KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY

G DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY

FTECH 152 - TECHNIQUES IN LABORATORY ANALYSIS II

LAB REPORT 4 - QUANTIATIVE ANALYSIS OF CARBOHYDRATES (ANTHRONE TEST)

INDEX: 9309819

70/85
CHARTEY - BLANKSON EMMANUEL APPIAH

09 - 03 - 2020

QUANTIATIVE TEST OF CARBOHYDRATES (ANTHRONE TEST) 3 Marks

ABSTRACT 13 marks

The aims of this lab work is to test for sugar samples and their concentrations with anthrones test and
Beer Lambert's Law. Carbohydrates are biomolecules made of carbon, hydrogen and oxygen usually
classified as monosaccharaides, disaccharides and polysaccharides. Quantitative tests are used to
quantitatively determine levels of molecules (sugar in this work) in substances. Anthrones test, a more
available and less expensive test was used in this work.

Anthrones reagent reacted with the sugar samples giving off a bluish - green color whose absorbances
were determined using a spectrophotometer and concentrations of unknown samples determined. A
curve was constructed which was compared to a standard curve and deductions made.

This work is limited as a result of some experimental errors which led to deviations of results obtained
form the standard results and Beer's principles. Precautions should really be put in place in experimental
works as such in order to achieve precise results.

INTRODUCTION 9 marks

This work aims at testing for the amount of carbohydrates in various samples/ substances using
Anthrones test, estimating the concentration, and applications of Beer Lambert's law in
analyzing results.

Carbohydrates are biomolecules consisting of carbon oxygen and hydrogen. It is basically

divided into monosaccharaides (pentose or hexose - one unit) , disaccharides( two
monosaccharaides) , oligosaccharides (more than two monosaccharaides) and
polysaccharides(more than ten monosaccharaides).

Quantitative tests are chemical tests used for the determination of the amount or level of an
element or molecule in a substance. Anthrones test is one among many tests that can be
qualitatively use to determine carbohydrates; but anthrone was used due to its availability and
lower cost as compared to Somogyi - Nelson or The Dinitrosalisyclic acid (DNSA) tests.

Anthrones test involves the use of Anthrones reagent (H2SO4 and anthrone - 10 - keto - 10,9-
dihydroanthracene), where H2SO4 dehydrates the sugar samples to form furfural which is
condensed into a colored complex (bluish - green complex) by the action of the anthrone. This
is calorimetrically measured with a spectrometer at 620nm.
PROCEDURE 12marks

A stock solution of sugar (glucose) sample of concentration 100ug/ml was serially diluted into 10 -
100ug/ml solutions. A blanc was set using distilled water. Two other unknown samples were also
considered. Eight test tubes were labeled respectively according to the samples present i.e 10ppm,
20ppm, 60ppm, 80ppm, 100ppm, blanc, Unknown 1and Unknown 2. Each sugar sample (0.5ml) was
taken with the pipette into thier respective test tubes including the blanc and unknown samples.
Anthrones solution (2ml) was added to each of the samples in the test tubes, sealed with parafilm and
vortexed for 10sec. It was then kept in a dark room for 30min to cool it to room temperature.

The absorbances of each sample was determined by a spectrophotometer at 620nm and recorded in
Table 1(see results). A graph of the values (absorbances against concentration) was plotted and the
concentration of the unknown samples determined.

RESULTS 11 marks

Table 1. Standard Calibration table for glucose

Concentration/ ppm Absorbance at 620nm

10 0.07589

20 0.13529

60 0.28715

80 0.35007

100 0.42408

Unknown 1 0.22525

Unknown 2 0.23327
Graph 1. Standard graph 0f absorbance against concentration

Unknown concentration 1 = 57.96

Unknown concentration 2 = 60.0

Table 2. Determined calibration table for glucose

Concentration/ ppm Absorbance at 620nm

10 0.03281

20 0.061

60 0.24557

80 0.7334

100 0.85837

Unknown 1 0.23271

Unknown 2 0.26399
Graph 2. Determined graph of absorbance against concentration

Unknown concentration 1 = 38. 10

Unknown concentration 2 = 41. 39

DISCUSSION 13 marks

According to Beer Lambert's law, the concentration of a substance is directly depending on its relative
absorbance as a wavelength of light is passed through the sample to check its absorbance. From the
above work based on Beer’’s principle, we had a calibration curve which is slightly deviated from the
standard given as shown above (see results - Graph 1 and 2). These deviations or limitations found in the
graph (Graph 2) can be attributed to some experimental errors that might have occurred during the
procedure which might have altered the concentration of the samples and the absorbancesabsorbance
as well.

Some experimental errors that may be as a result of the deviation are: time taken for incubation to take
place, environment in which incubation took place, impurities in sample as a result of improper rinsing
or washing of glasswaresglassware, and inaccurate measurements of samples

Incubation is one of the major parts of the procedure as it allows anthrone to combine and react well
with sample molecules. If the temperature for the incubation is not maintained constant, or time meant
for incubation to take place was not accurately kept, it can cause an alteration in the values.
Glasswares not properly rinsed or washed before use can also contribute since there may be some
impurities or different components which may mix with the solution altering its concentration.

Inaccuracy in sample measurement can produce either a higher results or lower results due to the either
a more measurement or lower measurement respectively. This is because anthrone’s test deals with the
number if monosaccharaides units present, the more the sugars, the higher the deeper the color
formation and vice versa. which also affects the absorbances and the concentration of the solution.

As a result of these errors, the values of the unknowns (concentrations) were also affected and hence
slightly deviated from that of the standard. But it was observed that both the standard curve and the
determined curve ( Graph 1 and 2) passed through the origin and thus can also be attributed to
experimental error (inaccuracy) in blanc preparation, the power of the wavelength of light that was used
in the spectrophotometer or the absorptive properties of the couvert used. Hence the equation of the
curves were altered from Beer's law; which expects the intercept of the curve to pass through the origin
hence an intercept of 0 on your equation. Unlike Beer's principle, the curves did not pass through the
origin and as a result the intercept of the equation was not zero which also contributed to a deviation in
our unknown concentrations.

CONCLUSIONN 4 marks

The experimental errors that occurred in this work can be curbed by taken key precautionary measures
such as properly washing and keeping of glasswares before and during work, proper time keeping and
incubation, use of proper equipment and careful reading of measured samples. The principles of Beer's
law must always be considered in each part of the work for a more accurate results.

REFERENCES 5 marks

Beer (1852). Determination of the absorption of red light in colored liquids. Annalen der Physik und
Chemie (in German). 86 (5): 78–88.

Thomas G. Mayerhöfer, Jürgen Popp (2019-05-15), "Beer's law derived from electromagnetic theory",
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (in German), 215, pp. 345–347,
doi:10.1016/j.saa.2019.02.103

Bien, H.-S.; Stawitz, J.; Wunderlich, K. (2005). "Anthraquinone Dyes and Intermediates". Ullmann's
Encyclopedia of Industrial Chemistry. Weinheim- Wiley

Fieser, Louis F.; Hershberg, E. B. (May 1939). "Inter- and Intramolecular Acylations with Hydrogen
Fluoride". Journal of the American Chemical Society. 61 (5): 1272–1281.