QUANTITATIVE ANALYSIS AND THIN LAYER CHROMATOGRAPHY OF CARBOHYDRATES

Elpa, Jose Fernando; Flores, Joan;

Francisco, Kaycee Allen;

Gallardo, Mario Emmanuel; Gan, James Viktor

Group 4 2-G Pharmacy Pharmaceutical Biochemistry Laboratory

ABSTRACT

Carbohydrates are one of the most important components in many foods. It is important to determine the

type and concentration of carbohydrates in foods for a number of reasons. Generally, the experiment was

performed in order to determine the components present in a given sample and to correlate those

standard sugars presented with that of the acid and enzymatic hydrolyzates using the thin layer

chromatography and the Nelson’s test. In the Thin layer chromatography performed, the glucose and the

acid hydrolyzate had the same Rf value, which means that they both travelled the same length of path

from the origin. Even the galactose and fructose had the same Rf value. The ribose travelled the farthest

while the galactose and fructose travelled the least. In the Nelson’s test, glucose standard curve was

plotted using the absorbance readings obtained against the concentrations of the standard solutions.

INTRODUCTION

A carbohydrate is an organic compound with

consisting only of carbon, hydrogen and oxygen

in which the last two is in the 2:1 atom ratio. It

can be viewed as hydrates of carbon. It is divided

into four chemical groupings: monosaccharides,

disaccharides, oligosaccharides, and

polysaccharides. In general, the monosaccharides

and disaccharides, which are smaller (lower

molecular weight) carbohydrates, are commonly

referred to as the sugars. Basically, its primary

function is to provide energy for the body,

especially the brain and the nervous system.

One of the methods used to analyze the sample

carbohydrates is the thin layer chromatography

(TLC). Generally, thin layer chromatography is a

chromatography technique used to separate

mixtures. This technique may be used for the

determination of the components a plant

contains, for monitoring organic reactions, for the

analysis of ceramides and fatty acids, for the

detection of pesticides or insecticides in food and

water, for the analysis of the dye composition of

fibers in forensics, for identifying compounds

present in a given substance, and for assaying

the radiochemical purity of radiopharmaceuticals.

In the interpretation of the results, the

components, visible as separated spots in the

chromatogram, are being identified by comparing

the distances they have traveled with those of

the known reference materials. The distance of

the start line to the solvent front (=d) is

measured, even the distance of center of the spot

to the start line (=a). The distance the solvent

moved is divided by the distance the individual

spot moved. The resulting ratio is called Rf-value.

Nelson's test for reducing sugar is a pretty old

test and is quite generic in its scope. It basically

uses the reduction of some dye compound and

then relies on spectrophotometry to determine

the level of chemical dye remaining at a specific

wavelength.

The objectives of the experiment were as

follows:

1. To perform thin layer chromatography on

the carbohydrate hydrolyzates.

2. To correlate the data obtained from the

color tests and thin layer chromatography

of the carbohydrate hydrolyzates.

3. To identify the monosaccharide present in

the polysaccharide sample.

4. To determine the amount of reducing

sugars using Nelson’s test and explain the

principle involved.

EXPERIMENTAL

A. Compounds used and tested

1. Thin-layer chromatography

Acid hydrolyzate 0.5 mL anisaldehyde

Enzymatic hydrolyzate 9.0 mL 95% CH3CH2OH

Galactose 0.5 mL H2SO4

Glucose 0.1 mL CH3COOH

Maltose Ribose

Fructose

9:6:3:1 n-butyl alcohol-acetic acid-ether-water

2. Quantitative analysis

Carbohydrate sample (hydrolyzates)

Nelson’s reagent A

Nelson’s reagent B

Arsenomolybdate reagent

Glucose standard

Distilled water

B. Procedure

1. Thin-layer chromatography

In the developing chamber, 40 mL of the

solvent system was placed. The chamber was

covered with inverted watch glass and was

equilibrated for 10 mins. At the same time, a

pencil line was drawn across one end of the TLC

plate, about 2 cm from the bottom. Then,

equidistant points were marked along the line for

the standards, acid, and enzymatic hydrolyzates.

