Characterization of Carbohydrates Using Thin-layer Chromatography and

Nelsons Method
Silva, J., Subijano, M., Sunglao, A., Supan, E., Tan, C., Tayag, P.
Group # 7, 2G Medical Technology, Faculty of Pharmacy, UST


Abstract
Carbohydrates are any of the group of organic compounds consisting of carbon, hydrogen and
oxygen, usually in the ratio of 1:2:1. In this experiment, thin-layer chromatography was used in partial
characterization and tentative identification of the unknown by comparing its Rf value to that of the
standard using a particular solvent system and Nelsons method was used to measure the amount of
carbohydrates present in a given sample which was based on the capacity of the free reducing groups of
sugars in a carbohydrate sample to reduce Cu
2+
in an alkaline solution. Results showed that dextrin had
the least distance travelled, which means it had the least Rf value, followed by maltose and glucose. All
three carbohydrates were contained in the acid hydrolysate. The data gathered for the Nelsons test was
plotted into a graph and showed the linear trend of the glucose standard curve having the best fit line.


Introduction
The objectives of the experiment were:
(1) to perform thin-layer chromatography on the
carbohydrate hydrolysate; (2) to determine the
carbohydrates present on the carbohydrate
hydrolysate; (3) to determine the amount of
reducing sugars using Nelsons test and explain
the principle involved; and (4) to generate a
curve that represents the data gathered.
When the word carbohydrate was
coined, it originally referred to compounds of
the general formula Cn(H
2
O)n. However, only
the simple sugars, or monosaccharides, fit this
formula exactly. The other types of
carbohydrates, oligosaccharides and
polysaccharides, are based on the
monosaccharide units and have slightly different
general formulas. Oligosaccharides are formed
when a few (Greek oligos) monosaccharides are
linked; polysaccharides are formed when many
(Greek polys) monosaccharides are bonded
together. The reaction that adds monosaccharide
units to a growing carbohydrate molecule
involves the loss of one H
2
O for each new linked
formed, accounting for the difference in the
general formula.
[1]
Many commonly encountered
carbohydrates are polysaccharides, including
glycogen, which is found in animals, and starch
and glucose, which occur in plants.
Carbohydrates play a number of important roles
in biochemistry. First, they are major energy
sources. Second, oligosaccharides play a key
role in processes that take place on the surfaces
of cells, particularly in cell-cell interactions and
immune recognition. In addition,
polysaccharides are essential structural
components of several classes of organisms.
Cellulose is a major component of grass and
trees, and other polysaccharides are major
components of bacterial cell walls.
[1]
The building blocks of all carbohydrates
are the simple sugars called monosaccharides. A
monosaccharide can be a polyhydroxy aldehyde
(aldose) or a polyhydroxy ketone (ketose). The
simplest monosaccharides contain three carbon
atoms and are called trioses. Glyceraldehyde is
the aldose with three carbons (an aldotriose),
and dihydroxyacetone is the ketose with three
carbon atoms (an ketotriose).
[1]
Aldoses with four, five, six and seven
carbon atoms are called aldotetroses,
aldopentoses, aldohexoses, and aldoheptoses,
respectively. The corresponding ketoses are
ketotetroses, ketopentoses, ketohexoses, and
ketoheptoses. Six-carbon sugars are the most
abundant in nature, but two five-carbon sugars,
ribose and deoxyribose, occur in the structures
of RNA and DNA, respectively. Four-carbon
and seven-carbon sugars play roles in
photosynthesis and other metabolic pathways.
[1]

