Faculty of Pharmacy
Department of Pharmacognosy
Cletus A. Ukwubile (Ph.D.)
Chromatography
1
Chromatography
Definition
No. of Phases involved Chromatography
Classification of Chromatography
Adsorption Chromatography
Partition Chromatography
Classification
of
Chromatography
Adsorption Partition
Chromatography Chromatography
S+L OR S/G L+L OR L+G
Adsorption
Chromatography
S+L OR S/G
Thin Layer
S+L
Column
Chromatography
Ion Exchange Gel
S+L
S+L Gas Solid Chromatograph
Chromatograp y
hy
S+G
Partition Chromatography
L+L OR L+G
Paper HPLC Gas Liquid
Chromatography
L+L L+L L+G
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:-
1) Preparation of the Paper.
2) Solvent system used
3) Preparation of the sample.
4) Application of the sample.
5) Development of the Chromatograms.
6) Identifying the Spots
The paper used for chromatography
Whatman no.1 is strong, medium fast, pure
cellulose paper that is widely used.
For the separation of polar substances special
ion exchange paper (containing ion
exchanging groups)
For the separation of the component which is
hydrophilic in nature, esters of cellulose can
be used.
Preparation of Paper
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.
• The solid sample is dissolved in organic
solvent having low boiling point. The
percentage of the sample in the solution should
be 0.1—1%. About 10 micro liter of the
sample are transferred to the paper by using
capillary or micro syringe. If the sample is of
biological origin, then proteins, lipids and
inorganic ions present in excess are to
removed for better separation.
Application of the sample.
• The point of the application of the
sample or the origin is marked with
pencil on the paper. The sample
should be applied by micro pipette or
capillary. 10-20 micro liter sample is
to be applied. After application of
sample on the marked spot solvents
associated with sample solution is
evaporated by using hair drier or
current of hot air.
Ascending
Descending
Development of Horizontal
the
Chromatograms 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
Petri dish for
Liquid mobile
mobile phase
phase
Separated
component
in terms of
Sample
concentric
Band
Solvent
Wick deep
in Solvent front
Two dimensional chromatography
Sample
A B A B
X X
AB AC
is is
Solvent immersed in immersed
mobile in mobile
phase phase
C
C
Solvent front
Identifying the Spots
Location of the substances:
The substances separated by paper
chromatography are colorless.
These separated substances are detected by
using visualizing agent.
The paper is dried at temperature 100 Oc.
The spots are detected by means of physical
or chemical method.
Physical Method :-
In this method paper is exposed to
ultraviolet light in the wave length range
240-260 nm.
The compound produces florescent
spots. Compounds which do not produce
florescent spots are exposed to high
pressure mercury vapour lamp.
Physical Method
Sun ra
ys
U.V.Lig
ht
Paper
Chromatogram
Chemical Method
Acid –base
H2S gas Iodine vapors indicators Ninhydrin
Carboxylic
Metal Unsaturated
ions Compounds
acids Amino
Cu+2 acids
Chemical Method
H2S Gas Separated
metal ions
Paper
Chromatogram
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:-
• The Rf value for each spot should be calculated. R f stands for
"ratio of fronts" and is characteristic for any given
compound. Hence, known Rf values can be compared to
those of unknown substances for the identifications.
•
• Distance traveled by solute
• Rf = ---------------------------------------------
• Distance traveled by solvent
• Note: Rf values often depend on the temperature, solvent,
and type of paper used in the experiment; the most
effective way to identify a compound is to spot known
substances next to unknown substances on the same
chromatogram.)
Calculation of Rf Value
Application of Paper Chromatography
• For the separation of organic, inorganic, biochemical and natural products
1
• Metal ions having same chemical properties or belonging to the same
2 group can be detected and separated
• The technique is useful for the separation of metal ligand chelate or
3 complexes, it’s also useful for the separation of isomer.
• The purity of a sample can be tested .In this case pure sample can produce
4. only one spot where as impure sample can produce two or more.
• It is useful in the field of biochemistry.
5
• It is very useful technique in the field of food, drugs and cosmetics.
6
•Thank
you