In those points, the standards and hydrolyzates

were applied five times and ten times,

respectively using capillary tubes with drying

after every application. Then, the TLC plate was

placed in the developing chamber and was

allowed to develop until the solvent was about 1

cm from the top of the plate. After the

development, the chromatoplate was removed

from the chamber and the solvent front was

marked with a pencil. The plate was air-dried and

was sprayed with p-anisaldehyde visualizing

agent. Then, the plate was heated on the hot

plate until the sugars became evident by the

presence of colored spots. The spots were lightly

circled with a pencil. Then, the Rf value was

computed. Lastly, the components of acid and

enzymatic hydrolyzates were identified.

2. Quantitative analysis

In this experiment, Nelson’s reagent was

prepared by mixing 12.5 mL Nelson’s A with 0.5

mL Nelson’s B. The 7 test tubes were labeled and

were filled with measured amounts of standard

glucose solution presented in the table below.

Table 1: Dilution of samples

test

tube

no.

Glucose

standard

(mL)

Distilled

water

(mL)

unknown

sample

(mL)

1 0 1.0 0

2 0.1 0.9 0

3 0.2 0.8 0

4 0.4 0.6 0

5 0.6 0.4 0

6 0.8 0.2 0

7 1.0 0 0

8 0 0.6 0.4

Then, 1.0 mL Nelson’s reagent was added into

each prepared tube, and was shaken well. The

tubes were heated simultaneously in a boiling

water bath for about 20 mins. Afterwards, the

tubes were removed simultaneously and were

cooled in a beaker of water. Then, 1.0 mL of

arsenomolybdate reagent was added into the

tubes. The tubes were shaken occasionally for 5

mins. or until the Cu2O precipitate was dissolved.

The absorbance of the standards and unknown

was taken against a reagent blank at 480 nm.

Standard curve was constructed by plotting

absorbance readings against concentrations of

standard solutions. Finally, concentration of

unknown in mg/tube and mg/mL was determined.

RESULTS AND DISCUSSION

Figure 1: Thin Layer Chromatography

The figure shows the result of the thin layer

chromatography performed. Visible spots

appeared on the plate as seen on the figure.

Distances travelled by the samples were also

illustrated on the sample.

Table 2: Thin Layer Chromatography

(standards)

GAL GLU MAL FRU RIB

distance

travelled

by

solvent

7

cm

7

cm

7

cm

7

cm

7

cm

distance

travelled

by solute

1.0

cm

1.3

cm

1.2

cm

1.0

cm

1.8

cm

Rf value 0.14 0.19 0.17 0.14 0.26

The table above shows the result of acid and

enzymatic hydrolyzates in the thin layer

chromatography conducted. The table points out

that the enzymatic hydrolyzate travelled farther

than that of the acid hydrolyzate.

Table 3: Thin Layer Chromatography (acid

and enzymatic hydrolyzates)

Acid enzymatic

distance travelled by

solvent (cm)

7 cm 7 cm

distance travelled by solute (cm)

1.3 cm 1.5 cm

The table above shows the result of acid and

enzymatic hydrolyzates in the thin layer

chromatography conducted. The table points out

that the enzymatic hydrolyzate travelled farther

than that of the acid hydrolyzate.

Table 4: Concentrations of Glucose and

Absorbance Readings

test

tube

no.

mg

glucose

std. per

tube

(mg/tube)

mg

glucose

std. per

mL

(mg/mL)

Absorbanc

e

1 0 0 0.049

2 0.03 0.01 0.462

3 0.07 0.02 1.606

4 0.13 0.04 2.170

5 0.20 0.06 2.200

6 0.27 0.08 2.266

7 0.33 0.1 2.95

8 0 0 - 0.598

The table above shows the different computed

concentrations of glucose per tube and per mL,

and the absorbance readings of each tube.

Spectrophotometer was used to get the

absorbance readings of each tube. The table

shows direct proportionality between the

concentration and the absorbance. As one

increases, the other also increases.

Graph 1: Absorbance vs Concentration

0 0.010.020.030.040.050.060.070.080.09 0.10

0.5

1

1.5

2

2.5

3

Concentration (mg/mL)

Abso

rban

ce

The graph shows the direct proportionality of

the concentration of glucose to the absorbance.

The higher the concentration of the glucose, the

higher is the absorbance.

REFERENCES

Flitsch, SL & Ulijn, RV (2003). Sugars Tied to the

Spot. Nature 421: 219–220.

Harwood, L. & Moody, C. Experimental Organic

Chemistry: Principles and Practice (Illustrated edition

ed.). pp. 159-173.

Vogel, A.I, Tatchell, A.R, Furnis, B.S, etc. Vogel's

Textbook of Practical Organic Chemistry (5th Edition).