Figure 1. The Structures of the Simplest
Carbohydrates, the Trioses
Thin-layer chromatography (TLC) is a
simple, quick, and inexpensive procedure that
gives the chemist a quick answer as to how
many components are in a mixture. TLC is also
used to support the identity of a compound in a
mixture when the R
f
of a compound is compared
with the R
f
of a known compound (preferably
both run on the same TLC plate).
[2]
A TLC plate is a sheet of glass, metal, or
plastic which is coated with a thin layer of a
solid adsorbent (usually silica or alumina). A
small amount of the mixture to be analyzed is
spotted near the bottom of this plate. The TLC
plate is then placed in a shallow pool of a
solvent in a developing chamber so that only the
very bottom of the plate is in the liquid. This
liquid, or the eluent, is the mobile phase, and it
slowly rises up the TLC plate by capillary
action.
[2]
As the solvent moves past the spot that
was applied, an equilibrium is established for
each component of the mixture between the
molecules of that component which are adsorbed
on the solid and the molecules which are in
solution. In principle, the components will differ
in solubility and in the strength of their
adsorption to the adsorbent and some
components will be carried farther up the plate
than others. When the solvent has reached the
top of the plate, the plate is removed from the
developing chamber, dried, and the separated
components of the mixture are visualized. If the
compounds are colored, visualization is
straightforward. Usually the compounds are not
colored, so a UV lamp is used to visualize the
plates. (The plate itself contains a fluorescent
dye which glows everywhere except where an
organic compound is on the plate.)
[2]


Figure 2. Thin-layer Chromatography Set-up

Sugars with reducing property (arising
out of the presence of a potential aldehyde or
keto group) are called reducing sugars. Some of
the reducing sugars are glucose, galactose,
lactose and maltose. The Nelson-Somogyi
method is one of the classical and widely used
methods for the quantitative determination of
reducing sugars.
[3]
Materials and Methods
A. Materials
In this experiment, the materials needed
are acid hydrolysate, n-butyl alcohol:acetic
acid:ether:water (9:6:3:1), standards (1%
concentration): dextrin, maltose and glucose,
p-anisaldehyde visualizing agent: 0.5 mL
anisaldehyde, 9.0 mL 95% ethanol, 0.5 mL
H
2
SO
4
, and 0.1 mL acetic acid, oven, watch
glass, capillary tubes, 5 x 10 cm TLC plate,
developing chamber, Nelsons reagent A,
Nelsons reagent B, Arsenomolybdate
reagent, glucose standard (100 mg in 1000
ml distilled water), distilled water,
spectrophotometer, cuvettes, test tubes,
pipettes, and beaker.
B. Methods

1. Thin-layer Chromatography
In the developing chamber, 40 mL of
the solvent system was placed. The chamber
was covered with an inverted watch glass
and equilibrated for 10 minutes. Pencil lines
were lightly drawn across both ends of the
TLC plate, about 1 cm each from the
bottom. Four equidistant points were marked
on one of the lines for the standards dextrin,
maltose, glucose and sample acid
hydrolysate. The standards were applied five
times and the hydrolysate ten times using
capillary tubes and dried after every
application. The TLC plate was then placed
inside the developing chamber and it was
ensured that the solvent was below the line
of origin. The chamber was covered and
developed until the solvent reached the line
near the top of the TLC plate. After
development, the chromatoplate was air-
dried and sprayed with a p-anisaldehyde
visualizing agent. The chromatoplate was
then placed inside the oven and heated at
100-150C for 10 minutes. The sugars
became evident by the appearance of dark
colored spots, which were then encircled
lightly with a pencil. The Rf values of each
spot on the chromatoplate were computed.
The obtained Rf values of the standards
were compared with the products of acid
hydrolysis and the components of the acid
hydrolysis were then identified.
2. Quantitative Analysis Using Nelsons
Method
To prepare the Nelsons reagent, 12.5
mL of Nelsons A was mixed with 0.5 mL
of Nelsons B. Eight test tubes were labelled
and measured amounts of standard glucose
solution were transferred into the test tubes
according to the following protocol (Table
1). 1.0 mL of Nelsons reagent was then
added to each test tube and shaken well. The
tubes were simultaneously heated in a
boiling water bath for 20 minutes. After 20
minutes, the tubes were removed and cooled
in a beaker of water. 1.0 mL of
arsenomolybdate reagent was added to each
of the test tubes and shaken occasionally for
5 minutes or until the Cu
2
O precipitate
dissolved. The solutions were then
transferred into cuvettes and the absorbance
of the standards and the unknown were read
against a reagent blank at 480 nm using a
spectrophotometer. The glucose standard
curve was constructed by plotting
absorbance readings against concentrations
of standard solutions and the concentration
of the unknown was determined in mg/tube
and mg/ml.




