Subject: Modern Analytical Techniques (MAT), Subject Code: BCY-252

Dr.C.L.Gehlot, Professor, Department of Chemistry

CHROMATOGRAPHY
Chromatography is relatively new techniques, first invented by M. Tswett a botanist in 1906
for separation of colored substances into individual components. Since then, technique has
undergone tremendous modifications so that now a day’s various types of chromatography
are in use to separate almost any given mixture, whether colored or colorless into its
constituents and to test the purity of theses constituents.
The name chromatography (Greek Chroma- color and Graphy- writing) means color writing
or color based separation.
Essentially, the technique is based on the difference in the rate at which the components of a
mixture move through a porous medium (called stationary phase) under the influence of some
solvent or gas (called mobile phase). Means chromatography consists of two immiscible
phases and thus different rate of movement of constituents of a mixture is the basis of
chromatography.
Phases of Chromatography:
(1) Stationary phase:Stationary or fixed phase may be a solid or liquid supported over
the solid.
(2) Mobile phase: Mobile phase may be a liquid or gas.
Manual Chromatography: Limited up to separation and identification of components of a
mixture. e.g. Paper chromatography, Thin layer chromatography (TLC) and column
chromatography.
Automated Chromatography: Determination of concentration of components of a mixture:
e.g. Gas Chromatography (Gas solid (GSC) and Gas liquid (GLC), Liquid Chromatography
(LC), High performance Liquid Chromatography (HPLC)
Classification of Chromatography on the basis of separation mechanism:
Stationary or fixed phase may be a solid or liquid and moving or mobile phase may be a
liquid or gas. Based on the nature of the fixed and moving phase, different types of
chromatography may be classified as follows:
(1) Adsorption chromatography: It is based on the differences in the adsorption
coefficients. In this the fixed phase is a solid e.g., silical gel, alumina, kisselghur and
cellulose etc. The solutes or components are adsorbed in different parts of the
adsorbent column. The adsorbed components are then eluted by passing suitable
(solvent or mobile phase) through the column. Example: paper chromatography, thin
layer chromatography (TLC), column chromatography etc.
(2) Partition chromatography: It operates by mechanism analogues to counter current
distribution. The fixed phase may be a liquid or gas strongly adsorbed on a solid
which acts as a support. In this case, the solute or sample gets distributed between the
fixed phase and the moving liquid (solvent). The technique is called partition
chromatography.
 Paper chromatography is a special type of partition chromatography in which the
adsorbent column is a paper strip.
(3) Gas chromatography: When the moving phase is a mixture of gases, it is called gas
chromatography or vapour phase chromatography (VPC)

PAPER CHROMATOGRAPHY

(1) Introduction: It was invented by A.J.P. martin and his co-worker Synge
(2) Principle: This technique is a type of partition chromatographyin which the
substances are distributed between two liquids i.e. one is the stationary phase
(usually) water which is held in the fibres of the paper and called the stationary
liquid phase (usually water) which is held in the fibres of the paper and called the
stationary phase; the other is the moving liquid or developing solvent and called
the moving or mobile phase. The components of the mixture to be separated
migrate at different rates and appear as spot at different points on the paper.
Paper chromatography was originally used to separate mixture of organic
substances such as dyes and amino acids only. But now this technique has been
perfected to separate cations and anions of inorganic substances as well.

In this technique, a drop of test solution is applied as a small spot on a filer paper
(Whatman 44) and the spot is dried. The filter paper is kept in a close chamber
and the edge of the filter paper is dipped into a solvent called developing agent.
As soon as the filer paper gets the liquid through its capillary axis and when it
reaches the spot of the test solution (a mixture of two or more substances), the
various substances are moved by the solvent at various speeds. When, the solvent
has moved theses cations to a suitable height (15-20 cm) the paper is dried and the
various spots are visualized by suitable reagent called visualizing reagents. The
movement of substances relative to the solvent is expressed in terms of Rf values.
i.e., migration parameters. Each substance has unique Rf value enable us to
differentiate among the substances or constituents of the mixture.
Migration parameters:
The position of migrated spots on the chromatograms are indicated by different
terms such as Rf, Rx, Rm and Rc. These parameters are also qualitative and
quantitative parameters, characteristics of ac substance.

