Chromatography its types and detail note on paper chromatography.

1. CHROMATOGRAPHY
➢ The first scientist to recognize chromatography as an efficient method of separation was the
Russian botanist Tswett, who used a simple form of liquid-solid chromatography to separate a
number of plant pigments.
➢ The name chromatography comes from the Greek word ‘Chroma’ means ‘colour’ and
‘Graphein’ means writing.
➢ This technique was originally confined to the seperation of coloured substances such as plant
pigments and dyestuffs. But the technique is now well applied to colourless substances also.
Definition: “Chromatography may be regarded as an analytical technique employed for the
purification and separation of organic and inorganic substance through the equilibrium
distribution between 2 phases, one of which is stationary and other is mobile phase.”
➢ The stationary phase can be solid or liquid while the mobile phase is a liquid or gas.
➢ The component which is strongly held by the stationary phase will tend to move slowly in the
mobile phase & vice versa.
➢ The component of small mass moves faster than the component having larger mass. As a
consequence of these differences in migration rates, sample components separates into discrete
bands or zones, that can be analyzed qualitatively or quantitatively.
➢ Area of use:
1. Separation
2. Identification
3. Purification
➢ In all chromatographic separations the sample is dissolved in a mobile phase which may be a
gas, liquid or a supercritical fluid. This mobile phase is then forced though an immiscible
stationary phase, which is fixed in a column or on a solid surface.
CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUE:
Chromatographic methods can be classified based on the physical means of bringing the
stationary and mobile phases into contact -
1. Column Chromatography - the stationary phase is held in a narrow tube through which the
mobile phase is forced either by pressure or by gravity.
Examples include:
• Simple column chromatography
• High pressure liquid chromatography (HPLC)
• Gas chromatography (GC).
Column chromatography can be further differentiated based on the types of stationary and
mobile phases and the kinds of equilibria involved in solute transfer between the phases.
There are two broad categories in this classification scheme:
• Liquid chromatography (for instance simple column or HPLC)
• Gas Chromatography (GC).

2. General
Classification
Special Method Stationary Phase
Type of
equilibrium
1
Gas
Chromatography
(GC)
a.
Gas-liquid chromatography
(GLC)
Liquid adsorbed or
bonded to a solid
surface solid
Partition b/w gas &
liquid
b. Gas-bonded phase
Organic species
bonded to a solid
surface
Partition b/w liquid
& bonded surface
c. Gas-solid chromatography (GSC) Solid Adsorption
2
Liquid
Chromatography
(LC)
a.
Liquid-liquid or Partition
chromatography
Liquid adsorbed or
bonded to a solid
surface solid
Partition b/w
immiscible liquid
b.
Liquid-solid or adsorption
chromatography
Solid Adsorption
c. Ion exchange chromatography Ion-exchange resin Ion exchange
d. Size exclusion
Liquid in
interstices of a
polymeric solid
Partition/sieving
e. Affinity chromatography
Group specific
liquid bonded to a
solid surface
Partition b/w
surface liquid &
mobile liquid
2. Planar Chromatography - the stationary phase is supported on a flat plate or in the fibres of a
paper. Here the mobile phase moves through the stationary phase by capillary action or by
gravity.
Examples include:
• Paper chromatography
• Thin layer chromatography (TLC).
Principle of Chromatography:

3. PAPER CHROMATOGRAHY
➢ Was developed by Consden et al (1944) as a technique for the separation of amino acids.
Advantages of Paper chromatography:
1) These are simple, rapid & inexpensive
2) Provide excellent resolving power.
Limitation of Paper chromatography:
1) Longer development times are required.
2) Zones are not always sharply defined.
3) Accuracy in quantitative analysis is only fair.
4) Development conditions are sometimes difficult to reproduce.
Definition: “Paper chromatography may be defined as the technique in which the analysis of an
unknown substance is carried out mainly by the flow of solvent on specially designed filter
paper. The separation takes place as a result of differences in partition-coefficient.
PRINCIPLE:
• This technique is a type of Partition /Adsorption chromatography in which the substance are
distributed b/w 2 liquid, i.e. one is stationary liquid (usually water) which is held in the fibres of the
paper & called the stationary phase, the other is the moving liquid or developing solvent & called
the mobile phase.
• The component of the mixture to be seperated migrate at different rates and appear as spots at
different points on the paper.
METHOD:
A drop of test solution is applied as a small spot on a filter paper
Dried the spot
Paper is kept in a closed chamber & the edge of the filter paper is dipped into a solvent called
developing solvent.
filter paper gets the liquid through its capillary action
when solvent reaches the spot of test solution
various substances are moved by solvent system at various speed
when the solvent has moved these cations to a suitable height (15-18cm)
dry the paper
various spots are visualized by suitable visualising agent.

