Paper Chromatography

Identification of Sugars
by Paper Chromatography
Section A4
WHAT IS CHROMATOGRAPHY?
a method of separating and identifying
the components of a complex mixture
differential movement through a twophase system, in which the movement is
affected by a flow of a liquid or a gas
(mobile phase) which percolates through
an adsorbent (stationary phase) or a
second liquid phase
WHAT IS CHROMATOGRAPHY?
Components of a mixture may be interacting
with the stationary phase based on charge,
relative solubility or adsorption
Basic Chromatographic Principles
All chromatographic systems contain:
A stationary phase
A mobile phase
Sample molecules
Type of Chromatography
Applications in the Real World
Why and What is it
Liquid Chromatography
test water samples to look for pollution,
Used to analyze metal ions and

organic compounds in
solutions. It uses liquids which
may incorporate hydrophilic,
insoluble molecules.
Gas Chromatography
detect bombs in airports, identify and

quantify such drugs as alcohol, used in
forensics to compare fibers found on a
victim
Used to analyze volatile gases.

Helium is used to move the
gaseous mixture through a
column of absorbent material.
Thin-Layer
Chromatography
Paper Chromatography
Uses an absorbent material on

detecting pesticide or insecticide residues in
flat glass plates. This is a simple
food, also used in forensics to analyze the
and rapid method to check the
dye composition of fibers
purity of the organic compound.
separating amino acids and anions, RNA

fingerprinting, separating and testing
histamines, antibiotics
The most common type of

chromatography. The paper is
the stationary phase. This uses
capillary action to pull the
solutes up through the paper
and separate the solutes.
PAPER CHROMATOGRAPHY
Paper Partition Chromatography
to emphasize the partitioning of solutes between
a mobile phase and water adhering to the filter
paper during chromatography
a method for testing the purity of compounds
and identifying substances
a useful technique because it is relatively quick
and requires small quantities of material
Carried out by:
placing samples to be
analyzed on sheets of
filter paper
allowing an organic
solvent to develop
the chromatogram
Mobile phase:
ORGANIC SOLVENT
Stationary phase:
THE WATER WHICH IS HELD TO THE CELLULOSE
FIBERS OF THE PAPER BY HYDROGEN BONDING
PROCEDURE
Prepare the developing chamber
Organic solvent: Butanol:Ethanol:Water (52:32:10)
Prepare the Stationary Phase

Spotting the Samples
Developing the Chromatogram
Spray the Chromatogram
Heat the Chromatogram
Identify Spots
PROCEDURE

Identify Spots
PREPARATION FOR THE STATIONARY PHASE

1. Draw a line, 2cm
away from the edge
of the filter paper
using a pencil.
2. Divided the baseline
into 6 spots.
3. Marked each spot and
labelled according to
the name of the
standard sugars.
PROCEDURE


Identify Spots
SPOTTING THE SAMPLES

1. Using a capillary
pipette, 4l each of
the given sugar
solutions were
applied.
2. Application is done 6
times for each spot
(drying spot in each
applications).
PROCEDURE

Identify Spots
DEVELOPING THE CHROMATOGRAM

The paper was
placed in the
chamber
containing the
solvent.
The spots should
not touch the
solvent.
PROCEDURE

Aniline acid oxylate spray

Identify Spots
PROCEDURE


Heat in a hot plate until spots become visible.
Identify Spots
PROCEDURE

Identify Spots
CHROMATOGRAM (1A4-1)
CHROMATOGRAM (1A4-2)
RESULTS
1A4-2
1A4-1
Sugar
Distance traveled
by sugar
Sugar
Distance traveled
by sugar
Galactose
2.6 cm
Galactose
3.6 cm
Glucose
3.3 cm
Glucose
3.9 cm
Fructose
5.1 cm
Fructose
5.5 cm
Maltose
1.0 cm
Maltose
1.4 cm
Sucrose
1.7 cm
Sucrose
2.2 cm
Unknown
1.5 cm
Unknown
2.1cm
Distance traveled by
farthest sugar = 5.1 cm
Distance traveled by
farthest sugar = 5.5 cm
COMPUTING THE Rf VALUE

Rf
distance traveled by unknown

distance traveled by solvent front
i.e. Galactose (1A4-1)

Distance traveled by the sugar = 2.6 cm
Distance traveled by farthest sugar = 5.1 cm
2.6 cm
Rf
5.1 cm
Rf 0.51
1A4-1
RESULTS
1A4-2
Sugar
Rf Value
Sugar
Rf Value
Galactose
0.51
Galactose
0.65
Glucose
0.65
Glucose
0.71
Fructose
1.00
Fructose
1.00
Maltose
0.20
Maltose
0.25
Sucrose
0.33
Sucrose
0.40
Unknown
0.29
Unknown
0.38
DISCUSSION
Mobile phase: Organic solvent system
Butanol: Ethanol: Water (52:32:10) solvent system
Stationary phase: Water molecules embedded in

Whatman filter paper
Color visualization: Reaction of reducing sugars
with aniline acid oxalate and the addition of
heat
Dehydration which produced fulfural from
hydroxymethyl-fulfural
DISCUSSION
Problem: Solvent ran off the paper
distance traveled by unknown
Rf
distance traveled by solvent front
Solution: Change the denominator to the

distance traveled by the farthest sugar
Unknown: Sucrose
SOURCES:
Jack, R. (1995). Basic Biochemical Laboratory
Procedures and Sampling. New York: Oxford
University Press, pages 84-85.
Furfural. Accessed on 07/08/09 at
http://en.wikipedia.org/wiki/Furfural
HMF. Accessed on 07/08/09 at http://
en.wikipedia.org/wiki/Hydroxymethylfurfural

Paper Chromatography

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Paper Chromatography

Uploaded by

Copyright:

Available Formats

Identification of Sugars

Applications in the Real World

Why and What is it

test water samples to look for pollution,

Used to analyze metal ions and

detect bombs in airports, identify and

Used to analyze volatile gases.

Uses an absorbent material on

separating amino acids and anions, RNA

The most common type of

Prepare the Stationary Phase

Prepare the Stationary Phase

Spotting the Samples

PREPARATION FOR THE STATIONARY PHASE

Prepare the Stationary Phase

Developing the Chromatogram

SPOTTING THE SAMPLES

Prepare the Stationary Phase

DEVELOPING THE CHROMATOGRAM

Prepare the Stationary Phase

Heat the Chromatogram

Prepare the Stationary Phase

Heat the Chromatogram

Prepare the Stationary Phase

COMPUTING THE Rf VALUE

distance traveled by unknown

i.e. Galactose (1A4-1)

Stationary phase: Water molecules embedded in

Solution: Change the denominator to the

You might also like