UST- FMS Department of Biochemistry, Molecular Biology and Nutrition

Academic Year 2022-2023

EXPERIMENT NO. 1

PAPER CHROMATOGRAPHY OF SUGARS

Name: LUGTU, Eiron John L.___________________ Facilitator: Dr. Jose Blas & Dr. Tiongson

Results

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

The actual chromatogram

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 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2022-2023
EXPERIMENT NO. 1
B. Compute for the Rf values of the standard sugars and your unknown sample.

Distance traveled
SUGARS COMPUTATION* Rf values
(cm)
Fructose 6.3 6.3 𝑐𝑚 1.0
𝑅𝑓 = = 1.0
6.3 𝑐𝑚
Galactose 4.5 4.5 𝑐𝑚 0.71
𝑅𝑓 = ~0.7
6.3 𝑐𝑚
Glucose 3.7 3.7 𝑐𝑚 0.59
𝑅𝑓 = ~0.59
6.3 𝑐𝑚
Maltose 1.0 1.0 𝑐𝑚 0.1
𝑅𝑓 = ~0.16
6.3 𝑐𝑚
Sucrose 2.0 2.0 𝑐𝑚 0.32
𝑅𝑓 = ~0.32
6.3 𝑐𝑚
Unknown 4.7 4.7 𝑐𝑚 0.75
𝑅𝑓 = ~0.75
6.3 𝑐𝑚
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
*Instead of using the typical formula of computing for Rf values (𝑅𝑓 = 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
),
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
the formula used was 𝑅𝑓 = 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑓𝑎𝑟𝑡ℎ𝑒𝑠𝑡 𝑠𝑜𝑙𝑢𝑡𝑒
. The distance traveled by fructose was

observed to be the farthest; hence, such was used for the Rf values computation. The rationale
behind the change in formula is stated in question 4.

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 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2022-2023
EXPERIMENT NO. 1

Discussion Questions:

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

- The experimental setup utilizes the principle of liquid-liquid partition chromatography. Both
phases (stationary and mobile) were in the liquid state. The partition was done by arresting one
liquid phase on the surface of the Whatman filter paper; the said cellulose paper functioned as
chromatographic support to better appreciate the partition. The liquid bound to the cellulose
paper served as the stationary phase. On the other hand, another liquid solution, the mobile
phase, was allowed to pass through the paper and the immobilized stationary phase.

The partitioning of the solutes was based on the partition coefficient of each solute. Such is
dependent on the affinity to the two phases of the solvent system. If a solute is more soluble in
the mobile phase, such has a higher affinity to the said phase (and a lower affinity to the
stationary phase), resulting in a faster and farther movement from the baseline. In contrast, if
the solute is more soluble in the stationary phase (higher affinity to the stationary phase, lower
affinity to the mobile phase), slower and shorter movement from the baseline is expected. The
two phases of the solvent system involved in the recently conducted paper chromatography are
as follow:
o Mobile Phase – refers to the organic solution, Butanol:Ethanol:Water (52: 32:10)
o Stationary Phase – refers to the aqueous, water.

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

- Carbohydrate by itself is colorless. Hence, a derivative of carbohydrates that demonstrates

distinct color must be produced for better visualization. In the experiment, the chromatogram
was sprayed with aniline acid oxalate uniformly and thoroughly. This subjected the sugars to
an acidic medium, leading to acid-catalyzed dehydration:

Figure 1. Acid-catalyzed dehydration of glucose

After the spraying of the acidic medium, the furfural derivatives (HMF) are then heated in the
hotplate until the spots become visible. The exposure to air leads to the further oxidation of the
derivatives, forming an acid (furoic acid) that imparts the brownish coloration of the sugar
analytes.

Figure 2. Oxidation reaction of 5-hydroxymethylfurfural

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 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2022-2023
EXPERIMENT NO. 1

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

- The affinity of the five sugars to the stationary and mobile phases is dependent on several
factors. The explanation for each sugar in relation to its Haworth projection is shown below.

Figure 3. The affinity of each carbohydrate analyte to the mobile phase. The Haworth projection of
each sugar provides information regarding the affinity patterns.

There were patterns observed based on the Rf values of all sugars. Though not related to
affinity, the molecular weight of the disaccharides (342.3 g/mol) contributed to the low Rf values
of such compared to monosaccharides (180.16 g/mol). Furthermore, a direct relationship was
observed in relation to the number of primary alcohol groups (-OH) present in each sugar.
As the number of 1° alcohol groups increased, the affinity of the sugar to the mobile phase
increased. Such explains why fructose (with two 1° alcohol groups) has a higher Rf value
compared to galactose and glucose (both with one 1° alcohol group); for disaccharides, sucrose
(with three 1° alcohol groups) has a higher Rf value compared to maltose (with two 1° alcohol
groups). The difference in affinity of galactose and glucose, both aldohexose, may be due to the
higher steric hindrance present in the former compared to the latter. The -OH group in the
carbon-4 of the galactose is in the same plane as its 1° alcohol, leading to a decreased affinity
with water (the stationary phase). The -OH group in the same carbon number of glucose is on
a different plane with its 1° alcohol group, allowing increased affinity with water.

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 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2022-2023
EXPERIMENT NO. 1

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

- If solvent run-over occurred, A change in the Rf computation must be done. The distance (from
baseline) traveled by the farthest solute must be used as the reference value (denominator) for
the computation of the Rf values. Hence, the formula would be:

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

- Comparing the obtained Rf values from all the sample sugars, the unknown sugar (Rf = 0.75)
has almost a similar retention characteristic as that of the galactose (Rf = 0.71); hence, the
unknown sugar is most likely a galactose sugar.

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

- Based on the clinical manifestation and the result of the paper chromatography, the patient has
a hereditary disorder known as galactosemia. Galactosemia is a disorder in the galactose
metabolism of the patient due to the autosomal recessive inheritance of the mutated GALT gene.
This mutation can lead to the reduction of galactose-1-phosphate uridylyltransferase (GALT)
enzyme activity which causes the accumulation of erythrocyte galactose-1-phosphate
concentration. A significant accumulation of galactose-1-phosphate can damage the liver – as
manifested by hepatomegaly and jaundice (yellowing of the skin) in the patient.

In addition, the galactose-1-phosphate is converted to other toxic byproducts via alternate

metabolic pathways. The enzyme aldose reductase can reduce galactose-1-phosphate into
galactitol which accumulates in the liver and brain leading to further liver toxicity and mental
retardation (developmental delays and speech issues). Furthermore, galactitol can accumulate
in the lens of the eye of the patient, increasing the pressure of the eye, which can cause swelling
and cataract formation.

To prevent further progression of the disorder, it is strongly advised that the patient undergo
strict dietary galactose restriction, eliminating all sources of galactose in the diet. This can
prevent possible acute toxicity caused by galactose accumulation and also reverse the clinical
manifestations presented by the patient.

References:

1. de Jongh, W. A., Bro, C., Ostergaard, S., Regenberg, B., Olsson, L., & Nielsen, J. (2008).
The roles of galactitol, galactose-1-phosphate, and phosphoglucomutase in galactose-
induced toxicity in Saccharomyces cerevisiae. Biotechnology and bioengineering, 101(2),
317–326. https://doi.org/10.1002/bit.21890
2. Masoodi, K. Z., Lone, S. M., & Rasool, R. S. (2021). To perform paper chromatography.
Advanced Methods in Molecular Biology and Biotechnology, 159–161. doi:10.1016/b978-0-
12-824449-4.00029-3

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