.0 0.5 2 0.8 0.0 3 0.5
0.5 4 0.8 0.8 5 0.2 0.4 6 0.8 0.5
6.8 ANSWERS
Self Assessment Questions 1. i) Capillary action
2. ii) 0.1 to 0.9
3. i) Because of impurities ii) Solvent will not rise because of
smooth surface iii) It has smooth surface on both sides. iv) It
also has smooth surface.
4. i) It has rough surface. ii) It has uneven surface. iii)
Besides uneven surface, solvent will be absorbed by the cardboard.
iv) It has uneven surface.
Terminal Questions 1. Measure Rf values in Fig. 6.12 and compare
two values obtained from PC and
TLC. Rf values obtained from TLC are likely to be more accurate
than those obtained by PC.
2. 100 ppm standards of each of U, Mg and Al show absorbances of
0.85, 0.80 and 0.65, respectively. Considering absorbance for
unknown mixture, concentration for different elements can be
calculated as follows:
Concentration of U = 0.39100/0.85 = 45.9 ppm Concentration of Mg
= 0.42100/0.80 = 52.5 ppm Concentration of Al = 0. 53100/0.65 =
81.5 ppm
3. i) In reverse phase chromatography applicable to
liquid-liquid chromatography, stationary phase is non-polar or
preferably long chain hydrocarbon and the solvent phase is polar
such as chloroform, acetone, tetrahydrofuran or alcohols etc. Its
applicability to PC and TLC are new developments where stationary
phase is modified accordingly so as to obtain better
separations.
ii) In reverse phase paper chromatography, paper is coated with
silicone or paraffin oil and the mobile phase is any polar organic
solvent. Similarly, in case of TLC, adsorbent is modified
accordingly and is coated on a smooth plate whereas the
chromatogram is developed using a polar solvent.
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76
Chromatographic Methods-I
4. In case of PC, paper acting, as stationary phase is difficult
to get in reproducible form. Further, it is difficult to get it in
humid free form. Also, there is no universal detector in PC unlike
in TLC where some common detectors like iodine and ninhydrin can be
used. In addition, PC takes longer time for analysis compared to
that for TLC. Only for these reasons very few attempts have been
made with regard to automation in PC.
Also, in case of PC, the particle size cannot be controlled
whereas it is an essential condition in the conversion to
high-performance form.
5. i) Cellulose filter paper used in PC should be free from any
impurities and humidity and it should not be wet.
ii) No. van Deemter equation can not be applied to TLC. iii) Br2
cannot be used for the detection of organic compounds because
it
reacts with the double bonds present in the compounds and it is
also hazardous to handle.
iv) Rf and tr.values are different. By definition, tr = tM (1+k)
where k is the capacity factor representing column characteristics.
However, Rf value is the ratio of distances travelled by the solute
to that of the solvent.
6. In Figure, the top line from right to left, three spots
correspond to compounds 2, 4 and 6, respectively. Second line from
top, one spot corresponds to compound 3
Third line from top, one spot corresponds to compound 5 Bottom
line, one spot corresponds to compound 1.
Further Readings 1. Vogels Textbook of Quantitative Chemical
Analysis by J. Menham, R.C.
Denney, J.D. Barnes and M.J.K. Thomas, 6th Edn, Low Price
Edition, Pearson Education Ltd, New Delhi (2000).
2. Quantitative Analysis by R. A. Day and A. L. Underwood, 6th
Edn, Prentice Hall of India, New Delhi (2001)
3. Instrumental Analysis, Editors, H. H. Bauer, G. D. Christian
and J. E. OReilly, 2nd Edn, Allyn and Bacon, Inc., Boston
(1991)
4. Principles of Instrumental Analysis by D. A. Skoog, F. J.
Holler and T. A. Nieman, 5th Edn, Thomson Brooks/Cole, Bangalore
(2004)
5. Fundamentals of Analytical Chemistry by D. A. Skoog, D. M.
West, F. J. Holler and S. L. Crouch, 8th Edn, Thomson Brooks/Cole,
Bangalore, 2004,
6. Analytical Chemistry by G. D. Christian, 6th Edn, John Wiley
& Sons Inc, Singapore (2003)
