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Department of Chemistry
T.Y.B.Sc. Analytical Chemistry
Paper-IV
Sem-V
Chromatography 1
• Introduction to Chromatography, Classification of Chromatographic
• Methods
• 2.2 Planar Chromatography (04 L)
• 2.2.4 Principle of Paper Chromatography(PC)
• 2.2.5 Techniques in paper Chromatography
• AC 31/08/2015
• Item No. 4.23
• A) Stationary phase
• B) Preparation of the Sample
• C) Application of the Sample
• D) Mobile phase Solvent
• System
• E) Development of paper chromatogram:
• Ascending Development, Descending Development ,
• Horizontal Development, Radial Development, Multiple
• Development, Two dimensional Development
• F) Detection and Quantitative analysisPhysical
• methods,
• Chemical methods, Enzymatic and biological methods of
• detection
• 2.2.6Applications of Paper chromatography
• 2.2.7 Comparison of PC and TLC
Chromatography
Definition
No. of Phases involved
Chromatography
Classification of Chromatography
Adsorption Chromatography
Partition Chromatography
Classification
of Chromatography
Paper
Chromatography
HPLC Gas Liquid
L+L L+L L+G
V
Sem.
Paper Chromatography
L+L
• Principle:
In paper chromatography stationary phase is liquid
as well as mobile phase is also liquid. In paper
chromatography solute undergoes partition between
the two liquid phases. The rate of transfer of solute
and its effective separation on paper will depend on
partition coefficient of the solute between the two
phases. The solutes from the original mixture will
have migrated along paper at different rates, forming
a series of separated spots. For identification
purposes spots are characterized by Rf values.
Distance traveled by solute
Solvent Front Rf = ---------------------------------------------
Distance traveled by solvent
S
Solute front (B)
O
8
L Rf (A) = -------=0.5
V 16
E
Solute front (A)
N
t 12
F Rf(B)= ---- = 0.75
L 16
O
W Original
Line
Solvent Front
S
O Solute front
L
V
E
N Solute front
t
F
L
O
W
Original
Line
EXPERIMENTAL PROCEDURE:-
15-20 Cm
Sample
application
3-5 Cm
Solvent system:-
• In paper chromatography the solvents used as stationary phase
and mobile phase should have following characteristics;
The solvents should not react with any component during separation.
The chemical compositions of the solvents should not change with time.
The Rf value for the component should be any where between 0.06 to 0.95.
The distribution ratio of the component should be independent of its
concentration.
The solvents used may be miscible or immiscible but one of the solvent
should be polar that can work as stationary phase.
The paper shows affinity with polar solvent that can work as
stationary phase.
If water is used as stationary phase then no special impregnation is
necessary. If polar solvent other than water is used then it is
necessary to remove the water from the paper.
Ex. Water and Ethanol
Mobile Phase
• Solvent system used: one main organic liquid
saturated with distilled water.
• Polar solvent which is adsorbed on paper is used
• The solvent should be cheap,
• very pure,
• should not volatile by temperature
• Its rate of flow should not affected by
temperature
Preparation of the sample.
Descending
Horizontal
Radial
Multiple
Ascending Paper
Chromatography
Mobile Phase
Solvent Front
S
O Solute front
L
V
E
N Solute front
t
F
L
O
W
Original
Line
Descending Paper Chromatography
Mobile Phase
HORIZONTAL DEVELOPMENT:-
The advantage of the technique is the space required is very small.
The Rf value is getting very sharp.
The tank used for separation is a shallow metal, glass or container.
The chromatogram is developed using paper enclosed between two
aluminum or glass plates.
Radial Paper Chromatography
Solvent
Wick deep
in Solvent front
Two dimensional chromatography
Sample
A B A B
X X
AB AC
is is
Solvent immersed immersed
in mobile in mobile
phase phase
C
C
Solvent front
Identifying the Spots
Paper
Chromatogram
Chemical Method
Carboxylic
Metal Unsaturated
ions Compounds
acids Amino
Cu+2 acids
Chemical Method
Detection of
Iodine unsaturated
Vapours organic
Paper compounds.
Chromatogram
Chemical Method
Acid-Base Detection of
Indicators Carboxylic acids
Paper
Chromatogram
Qualitative Analysis Or Interpreting the Data:-
2 •Metal ions having same chemical properties or belonging to the same group can be detected and separated
3 •The technique is useful for the separation of metal ligand chelate or complexes, it’s also useful for the separation of isomer.
4. •The purity of a sample can be tested .In this case pure sample can produce only one spot where as impure sample can produce two or more.