UNIT 6 PLANAR CHROMATOGRAPHY

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
6.5
6.6
6.7
6.8

6.1

Quantitative Aspects of PC and TLC

Comparison of PC and TLC
Summary
Terminal Questions
Answers

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
53

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

54

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.

6.2

Planar
Chromatography

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

Rf =

Distance traveled by the solute

Distance traveled by the solvent

ds
dm

(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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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

...
...
...

56

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

Planar
Chromatography

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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Chromatographic
Methods-I

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;
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

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.

58

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

59

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

60

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?

Solution:

Here,

Rf value for Ag+ = 15.8/18.4 = 0.86

+

Rf value for Pb = 12.1/18.4 = 0.66

Planar
Chromatography

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:

61

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
Mn
Co
Cu

ii)

- 0.09
- 0.21
- 0.43
- 0.61

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-isopropanol-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.

62

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

63

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 Dowex50W-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.

64

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.

Planar
Chromatography

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.
65

Chromatographic
Methods-I

Table 6.1: Eluotropic series of mobile phase

Solvent
n- Pentane
Cyclohexane
Carbon tetrachloride
Toluene
Chloroform
Methylene chloride
Tetrahydrofuran
Acetone
Methyl acetate
Acetonitrile
n- and iso-propanol
Ethanol
Methanol
Water
Ethylene glycol

Solvent strength
o
0.00
0.04
0.18
0.29
0.40
0.42
0.45
0.56
0.60
0.65
0.82
0.88
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 <5 m. As the name implies, high performance plates provide fast and sharp
separations. In terms of plate theory, the conventional TLC will have about 2000
plates in 12 cm with development time of less than 30 minutes. Correspondingly, the
number of plates in high-performance TLC will be about 4000 in 3 cm requiring 10
minutes development time. However, the high performance TLC has much less
sample capacity.
The sample application in TLC is the most critical aspect and particularly so in
quantitative measurements. Usually the sample is dissolved in a suitable solvent with a
concentration of less than 0.1% and applied as a spot of about 1 cm from the edge of
the plate. The spot should be as small as possible and dried in a manner similar to PC,
(Fig. 6.2). In case of dilute solutions, 3 to 5 drops are applied. The spotting may be
done with a capillary tube or by use of a hypodermic syringe. However, mechanical
dispensers with increased precision and accuracy are available commercially. After the
sample solvent has evaporated, the plate is placed in a closed container presaturated
with the developing solvent. Typical developing chambers used for ascending and
horizontal thin layer chromatography are shown in Fig. 6.7.

Fig. 6.7: Typical developing chambers used for (a) ascending and (b) horizontal thin
layer chromatography. In the later case, the sample is placed on both the sides
of the plate and developed towards the centre

66

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.

Planar
Chromatography

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
2,4-dinitophenylhydrazine
H2S water, diphenylcarbazide
Rubeanic acid
Aniline phthalate
Chloroplatinic acids
Bromothymol blue
Antimony trichloride

Amino acids
Ketones and aldehydes
Metals
Metals
Sugars
Alkaloids
Lipids
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
67

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

68

For the mobile phase, tm may be defined as the distance divided by its linear velocity, u
tm

ds /u

(6.2)

Planar
Chromatography

The solvent does not reach the same point until the mobile phase has travelled the
distance dm. Therefore,
tr

dm /u

Substituting the values of tr and tm in equation

(6.3)
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 highperformance liquid chromatography (HPLC), mass spectrometry (MS), Fouriertransform 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.
69

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
70

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.

Planar
Chromatography

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.
71

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-isopropanol (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

72

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.

Planar
Chromatography

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.

73

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
74

Planar
Chromatography

values.
Compound
1
2
3
4
5
6

6.8

Rf (solvent )
0.0
0.8
0.5
0.8
0.2
0.8

Rf (solvent B)
0.5
0.0
0.5
0.8
0.4
0.5

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.

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.

4.

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.
75

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.

6.

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.

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

76

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)

INDEX

Planar
Chromatography

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

77

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

78

Liquid-liquid partition chromatography 43 Stationary phases 42

Particle Size 42
Polar Adsorbents 41
Regeneration 42
Stationary phases 41, 42

Planar
Chromatography

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

79

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

80