Retention factor (Rf) = Distance travelled by the solute from the origin line
Distance travelled by the solvent from the origin line
 R is a function of the partition coefficient. It is a constant for a given substance,
provided the conditions of chromatographic system are kept constant with respect
to temperature, type of paper, duration and direction of development, nature and
the shape and the size of the wick used (i.e., Radial chromatography), the amount
of liquid in the reservoir humidity etc.
The Rf defines the movement of the substance relative to the solvent front in a
given chromatographic system as shown in figure 1

Figure 1
The Rf value of a substance depends upon a number of factors, which are (i) The
solvent used (ii) The medium used for separation i.e., the quality of paper in case
of paper chromatography (iii) The nature of the mixture to be separated (iv) The
temperature (v) The size of the vessel in which the operation is carried out
Keeping the above factors constant, it is possible to compare the R f values of
different substances.
In some cases, the solvent front runs off the end of filter paper, the movement of a
substance in such cases is expressed as Rx but not Rf

Rx = Distance travelled by the substance from the origin line

Distance travelled by the standard substance from the origin line
RM: The Rf values of chemically related compounds are very close. The influence of
individual functional groups was presumed to be added to a rough approximation.
RM is defined as:
RM= log (1/Rf -1)
The term RM is additive and is composed of the partial R M values of the individual
functional groups or other groupings of atoms in the molecules.
Types of Paper Chromatography:
(1) Ascending Chromatography: Whenthedevelopment of the paper is done by
allowing the solvent to travel upward the paper, it is known as ascending
techniques.
In ascending chromatography, the mobile phase(solvent) is placed in a suitable
container at the bottom of the chamber or in the chambers itself. The samples are
applied a few centimeters from the bottom edge of the paper suspended from a
hook. Alternatively,the paper may be rolled into a cylinder, held together by
staples strings or plastic clip.

(2) Descending Chromatography: When the development of the paper is done by

allowing the solvent to travel downward the paper, it is known as descending
techniques. The advantage of descending technique is that the development can be
 continued indefinitely even through the solvent runs off at the other end of the

paper.
Both ascending and descending techniques have been used for separation of organic
and inorganic substances. But the descending technique is preferred, if R f values of
various constituents are almost same.
(3) Ascending-Descending Chromatography: It is the hybrid of the above two
techniques. In this technique, the upper part of ascending chromatography can be
folded over a glass rod allowing the ascending development to change over into
the descending after crossing the glass rod.

(4) Radial Paper Chromatography: Also known as circular paper chromatography.

This makes the use of radial development. In this technique a circular filter paper
is used and various substances to be analyzed are placed at its center. After drying
 the spot, the paper is fixed horizontally on the petri-dish possessing the solvent so
that the tongue or wick of the paper dips into the solvent. Cover the paper by
means of petri-dish cover. The solvent rises through the tongue or the wick. When
solvent front has moved through a sufficient large distance, the components get
separated in the form of concentric circular zones.

(5) Two dimensional chromatography: In this technique, a square or rectangular

paper is used. The sample is applied to one of the corners. The second
development is performed at right angle to the direction of the first run. This type
of techniques can be carried out with identical solvent systems in both the
directions or by two solvent systems.

Experimental procedure of paper Chromatography:

