EXPERIMENT 1

Identification of Sugars by Paper

Chromatography
BIOCHEMISTRY 1D3
Identification of Sugars by Paper
Chromatography
Introduction & Objectives
Materials
Procedures
Results
Discussion
Conclusion
Identification of Sugars by Paper
Chromatography
Introduction & Objectives
Materials
Procedures
Results
Discussion
Conclusion
Chromatography
used for separating mixtures into their
components in order to
analyze
identify
purify
quantify the mixture or components.
Introduction
Paper Chromatography
A type of chromatography that uses high
quality filter paper to separate substances
that can or are coloured, especially pigments

Among all types of chromatography, it
provides a inexpensive and rapid method that
produces clear and graphic results
Introduction
Uses of Paper Chromatography
Separating coloured pigments (e.g. plants)
Obtaining pure compounds (e.g. proteins)
Qualitative analysis (e.g. antibiotics)
Pathology and forensic science (e.g. DNA and
RNA in fingerprinting or chemicals in blood)
Analyzing complex mixtures (e.g. amino acids
and anions)
Introduction
Objectives
Compute the Rf value of each sugar using the
formula:
Rf = distance traveled by unknown /
distance traveled by solvent front

To identify the chemical reactions involved

To identify the unknown through paper
chromatography
Identification of Sugars by Paper
Chromatography
Introduction & Objectives
Materials
Procedures
Results
Discussion
Conclusion
Whatman filter paper no. 1
Standard solution of galactose,
glucose, fructose, maltose, sucrose
(60mg/ml conc.) and the unknown
sugar
Fructose
Galactose
Glucose
Maltose
Sucrose
Solvent system Butanol: Ethanol:
Water (52:32:10) in the
Chromatography chamber
Aniline Acid Oxalate spray
Identification of Sugars by Paper
Chromatography
Introduction & Objectives
Materials
Procedures
Results
Discussion
Conclusion
Measuring the distance travelled by the known and
unknown sugars as well as the distance travelled by
the solvent front to compute for the Rf values.
Identification of Sugars by Paper
Chromatography
Introduction & Objectives
Materials
Procedures
Results
Discussion
Conclusion
Calculate Rf value of each sugar using the formula:
Rf = distance traveled by unknown
distance traveled by solvent front
SUGAR
DISTANCE
TRAVELLED BY
SUGAR (cm)
DISTANCE
TRAVELLED BY
SOLVENT (cm)
Rf VALUE
A
Fructose
5.5 15
0.37
B
Galactose
2.7 15
0.18
C
Glucose
3.2 15
0.21
D
Unknown
3.2 15
0.21
E
Maltose
0.9 15
0.06
F
Sucrose
2.2 15
0.15
1
st
unknown
SUGAR
DISTANCE
TRAVELLED BY
SUGAR (cm)
DISTANCE
TRAVELLED BY
SOLVENT (cm)
Rf VALUE
A
Fructose
8.3 15
0.55
B
Galactose
4.6 15
0.31
C
Glucose
6.5 15
0.43
D
Unknown
6.5 15
0.43
E
Maltose
1.4 15
0.09
F
Sucrose
3.0 15
0.20
2
nd
unknown
Identification of Sugars by Paper
Chromatography
Introduction & Objectives
Materials
Procedures
Results
Discussion
Conclusion
1. Describe the specific mobile and stationary
phases involved in the chromatographic
system.
Mobile phase:
phase which moves in a certain direction
consists of the sample being separated
flows through the stationary phase
a liquid or a gas
solvent system-Butanol:Ethanol:Water (52:32:10)


Stationary phase:
does not move with the sample
a solid, or a liquid supported on a solid
Water absorbed by the filter paper
Cellulose fibers- hold moisture tightly
Filter paper- weak ion-exchange and absorptive
property

2. What is the principle behind the visualization
of color of the spots on the chromatogram?
Give the chemical reaction involved.
Dehydration reaction

Pentose + acid + heat furfural

Hexose + acid + heat hydroxymethylfurfural

Pentose + acid + heat furfural

Pentose + aniline acid oxalate + heat
furfural + 3 H
2
O

Hexose + acid + heat hydroxymethylfurfural

Hexose + aniline acid oxalate + heat
hydroxymethylfurfural + 3 H
2
O


Insert PICTURE HERE



Oxidation of aniline

3. In case the solvent ran off the paper
how will you compute for the Rf values?

Rf =
distance travelled by the unknown
--------------------------------------------------
distance travelled by the solvent front distance travelled by the farthest sugar
4. Based on the Rf values obtained, which
sugar is most likely your unknown?
Based on the computed values of Rf, the
unknown was found out to be glucose.
Identification of Sugars by Paper
Chromatography
Introduction & Objectives
Materials
Procedures
Results
Discussion
Conclusion
Based on the computed values of Rf, the
unknown was found out to be glucose.