Paper-chromatography -latest technique of separation

2. • Paper Chromatography (PC) was first introduced
by German scientist Christian Friedrich
Schonbein (1865).
2
What is Chromatography?

3. Principle of Paper Chromatography
The principle involved is partition chromatography
wherein the substances are distributed or partitioned
between liquid phases.
One phase is the water, which is held in the pores of the
filter paper used;
and
other is the mobile phase which moves over the paper.
The compounds in the mixture get separated due to
differences in their affinity towards water (in stationary
phase) and mobile phase solvents

4. Principle of Paper Chromatography
• Capillary Action – the movement of liquid within the spaces
of a porous material due to the forces of adhesion, cohesion,
and surface tension. The liquid is able to move up the filter
paper because its attraction to itself is stronger than the
force of gravity.
• Solubility – the degree to which a material (solute) dissolves
into a solvent. Solutes dissolve into solvents that have similar
properties. (Like dissolves like) This allows different solutes
to be separated by different combinations of solvents.
Separation of components depends on both their solubility in
the mobile phase and their differential affinity to the mobile
phase and the stationary phase.

5. • The chromatogram can be analysed by measuring the distance
travelled by the solvent front, and the distance from the
origin to the centre of each spot.
• This is used to calculate the Rf (relative front) value for each
spot:
• An Rf value is characteristic of a particular solute in a
particular solvent. It can be used to identify components of a
mixture by comparing to tables of known Rf values.
Rf 
distance moved by spot
distance moved by solvent

6. origin
Solvent
front
solvent
Amino acid spots
Purple spots
develop located at
different distances
from the origin line
Mark the solvent
front & allow
paper to dry

7. Factors which will affect Rf value
1. Type of paper
2. Solvent composition
3. Temperature
4. Chamber saturation
Factors which will NOT affect Rf value
1. Solvent volume
2. Size of paper
3. Sample size

8. Partition versus Adsorption
Chromatography
• Chromatographic methods are divided into two types
according to how analytes bind to or interact with
the stationary phase.
•
• Partition
• Adsorption

9. Types of Paper chromatography
Development methods based on direction of flow of mobile
phase
• Ascending Paper Chromatography
• Descending Paper Chromatography
• Circular / Radial Paper Chromatography
Ascending Descending Circular

10. TWO DIMENSIONAL DEVELOPMENT
• Sometimes chromatography with a single solvent is
not enough to separate all the constituents of a
mixture.
• In this case the separation can be improved by two-
dimensional chromatography, where the
chromatography paper is turned through 90° and run
a second time in a second solvent.

11. first run second run
original
spot
original
spot
rotate
through
90°

12. Mixture of
amino acids
on origin line
Paper dried and
rotated clockwise
through 90o
Solvent
front
First solvent
Second solvent
Two-way chromatography provides better separation of substances that behave
in a similar fashion in the first solvent.
A second run in a different solvent resolves two very close spots more clearly
Solvent
front

13. 1) Destructive techniques
• Specific spray reagents, samples destroyed before
detection e.g. – ninhydrin reagent
2) Non-destructive techniques
• uv chamber, iodine chamber
Following detecting tech. can also be
categorized as

14. Methods of detection
• UV chamber
• Spraying of chemical indicators: Ninhydrin,
H2SO4, Rubeanic acid, Ammonium sulphide.
• Iodine vapours
• densitometer
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15. Uses for Chromatography
Real-life examples of uses for
chromatography:
• Pharmaceutical Company – determine amount of
each chemical found in new product
• Hospital – detect blood or alcohol levels in a
patient’s blood stream
• Law Enforcement – to compare a sample found at
a crime scene to samples from suspects
• Environmental Agency – determine the level of
pollutants in the water supply
• Manufacturing Plant – to purify a chemical
needed to make a product