(1) Selection of the proper chromatographic technique: The first job is to select
the mode of paper chromatographic technique i.e., ascending, descending,
ascending-descending, radial or two-dimensional technique.
(2) Choice of filter paper:Filter paper plays an important role in the success of paper
chromatography. Prime factors that govern the choice are (i) Whether, the paper is
being used for quantitative or qualitative analysis (ii) Whether, it is used for
analytical or preparative chromatography. (iii) Whether, the substances used are
hydrophobic or lipophilic or neutral or charged species.
Various types of Whatmann chromatography papers are available. The choice of
particular Whatmann chromatography paper depends upon the type of separation.
Generally, coarser and faster papers i.e., Whatmann 31, are used when substances
to be separated have sufficiently wide apart Rf values. Slow papers are rarely
used. Slow papers are Whatmann 20, Whatmann 3MM. Filter paper of the size
10x05, 20x10, 15x10and 25x10 cm are used.
Whatmann filter papers commonly used for chromatographic purposes have a
content of 99% of α-cellulose. The rest is mineral content.
(3) Selection of proper developing solvent or mobile phase:The best possible
developing solvent is generally selected for the separation of substances under
examination. The choice depends upon the simple fact that Rf values should be
different for different constituents present in a mixture. Generally, a solvent or
solvent mixture which gives a Rf of 0.2-0.8for the sample should be selected.
Solvents used in paper chromatography are listed in the Table: 1. The solvents are
listed in order of increasing polarity. An indication of polarity can be obtained
from the dielectric constant. If a pure solvent is not satisfactory, solubility of
suitable polarity may be obtained by trying out mixtures in various proportions of
solvents listed below. The eluting power of a solvent (mobile phase) is determined
by (i) its overall polarity (ii) the polarity of stationary phase (iii) the nature of the
sample components.
List of Solvents or mobile phases in the order of their eluting power known as
Elutropic Series of Solvents. Least polar solvents are used for better separation
and for optimum separation, the mixture of solvents is often used.
n-hexane
Cyclohexane
Carbon Tetrachloride
Benzene
Toluene
Trichloroethylene
Diethyl ether
Chloroform
Ethyl acetate
n-butanol
n-propanol
Acetone
Ethanol
Methanol
Water
(4) Preparation of samples: If sample is solid then it is dissolved in highly volatile
solvent to prepare the solution. If sample is liquid then it may be directly used.
(5) Application of Sample or spotting of sample:For application of the sample, a
horizontal line or base line is drawn using a pencil or marker on filter paper. This
line is known as originline.Cross marks (x) are made on the origin line with a
pencil in such a way that each cross (x) is at least 2 cm away from each other. The
liquid samples are applied on cross marks(x) using a syringe or micro pipette and
spot are then dried to remove the solvent by keeping in the oven for some time.

Fig. Spotting on the chromatographic paper

In This figure A, B, C and D are four cross(x) marks, On the first three marks,
solutions of pure substances are applied, whereas on D point, the mixture of A, B,
C is applied.

(6) Drying the chromatograms: The wet chromatograms after development are
dried in special drying cabinets, which are being heated electrically with
temperature controls.
(7) Application of mobile phase or development of spots:Dry filter paper
(chromatogram) is dipped in to a solvent taken in a chamber selected from the list
of Solvents or mobile phases in the order of their eluting power known as
elutropic series. Least polar solvents are used for better separation and for
optimum separation, the mixture of solvents is often used. After development the
paper removed from development tank or chamber.
(8) Detection or visualization of the spots: Visualization of the spot can be done in
two ways
(i) Either by Chemical methods or
(ii) By Physical methods
Chemical Methods:Chemical treatment can develop the color of colorless
solvents on the paper. The reagents used for visualizing the spot are known as
chromogenic reagent or visualizing reagents.
 Visualizing reagents are applied on one of the two ways, either by the pressure
spraying or by dipping the chromatogram. Reagents in organic solvents are more
suitable for spraying than aqueous solutions. Then, the spots are dried. Ninhydrin
spray are generally used.
Physical Methods: Some colorless spots, when held under a UV lamp
fluorescence and reveal their existence. When, the substance is colored, the spots
can be observed either by reflected or transmitted light. The former is more
selective but the later more sensitive.
(9) Calculation of Rf values:
Retention factor (Rf) = Distance travelled by the solute from the origin line
Distance travelled by the solvent from the origin line
 Classification of Chromatographic Techniques
Chromatographic techniques can be classified on the basis of types of mobile &
stationary phases and the mechanism of separation.