7. Principles and Practice of Analytical Chemistry by F.W.
Fifield and D. Kealey, 5th Edn, Blackwell Science Ltd, New Delhi
(2004)
8. Handbook of Instrumental Techniques for Analytical Chemistry,
Editor, F. Settle, Low Price Edition, Pearson Education Inc, New
Delhi (2004)
9. Instrumental Methods of Chemical Analysis by G. W. Ewing, 5th
Edn, Mc-Graw Hill, Singapore (1985)
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77
Planar Chromatography INDEX
A. J. P. Martin 55 Activated alumina 41 Adsorbent type 41
Adsorption 6, 7, 8, 36 Affinity chromatography 6, 8 Applications of
liquid column Applications
Autoradiography 62 Calvin 62
Separation of pesticides 62 Speciation 62
Reverse-phase PC 62 Ascending chromatography 59 Auto radiography
62 Carrier gas velocity (u) 24 Chromatogram 14 Chromatographic
columns 11 Chromatography 5, 6, 7, 49
Classification 7 Mechanism responsible for separation 7 Nature
of mobile phase 7 Shape of the solid support 7
Chromogenic reagents 67 Circular paper chromatography 55
Cross-linked dextrans 9 Dead time 16 Detection methods 60, 67
Chromogenic reagents 67 Dithizone 60 Development of chromatogram
58
Ascending chromatography 8, 59 Development chamber 59, 67
Development techniques 45 Discplacement development 45, 46
Displacer 46 Elution analysis 45, 46 Gradient elution analysis
47 Frontal analysis 45
Development time 56 Distribution coefficient 37 Distribution
constant 15 Displacement development 46 Displacer 46 Dithizone 60
Durapck 42 Eddy diffusion 20 Eddy diffusion term (A) 21
Multiple flow path term 20, 21 Elution 13 Elution analysis 46
Equipment Column 40
Column packings 40 Detectors 40, 43, 48 Bulk property 40 General
detectors 40
Selective detectors 40 Solute property 40
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78
Chromatographic Methods-I
Mobile phase reservoir 39 Frontal analysis 45 Gas chromatography
7, 10, 13
Gas- liquid chromatography (GLC) 10 Gas- solid chromatography
(GSC) 10
Gas-liquid chromatography 5 Gel filtration chromatography 6, 9
Gel permeation chromatography 9 Gradient elution analysis 47 Height
equivalent to theoretical plate (HETP) 18 Henrys law 23 High
performance liquid chromatography 6, 9 High-performance thin layer
chromatography HPTLC) 10, 69
CAMAG 70, 72 Detection 70 Platinum-iridium capillary 69
HPLC Basic aspects of 48
Ion exchange 5, 6 Ion exchange chromatography 5, 9
Amphoteric exchangers 9 Anion exchangers 9 Cation exchangers
9
Ion pair chromatography 9 Linear chromatography 15 Liquid
chromatography 7, 8, 13, 20, 38
Adsorption phenomenon 36 Affinity chromatography 6, 8 Ascending
chromatography 8 Descending chromatography 8 Extraction
chromatography 8 Gel filtration chromatography 6, 9 Gel permeation
chromatography 9 High performance or high pressure liquid
chromatography 9 High pressure thin layer chromatography (HPTLC) 10
Ion exchange chromatography 9 Ion-pair chromatography 9 Liquid-
liquid partition chromatography 8 Liquid- solid adsorption
chromatography 8 Normal-phase chromatography 8 Reversed-phase
chromatography 8 Size exclusion chromatography 9 Separation factor
27, 37
Liquid column chromatography 35, 38, 43 Applications 49
Experimental set up 38
Liquid-liquid partition chromatography 35 Liquid, gas and
supercritical fluid Liquid-liquid chromatography 37
Capacity 37 Differential partitioning 37 Distribution
coefficient 37 Partition coefficient K 37 Retention factor, k 37
Reversed phase partition chromatography 37
Liquid-liquid partition chromatography 8, 35, 43, 55
Liquid-solid adsorption chromatography 5, 8, 35 Liquid-solid
chromatography 36 Liquid-solid column chromatography
Durapak 42
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79
Planar Chromatography
Liquid-liquid partition chromatography 43 Stationary phases 42
Particle Size 42 Polar Adsorbents 41 Regeneration 42 Stationary
phases 41, 42 Activated alumina 41 Silica gel 41 Surface Area
41
Longitudinal diffusion 20, 22 Longitudinal diffusion term (B/u)
22 M. Tswett 5 Martin and Synge 5 Mass transfer between phases 20
Mass transfer term (CS) 24 Migration rates 15
Distribution constant 15 Linear chromatography 15 Non- linear
chromatography 16
Mobile phase 6, 43 Retention factor 17 Retention time 16
Selectivity factor 18 Shapes of peaks 18 Mobile phase mass transfer
coefficient (CM) 24 Nature of the mobile phase 7 Ninhydrin 67
Non-equilibrium in mass transfer term (Cu) 23
Henrys law 23 Non-linear chromatography 16 Number of plates (N)
18 Optimum carrier gas velocity (Uopt.) 24 Paper chromatography
(PC) 5, 7, 53, 55 Applications 61 Detection methods 60 Development
of chromatogram 58 Principle 55 Solvent systems 58 Stationery phase
55
Stationery support 57 Particle size 42 Partition 6, 10 Partition
coefficient, K 37 PC and TLC
Comparison 72 Physical measurement of coloured spots 71
Quantitative aspects 71 Radioactivity measurements 72 Removal of
spots 72 Separation of nucleotides 72 Spot area measurements 72
Pesticides separation of 62 Physical adsorption 10 Planar
chromatography 53 Plate concept 68 Plate count 18 Plate height (H)
18 Platinum-iridium capillary 69 Polar adsorbents 41
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80
Chromatographic Methods-I
Polyacrylamide 9 Rate theory 20 Regeneration of adsorbent 42
Resolution 26 Retardation factor (Rf) 54, 55 Retention factor 17
Retention time 16
Dead time 16 Reverse phase PC 62 Reversed phase partition
chromatography 37 Selectivity factor (separation factor) 18, 27
Separation factor 37 Separation medium 55 Shapes of peaks 18 Silica
gel 41 Size exclusion 6 Size exclusion chromatography 9 Solvent
strength 44 Solvent systems 58
Paper chromatogram 58 Solvents 56, 58, 65, 67, 68
Speciation 62 Stationary phase 6, 42, 55 Stationary phase mass
transfer coefficient (CS) 24 Stationary support 57 Supercritical
fluid chromatography 6, 7, 11, 13
Critical point 11 Critical pressure 11 Critical temperature 11,
20 Supercritical fluid 11
Surface area 41 Theoretical plates 18, 20 Thin layer
chromatography (TLC) 7, 53, 63, 68
Apparatus and requirements 66 Plates 66
Conventional 66 High-performance 66
Developing chambers 66 Applications 70
Gallic acid 70 Trifala 70
Detection methods 67 Chromogenic reagents 67
Mobile phase 65 Eluotropic series 66
Plate concept 68 Stationary Phases 64
Alumina 64 Sephadex 64 Slurry 64
Three-dimensional chromatography 7 Two-dimensional
chromatography 7 van Deemter plot 24 van Deemter equation 20 Zone
broadening 24