Paper

Chromatograp
hy

of

Amino
Acids
Experiment
No. 6

Objectives
To separate and detect the amino
acid present in a given mixture of
unknown solution by paper
chromatography
To discuss factors affecting Rf
value
To differentiate paper
chromatography from other
types of chromatography

Chromatography
is usually introduced as a
technique for separating and/or
identifying the components in a
mixture

Chromatography
To write with colors -- literally
translated from its Greek roots
chroma and graphein
Chromatography was first
developed by Mikhail Tswett in 1903
as he produced a colorful
separation of plant pigments
through a column of calcium
carbonate.

Chromatography
Chromatography can also be used in
different sectors such as in:
Forensic Testing - gas chromatography
is often used to investigate criminal
cases. This can take the form of crime
scene testing, arson verification or
blood testing after
death to determine
levels of alcohol,
drugs or poisonous
substances in the
body.

Chromatography
Chromatography can also be used in
different sectors such as in:

Performance Enhancing Drug

Testing - identifies substances in
the bloodstream for tests on
performance enhancing drugs in
athletes

Materials
and
methodolog
y

Materials and Methodology

Sources of possible
error

Results and
discussion

Result
AMINO ACID DISTANCE Rf VALUE
MIGRATE
D
A.Pure

1.Alanine
3.5 cm
0.39
2.tryptopha
4.1 cm
0.46
n
3.5 cm
0.39
3.Alanine
Distance from Origin to Solvent Front:
8.9

Why Ninhydrin?
Amino acids have no colour .
Ninhydrin, a revealing agent, is
sprayed on the resulting
chromatogram.
It is originally yellow that when
reacted with amino acid, it will
turn to deep purple color.

Why don't all of the amino

acids travel up the paper at the
same rate?
Different amino acids will move
different distances up the paper
depending upon their relative
solubilities.
Non-polar amino acids will have a small
Rf value and the polar amino acids will
have greater Rf value.

Answers to
Questions

CHromatography
Definition
It is usually introduced as a
technique for separating and/or
identifying the components in a
mixture.

Use
It is a powerful
method in
industry,
where it is used
on a gauge scale to separate and
purify the intermediates and products
in various syntheses.

Types of
Chromatography

Adsorption Chromatography
- utilizes a mobile liquid or gaseous
phase that is
adsorbed onto
the surface of a
stationary solid
phase. The equilibration between
The mobile and
stationary Phases
accounts for
the separation of
different solutes.

Types of
Chromatography

Partition Chromatography
- based on a film formed on the
surface of a solid support by a
liquid stationary phase. Solute
equilibrates between the
mobile phase and
the stationary
liquid.

Types of chromatography
Ion Exchange Chromatography
- the resin (the stationary solid phase) is
used to covalently attach anions or
cations onto it. Solute ions of the
opposite charge
in the mobile
liquid phase are
attracted to the
resin by
electrostatic
forces

Types of chromatography
Affinity and Chromatography
most selective type employed. Utilizes
the specific interaction between one
kind of solute molecule and a second
molecule that is immobilized on a
stationary phase.

Types of chromatography
Size-Exclusion
Chromatography
- also known as gel filtration, this type of
chromatography lacks an attractive
interaction between the stationary and the
solute. The liquid or gaseous phase passes
through a porous gel
which separates the
molecules according
to its size. This causes
the larger molecules
to pass through the
column at a faster

Retention factor (Rf)

It is defined as the ratio of the
distance moved by the solute and
the distance moved by the solvent
along the paper, where both
distances are measure from the
common origin or application
baseline, that is the point where
the sample is initially spotted on
the paper. It is used to quantify the
movement of the materials along
the plate.

Retention factor (Rf)

Factors affecting Rf value:
Chromatography Medium
Temperature
Solvent concentration and purity

Chromatogram
the pattern formed on an adsorbent
medium by the layers of
components separated by
chromatography.

Solvent front
the wet moving edge of the solvent
that progresses along the surface
where the separation of the mixture
is occurring.

Stationary phase
the solid or liquid phase of a
chromatography system on which
the materials to be separated are
selectively adsorbed.

mobile phase
the liquid or gas that flows through
a chromatography system, moving
the materials to be separated at
different rates over the stationary
phase

happen

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