CHROMATOGRAPHY
↓
……………………………………………………………………………….
↓ ↓
Liquid Chromatography Gas Chromatography
↓ ↓
…………………………………………………… ……………...
↓ ↓ ↓ ↓ ↓ ↓
LLC LSC IEC EC GLC GSC
LLC (Liquid-Liquid Chromatography): In this technique separation involves a simple
distribution or partition between the immiscible liquid phases, one stationary and the other
mobile phase.
LSC (Liquid-Solid Chromatography): In this technique, the stationary phase is solid and
the mobile phase is liquid. The retentive ability of the stationary phase is due to the physical
surface forces.
IEC (Ion Exchange Chromatography):Counter ions of the stationary phase are selectively
exchanged with the ionic components of the sample.
EC (Exclusion Chromatography): Solute molecules are separated based on molecular
geometry and size. Here, stationary phase has porous, gel structure which is used in the form
of exclusion packing.
GLC (Gas Liquid Chromatography):the mobile phase is a gas and the stationary phase is a
liquid and
GSC (Gas Solid Chromatography):the mobile phase is a gas and the stationary phase is a
solid.
MECHANISM OF CHROMATOGRAPHIC SEPARATIONS
Solute molecules are continuously moving back and forth between the stationary and mobile
phase during chromatographic separation. During the time, the solute spends in the stationary
phase, they remain virtually stationary but when they are in the mobile phase, they are carried
forward with it. Hence, the proportion of time solute spends in the mobile phase determines
its rate of migration.
The solute is transferred from a mobile to a stationary phase by a process known as sorption.
There are three different sorption mechanisms, which are the basis of chromatographic
techniques.
(i) Surface Adsorption: The rate of movement of the solute through a column or
across a surface is determined by the relative polarities of solute and solid
stationary phase.
(ii) Partition or distribution:The sorption is known as partition, when the liquid is
coated onto the surface of an inert solid support and the mobile phase is also
liquid. The relative solubility of solute on both the phases determines the rate of
movement of the solute. But, if mobile phase is a gas, movement of solute is
determined by its volatility.
(iii) Ion-exchange: A permeable polymeric solid having fixed charged groups and
mobile counter ion acts as a stationary phase. When, the mobile phase carries
solute molecules through the permeable structure of stationary phase, exchange of
ions of the solute with the mobile counter ions takes place.
(iv) Exclusion: Stationary phase is gel, which has large number of small sized pores
into which small solute molecules (upto a certain critical size) can diffuse. Bigger
solute molecules move unhindered through the column or layer as they are
excluded from the gel. Separation occurs due to variations in the extent to which,
the solute molecules can diffuse through an inert and porous stationary phase.

Terminology used in chromatography:

(i) Distribution Ratio (D): It is defined as the ratio of the concentration of
solute in stationary phase to that in the mobile phase.

D= Solute (stationary phase)

Solute (mobile phase)
Distribution ratio determines the rate of movement of a solute. The smaller the value
of D, faster will be the progress of the solute through the system.
(ii) Retention volume (VR): It is defined as the volume of the mobile phase
passing through the column required to move the solute from one end of
the column to the other. may be expressed as:
VR=VM+KVM
Where VR=retention volume
VM= dead or void volume (vol. of mobile phase in the column)
K= retention factor
(a) If K=0 VR=VMi.e solute is eluted without being retarded or retained by the
stationary phase.
(b) Large values of K (or D) results in very large retention volumes and hence,
long retention times.
(iii) Retention Time (t R):It is the time taken by the solute between the sample
injection point to reach the detector.
t R= Length of the column
Linear flow rate of solute migration (V)
VR=FtR
Where VR = Retention volume
F= Constant rate of flow of mobile phase
 t R can be used as a measure of VR in gas chromatography and high-performance
liquid chromatography, whereby the flow of mobile phase is monitored by a
detector and recorder
(iv) Retention or Retardation factor ( Rf):
(Rf) = Distance travelled by the solute from the origin line
Distance travelled by the solvent from the origin line

THIN LAYER CHROMATOGRAPHY (TLC)

TLC is often named by other names such as drop, strip, spread layer, surface
chromatography and open column chromatography.
Superiority aspects / advantages of TLC over other chromatographic
techniques
TLS not only combines the advantages of paper and column chromatography but
in certain respect it is superior to either of the two.
(1) Simple equipment: TLC requires very simple equipment.
(2) Short Developing time: TLC development time is only about 1 hr. other
require several hours.
(3) Wide choice of stationary phase: TLC may be used for adsorption, partition
including reversed phase or ion exchange chromatography.
(4) Early recovery of separated components: It is possible to remove the
powdery coating of the plates by scraping. It means zones or spots of
separated components may be removed/recovered quantitatively.
(5) Separation effect: Separation effect achieved by TLC are usually superior
than other techniques.
(6) Easy detection /visualization of separated components: Detection of
fluorescence compounds under UVlight is easier than other techniques.
(7) Sensitivity: Extremely sharp spots are obtained in TLC.Small amounts of
sample can be detected and separated. This increases the sensitivity in the
order of 10-100 folds as compared to other techniques.
(8) Variable thickness of thin layer: Thickness of thin layer are changeable.
(9) Chemically inert stationary phase:

Experimental procedure of TLC

(1) Coating materials used in TLC:

Adsorbent Acidic or Activity Separation Components to be

basic mechanism separated
Silica Gel Acidic Active Adsorption, Acidic and neutral
partition substances
Alumina Basic Active Adsorption, Basic and neutral
partition substances
Kieselguhr Neutral In active Partition Strongly hydrophilic
substances
Cellulose Neutral None Partition Water soluble
power compounds
To adhere tightly adsorbents with glass plate some binders such as gypsum
(calcium sulphate, starch, hydrated silicon oxide etc. are used. Silica gel ‘G’
means it contains binder
(2) Preparation of thin layer on glass plates: Slurry of the substances
prepared by dissolving the substances in highly polar solvents. The
various methods of preparing thin layers are as follows:
(i) Pouring (ii) dipping (iii) spreading (iv) spraying (v) precoated plates
(3) Activation of Adsorbent: activation of adsorbent is required to remove
the liquid(solvent) associated with the thin layer. Activation can be done
by drying the thin layer for 3o mins. In air and then in an oven at 111°C
for another 30 mins. This drying makes the adsorbent layer active. In order
to obtain very active layers silica gel and alumina plates can be heated to
150°C for about 4 hours.
(4) Application of sample or spotting: sample are applied using micropipette
or microsyringe keeping a distance of 2 cm between two spots. Solvents
used for sample solutions should be volatile and as non-polar as possible.
(5) Development of TLC plate or application of mobile phase:

Ascending mode of development is most widely used. The TLC plate is

placed in development tank at an angle of 45° and the bottom of the
chamber is covered upto nearly 1 mm by the solvent.
(6) Solvent systems: stahl’s triangle can be used to know/find a suitable
solvent to be used for the development of TLC pate.
Eluotropic series of one and two component solvent systems with increasing
eluting power or polarity:
High boiling paraffins
Cyclohexane
Benzene
Cyclohexane, ethyl acetate (95:5)
Benzene, ethyl acetate (95:5)
Chloroform
Cyclohexane, ethyl acetate (85:15)
Benzene, ethyl acetate (85:15)
Chloroform, acetone (9:1)
Benzene, methanol (95:5)
Cyclohexane, ethyl acetate (1:1)
Benzene, ethanol (9:1)
Chloroform, acetone (85:15)
Benzene, ethyl acetate (1:1)
Cyclohexane, ethyl acetate (1:4)
Diethyl ether
Ethyl acetate
Ethyl acetate, methanol (99:1)
Benzene, acetone (1:1)
Acetone
Methanol
Dimethyl formamide
Dimethyl sulphoxide
Water
(7) Detection/visualization of components: Colorless components can be
detected either under UV lamp or by treating the plate with a visualizing
reagent (e.g.ninhydrine). Corrosive reagents such as chromic acid and
sulphuric acid can also be used to detect the components of mixture. While
these cannot be used in paper chromatography.
(8) Evaluation of chromatogram: After detecting the separated solutes on
the TLC plate and marking their positions, their evaluation may be either
qualitative or quantitative.Quantitative evaluation can be done by Rf
determination. However, Rf values for TLC show inferior reproducibility.
Quantitative evaluation may be done using direct methods or indirect
methods. Direct methods involve direct spectrophotometry, IR,
reflectance spectroscopy, measurement of spot area and a relation between
spot area and the amount of the substance present can be used. While,
indirect methods involve Gravimetric method, UV
spectrophotometry,colorimetry and fluorimetry, polarography, radiometry,
flame photometry, GC, LC, HPLC etc.
Applications of TLC:
(1) As a check on process: separation, purification, distillation etc.
(2) In organic chemistry: isolation and separation (a)for checking the
purity of samples (b) as a purification process(c) examination of
reactions(d) identification of organic compounds has been successfully
used for isolation and separation of the following organic compounds
such as Acids ,Alcohols, Glycols, Alkaloids, Amines, Aminoacids,
proteins, peptides, Antibiotics such as: Tetracyclines, penicillin’s,
neomycin sulphates etc.