4. RF : Defines the movement of substances relative to the solvent is expressed in terms of RF value i.e.
migration parameter.
➢ Also called as Retardation factor or Relative front value.
➢ The R is related to the migration of the solute front relative to the solvent front as:
RF = Distance travelled by the solute from the origin line
Distance travelled by the solvent from the origin line
Its value should be b/w 0.3 – 0.8.
Factors affecting RF value:
1) The solvent employed.
2) Quality of paper used.
3) Nature of mixture to be separated.
4) Temperature.
5) Size of vessel in which the operation is carried out.
TYPES OF PAPER CHROMATOGRAPHY:
Sr
no.
Basis of
classification
Types Description
I.
On the basis of
Principle of
chromatography
Paper partition
chromatography
Paper is used as an inert support with one solvent as
mobile phase & other solvent as stationary or immobile
phase.
Paper adsorption
chromatography
A modified paper (first impregnated with an adsorbent
like silica (or alumina) is used as an adsorbent) & the
single solvent is allowed to flow over the unknown
components.
II.
On the basis of
development of
paper
Descending
chromatography
Development of paper is done by allowing the solvent
to travel down the paper is known as descending
chromatography.
Advantage: development can be continued indefinitely
even though the solvents runs off at the other end of
paper.
Ascending Development of paper is done by allowing the solvent

5. chromatography to travel up the paper is known as ascending
chromatography.
Advantage: ascending technique is preferred if the RF
values of various constituents are almost same.
Ascending-
Descending
chromatography
It is a hybrid of ascending & descending
chromatography. In this technique the upper part of the
ascending chromatography can be folded over a glass
rod allowing the ascending development to change over
into the descending after crossing the glass rod.
Radial paper
chromatography
Also known as circular paper chromatography.
In this technique sample is placed at the centre of
circular filter paper having a wick or tongue. After
drying the sample spot, paper is horizontally fixed on
petri-dish having solvent. The solvent ascends or
descends through the tongue & flows radially through
the paper. In this technique the components get
separated in the form of concentric circular zones.
Fig: Descending Chromatography
Fig: Ascending Chromatography

6. Fig: Ascending-Descending Chromatography
Fig: Radial Paper Chromatography
Methods /Techniques of Paper chromatography:
The method of chromatography may be:
1. One dimensional chromatography
2. Two dimensional chromatography
1. One dimensional chromatography: in this method development of paper is carried out along the
one axis.
2. Two dimensional chromatography: in this method 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 first run.
The solvent may be identical in both the directions or may be 2 different solvent systems.
Method :
Spot the sample at lower corner of a rectangular sheet of filter paper and dried
Paper is then kept with its edge in solvent and developed by either ascending or descending technique
When solvent reached the opposite edge of paper
Removed the paper and dry
Solvent system is now changed to second liquid
Filter paper is rotated 90˚ so that the edge having series of spot is now at bottom
Then chromatogram is run as before
Now chromatogram is having spots of solute scattered all over the paper.

7. Advantage:
• Suitable for those substances which cannot be separated by one dimentional paper
chromatography because of very close and nearly same Rf value.
Experimental details of Paper chromatography:
1. Choice of Proper Chromatographic Technique: the first job is to select the mode of paper
chromatographic technique, i.e. ascending, descending, ascending-descending, radial or two-
dimentional technique. The choice of technique depends on the nature of the substance to be
separated.
2. Choice of filter paper: A wide variety of Whatmann chromatography paper are available
commercially in different sizes, shapes, porosities, thickness & chemical treatment (acid base
washed).
Whatmann filter paper (made up of α- cellulose = 98.99%, β- cellulose = 0.3-1.0%, pentose =
0.4- 0.8%, ether soluble matter = 0.015—0.02% & ash= 0.07- 0.01%) has extensively been
used in paper chromatography.
The prime factors that governthe cchoice are as follow:
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 substance used are hydrophilic or lipophilic, neutral or chargrd species.

