UST- FMS Department of Biochemistry, Molecular Biology and Nutrition

Academic Year 2021-2022

EXPERIMENT NO. 1

IDENTIFICATION OF SUGARS BY PAPER CHROMATOGRAPHY

CASE:
Dr. CHO, an aspiring geneticist, wants to determine the medical condition of a 2-year-old child with
ocular cataracts, hepatic enlargement, yellow skin, and global developmental delay.

He collected a urine sample from the child, which tested positive for the presence of an unknown
sugar. He used the rest of the sample for ascending paper chromatography in order to identify the
sugar being excreted.

INTRODUCTION:
Chromatography covers a variety of techniques that resolve solutes by differential migration
during passage through a porous medium. The components of a solution are separated as a result of
differential affinity for a stationary phase (solid or liquid) or for a mobile phase (gas or liquid). Paper
chromatography is a type of partition chromatography that uses paper as support for the stationary
phase. When a number of standard sugars are separately applied as spots on paper, simultaneously
with an unknown sugar solution, the extent of migration of the unknown expressed as the Rf, can be
compared with those of the standard sugars. From the Rf value, the unknown sugar can be identified.

Materials/Reagents:
Whatman filter paper no.1
Standard solutions of galactose, glucose, fructose, maltose, sucrose (60 mg/ml concentration)
“Unknown” sugar solution
Solvent system: Butanol:Ethanol:Water (52: 32:10)
Aniline acid oxalate spray
Chromatography chamber/jar

Procedure:
1. Measure about 2 cm from the edges of the Whatman filter paper (horizontal and vertical sides),
and mark using a pencil. Draw a line across the paper to connect the marks. Assign one side of
the paper as baseline.

2. Divide the baseline such that 6 spots can be accommodated within the whole length. Allow about
3 cm distance between spots. Mark each spot with pencil and label accordingly with the name of
the standard sugars to be used. Leave one spot for the unknown.

3. Insert the Whatman paper between pages of a thick book, such that the baseline mark is
exposed. Place the book in an inclined position on the table taking care that the edge of the
paper does not touch the table.

4. Using a capillary pipette, apply about 4μl each of the unknown sugar solution and the standard
sugar solutions (roughly 240 μg sample) on corresponding spots on the paper. Application should
be done 6 to 7 times for each spot, drying the spot in between applications. Make sure the spot is
as small as possible not exceeding 3 mm. In diameter. Too big spots will cause the samples to
overlap and influence the movement of solute.

5. While drying the spots, after all sugar solutions have been spotted, introduce the solvent into the
chromatography chamber or jar. Cover the jar and allow the solvent to equilibrate for 5 minutes.

7. When the spots have dried, place the paper in the trough containing the solvent (or alternatively,
roll the paper, spots facing out, and staple the ends before placing in the chromatographic jar).
The edge of the paper where the sugars have been spotted should be placed nearer the trough,
but the spots should not touch the solvent.

8. Allow the solvent to permeate through the paper until it rises to about 1 cm. from the top edge
of the paper. Mark the position of the solvent front (or allow chromatography to proceed
overnight, taking care not to run the spots off the paper).

9. Remove the chromatogram from the jar and dry in air.

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 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2021-2022
EXPERIMENT NO. 1
10. Spray the chromatogram with aniline acid oxalate uniformly and thoroughly (but not dripping wet)
and heat in the hotplate until the spots become visible.

11. Calculate the Rf value of each sugar using the formula:

Result of the Chromatogram is as follows:

FRUCTOSE GALACTOSE GLUCOSE MALTOSE SUCTOSE UNKNOWN

*Please do not resize the paper chromatography (standard size of the paper chromatogram:
width 17.0 cm, height 11.55 cm)

*Please use the ruler provided to measure distance travelled. (you can rotate the ruler, but
please do not resize – standard size of ruler: width 14.25 cm, height 3.03 cm). Suggestion: you
can measure the distance using powerpoint, as the paper chromatogram and the ruler can
overlap.

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 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2021-2022
EXPERIMENT NO. 1

PAPER CHROMATOGRAPHY OF SUGARS

Name: _________________________________ Facilitator: ______________

Results

A. Draw the actual chromatogram showing the different spots. Label all important parts. Tabulate the
sugars and the corresponding Rf values obtained.

B. Compute for the Rf values of the standard sugars and your unknown sample.

SUGARS Distance travelled COMPUTATION Rf values

(cm)

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 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2021-2022
EXPERIMENT NO. 1

Discussion Questions:

1. Differentiate the two phases of the solvent system involved in paper chromatography.

2. Describe the principle behind the visualization of color of the spots on the chromatogram. Illustrate
the chemical reaction involved.

3. Contrast the affinity of the solutes to the two phases of the solvent system by illustrating the
Haworth projection of the sugars.

4. In case the solvent ran off the paper, discuss how you will compute for the Rf values.

5. Based on the Rf values obtained, explain which sugar is likely the unknown.

6. Explain to Dr. CHO which medical condition the child would likely have.

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