Table 1. Preparation of Standard Solution
Tube Number Glucose Standard
(mL)
Distilled Water (mL) Unknown Sample
(mL)
1 0 1.0
2 0.1 0.9
3 0.2 0.8
4 0.4 0.6
5 0.6 0.4
6 0.8 0.2
7 1.0 0
8 0.6 0.4

Results and Discussion
1. Thin-layer Chromatography
Thin-layer chromatography is based on
the adsorption phenomenon. It gives better
resolution and entails faster development
than paper chromatography in separating
and identifying unknowns.
Figure 3 shows the chromatoplate
obtained after performing thin-layer
chromatography.

Figure 3. Chromatoplate
In the above chromatoplate, dextrin has
the least travelled distance, and therefore,
has the lowest Rf value. It was followed by
maltose and the glucose, which travelled
higher than the solvent font and has the
highest Rf value. The stationary phase for
this type of chromatography was the silica
gel coating while the mobile phase was the
solvent system consisting of 9:6:3:1 ratio of
n-butyl alcohol, acetic acid, ether and water.
Dextrin, which has the least Rf value, has
the greatest affinity to the stationary phase
and was the most polar of the three
standards. On the other hand, glucose, which
has the highest Rf value, has the greatest
affinity to the mobile phase and was the
least polar of the three standards. The Rf
values obtained for the acid hydrolysate
were then compared to that of the three
standards. It showed that the acid
hydrolysate contained all of the three
standards since it has the same results for the
obtained Rf values of the given standards.
2. Quantitative Analysis Using Nelsons
Method
In Nelsons method the reducing sugars,
when heated with alkaline copper tartrate,
reduced the copper from the cupric to
cuprous state and thus cuprous oxide was
formed. When cuprous oxide was treated
with arsenomolybdate reagent, the reduction
of molybdic acid to molybdenum blue took
place. The blue color developed was
compared with a set of standards in a
colorimeter at 480 nm.
In solving for the concentration of
glucose (mg/mL), the formula C
1
V
1
= C
2
V
2

was used. Using the formula, the following
values were obtained:

= 0 mg/mL

= 0.01 mg/mL

= 0.02 mg/mL

= 0.04 mg/mL

= 0.06 mg/mL

= 0.08 mg/mL

= 0.1 mg/mL
Table 2 shows the amount of glucose
and the corresponding absorbance reading.

Table 2. Amount of Glucose and the Corresponding Absorbance Reading
Test tube
Number
Concentration, mg/mL Absorbance, 480 nm
1 0.00 0.276
2 0.01 0.476
3 0.02 0.384
4 0.04 0.422
5 0.06 0.570
6 0.08 0.524
7 0.1 0.500
8 0.411


Figure 4. Absorbance VS. Concentration Glucose Standard Curve
y = 7.0335x
R = -5.576
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.00 0.02 0.04 0.06 0.08 0.10 0.12
Absorbance, 480
nm
Concentration, mg/mL
Glucose Standard Curve
Figure 4 shows a straight line, the
standard curves best fit line and the plotted
points which indicate amount of glucose and
the corresponding absorbance. The line was
computed through the linear regression
function of a scientific calculator and the
slope-intercept form calculated was found to
be y = 7.0335x. It does not show a linear
trend, which indicates inaccuracy of the data
gathered. This may be due to the darker
color of the solutions which may have a
higher absorbance reading or the
spectrophotometer encountered some
problems while reading the absorbance.
Through the standard curve, the glucose
concentration of the unknown sample can be
obtained and it is read as having the
concentration of 0.06 mg/mL.
References:
Book:
[1]
Campbell, M., Farrell, S. (2012).
Biochemistry 7th edition (International edition).
USA: Brooks/Cole, Cengage Learning. 451.
Internet:
[2]

http://orgchem.colorado.edu/Technique/Procedu
res/TLC/TLC.html
Date and time retrieved: February 25, 2014, 1:36
pm
[3]

http://www.newagepublishers.com/samplechapt
er/000091.pdf
Date and time retrieved: February 25, 2014, 2:09
pm
Images:
Figure 1:
Campbell, M., Farrell, S. (2012). Biochemistry
7th edition (International edition). USA:
Brooks/Cole, Cengage Learning. 452.
Figure 2:
http://orgchem.colorado.edu/Technique/Procedu
res/TLC/Images/TLC7.jpg
Date and time retrieved: February 25, 2014, 1:38
pm