8. Types of Whatmann Paper
_____________________________________________________
Modification of Paper: Some times paper is modified to achieve the desired properties like:
a. Impregnated with diatomaceous earth, alumina, silica gel and ion exchange resins to
get the different techniques of separation;
b. Increase of carboxyl content by partial oxidation to increase the exchange capacity
during separation of polar substances;
c. Acetylated or treated with silicone or impregnated with inert nonpolar type organic
polymers which gives hydrophobic property to paper which helps in retaining of
hydrophobic type solvent rather hydrophilic type (a basis of reverse phase
chromatography).
3. Selection of Mobile phase (developing solvent): the choice of this depends on the simple fact
that the Rf value should be different for different constituents present in a mixture. The
following are gensral criteria for a good solvent system:
i. The Rf value of the sample should lie between 0.05-0.85 in the system.
ii. The difference between the Rf values of ant two component must be 0.05.
iii. The solvent should not undergo chemical reaction with any of the components of the
sample mixture.
iv. The solvent should not interfere with the detection of the spots on the developed
chromatogram.
v. The composition of the solvent system should not alter with time.
vi. The distribution ratios of the components in the solvent system should be independent
of concentration.
Pure solvents, buffer solutions or mixture of solvents are used. Some of the examples are as
follows:
a) For hydrophilic substances
Isopropanol : Ammonia : Water (9 : 1 : 2)
n-Butanol : Acetic acid : Water (4 : 1 : 5)
Methanol: Water (3:1)
t-Butanol : Water : Formic acid (40 : 20 : 5)
b) For intermediate hydrophilic substances
Formamide : Chloroform
Coarser & Faster Paper Slow Paper Heavy Paper
Used when substances to be
separated have sufficiently
wide- apart Rf values.
e.g. Whatman 31 ET
Used for better resolution
of substances with closer Rf
values.
e.g. Whatman 20
Used for Preparative
purposes.
e.g. Whatman 3MM

9. c) For hydrophobic substances
DMF : Cyclohexane
4. Preparation of samples: there is no any standard procedure for preparation of samples. Ideally
sample volume of 10-20 µ having as many µg of the substance is the ideal quantity to be
spotted.
5. Spotting procedure: For ascending technique,
i. a strip of Whatmann filter paper of suitable size 25 cm is generally used
ii. Mark a line (origin line) on the bottom edge of paper approx. 2 cm from the rim
iii. On the origine line cross marks (x) are made with a pencil in such a way that each x mark
is atleast 2 cm away from each other
iv. Put a spot of sample on x marks of origin line with the help of graduated micropipette
v. Spots are dried
vi. Select an air tight development chamber.
vii. Add mobile phase to the chamber so that it is about 2 cm deep.
viii. Seal the chamber and wait for saturation of atmosphere of development chamber.
ix. Suspend the piece of spotted paper using thread in the air for 10 min.
x. Finally immerse the paper’s bottom edge into the developing chamber.
xi. Remove paper when solvent has travelled 3/4th length of paper.
6. Drying the Chromatogram: the wet chromatogram after development are then dried.
7. Visualisation: the spots can be visualised by 2 ways:
a. By using physical methods (e.g. UV chamber) or
b. Chemical detection
a. By using physical methods: some colourless spots can be observed when they are held
under a UV lamp. Coloured spots can be either observed either by reflected or
transmitted light.
b. Chemical detection: The chemicals used for visualization is known as chromogenic
reagents or visualizing reagents. Some of the common visualizing reagents are:

10. Iodine chamber method: Brown or amber spots are observed when the paper is kept in
a jar containing few iodine crystals.
Some of the chemicals can be used for spraying for identification of specific
compounds:
Ninhydrin reagent: for amino acids,
Dragendorff reagent: for alkaloids,
2,4-dinitrophenyl hydrazine: for aldehydes, ketones, etc.
8. Calculation: The Rf value for every spot should be calculated. It is specific for any given
compound for the same set of stationary and mobile phase.
Rf value = Distance from the origin to the centre of spot
Distance of the solvent front from the origin line
Quantitative Analysis:
1. Isolation of Separated components: cutout the appropriate part of the filter paper having spot and
soak it in the minimum quantity of solvent. The separation of substance from the spot is known as
eluation.
2. Determination: the analysis of the resultant eluate can be performed by adopting one or more of
the following technique:
a. Gravimetric estimation
b. UV Spectrophotometry
c. Colorimetry
d. Polarography
e. Coulometry
f. Radioactivity
g. Flame photometry
These techniques are selected on the basis of following information:
• The nature of the substance to be assayed.
• The scientific equipment available and its sensitivity.
• The time available.
• The alternative method available, if any, and their relative accuracies.
Application:
1. for qualitative and quantitative analysis.
2. Determination of indols in whole urine.
3. Study of barbiturates, antibiotics, carabamyl phosphates, hormones & amino acids among
others.
4. Study of inorganic metal, salts and complex ions.
5. For analysis of mixture of sugars.