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Experiment 6
AIM
To characterize carbohydrates present in an unknown solution on the basis of various chemical assays.
INTRODUCTION
Most of the chemical compounds present in aldehydes and ketones. Aldehydes (CHO) and
living organisms contain skeletons of ketones ( = CO) constitute the major groups in
covalently bonded carbon atoms (C-C-C-C). carbohydrates.
These compounds are known as organic
compounds, because most of these are either
present in, or produced by living things.
Organic compounds are the major components
of cells and tissues. They provide energy for life
processes, participate in and regulate
metabolic reactions, and transmit information.
Organic macromolecules in living organisms
can be classified as either carbohydrates, (A carbohydrates)
proteins, lipids, or nucleic acids, among others.
These macromolecules are always made of Heating of monosaccharides under acidic
smaller subunits. The subunits of
conditions (concentrated sulphuric,
macromolecules are held together with
hydrochloric acids) leads to their dehydration.
covalent bonds, and have different structures
From pentoses the furfural is generated, and in
and properties. For example, lipids made of case of hexoses 5-hydroxymethylfurfural.
fatty acids have many C-H bonds and relatively Pentoses more easily undergo dehydration
little oxygen, while proteins made of amino compering to hexoses and ketoses undergoes
acids have amino groups (-NH2) and carboxyl (- dehydration more easliy than aldoses.
COOH) groups. These characteristic groups Disaccharides react more slowly in comparison
impart different chemical properties to to all monosaccharides. This phenomenon is
macromolecules for example, due to the fact that oligo- and polysaccharides
monosaccharides such as glucose are polar and have to be hydrolysed first and then undergo
soluble in water, while lipids are nonpolar and dehydration.
insoluble in water. Carbohydrates are
compounds that contain carbon, hydrogen and
oxygen. Carbohydrates include a variety of
compounds, such as sugars, starches, and
celluloses. While sugars and starches serve as
energy sources for cells; celluloses are
structural components of the walls that
surround plant cells. The term carbohydrate
literally means "hydrated (H20) carbon"
Carbohydrates may contain one sugar
molecule (monosaccharides), two sugar
molecules (disaccharides), or many sugar units
(polysaccharides). Carbohydrates are
polyhydroxy aldehydes and ketones or
substances that hydrolyze to yield polyhydroxy
(Heating of saccharides with concentrated sugars gets reduced to yellow or red cuprous
acids leads to the loss of one water molecule oxide and is precipitated. Hence, formation of
in the first phase and then two water the yellow or brownish-red colored precipitate
molecules. As a result furfural is formed a helps in the detection of reducing sugars in the
heterocyclic aldehyde or its derivatives.) test solution.
This test is used for the detection of starch in Reducing Sugar Reactions (Experimental):
the solution. The blue-black colour is due to the
formation of starch-iodine complex. Starch A. Reaction with Benedicts Reagent
contain polymer of -amylose and amylopectin
which forms a complex with iodine to give the Place 1 drop of carbohydrate on the
blue black colour. spotting plate and add 10 drops of
Benedicts reagent (an alkaline solution
Osazone Test: containing the cupric ion). Mix well and
then heat on a hot late for about 5 minutes.
Observe the results.
The ketoses and aldoses react with
phenylhydrazine to produce a
B. Reaction with dinitrosalicylic acid
phenylhydrazone which further reacts with
another two molecules of phenylhydrazine to
Dilute 1 drop of carbohydrate solution with
yield osazone. Needle-shaped yellow osazone
5 drops of water. Then add 2 drops of
crystals are produced by glucose, fructose and
dinitrosalicylic acid (DNS) reagent (an
mannose, whereas lactosazone produces
alkaline solution containing dinitrosalicylic
mushroom shaped crystals. Crystals of
acid). Mix well and then heat on a hot plate
different shapes will be shown by different
for about 5 minutes. Observe the results.
osazones. Flower-shaped crystals are produced
by maltose.
C. Quantitative application of a reducing
sugar reaction
MATERALS AND METHODS Reducing carbohydrates can be
determined by a variety of methods, one of
96 well plate, 0.5% solutions (of ribose, which is the socalled Nelson method. In
glucose, fructose, lactose, sucrose and this method, an alkaline solution
starch), anthrone, H2SO4, orcinol, sodium containing the cupric ion is first used to
bicarbonate, DNS oxidize the carbohydrate. The cuprous
oxide formed as a result of this is in turn Color Change
used to reduce the arsenomolybdate ion Lactose -
and give rise to a deep bluegreen Ribose light green
chromophore that can be determined
Starch -
colorimetrically. The purpose of this
experiment is to acquaint you with the Sucrose blue
Nelson method as an example of a Glucose -
quantitative application of a reducing Fructose blue-green
sugar reaction. Over the years, methods
such as this have been used widely used in
both industrial and clinical laboratories.
While they are losing popularity in
laboratories concerned with clinical
chemistry, they continue to be popular in
laboratories concerned with starch
chemistry. You will make use of this
method in a subsequent experiment Figure 1
involving the action of amylase on starch.
Before starting the experiment outlined
below, be sure all of the glassware you
intend to use is thoroughly clean since the
experiments you performed in parts A and
B used %0,5 solutions of carbohydrates
and your pipettes and test tubes might still
contain enough carbohydrate to ruin your
assay.
Figure 2
Tube No ml Standard ml water ml Unknown DNS Reaction with orcinol and hydrochloric acid:
0 0 1.0 0 0.5
1 drop of carbohydrate solution on the spotting
1 0.2 0.8 0 0.5
plate and 2 drops of orcinol reagent.
2 0.4 0.6 0 0.5
3 0.6 0.4 0 0.5 Color Change
4 0.8 0.2 0 0.5 Lactose -
5 1.0 0 0 0.5 Ribose -
6 0 0 1.0 0.5 Starch -
Sucrose -
Add to each tube 500 L DNS. Place the tubes Glucose -
in an actively boiling water bath for 5 minutes. Fructose -
Cool the tubes in a beaker of cold water (1-2
min).Dilute to the graduation mark and mix
well. Determine the absorbance of each tube at Reducing Sugar Reactions (Experimental):
540 nm. Set tube 0 at zero absorbance.
Reaction with Benedicts Reagent:
Reaction with anthrone and sulfuric acid: Lactose Ribose Starch Sucrose Glucose Fructose
Benedicts (10 drops) blue-green red red - blue-green -
We dropped a drop ribose, glucose, fructose, DNS (2 drops) red red - - red red
lactose, sucrose, starch and 10 drops anthrone
in each tubes.
Tube No ml Standard ml water ml Unknown DNS
0 0 1.0 0 0.5
1 0.2 0.8 0 0.5
2 0.4 0.6 0 0.5
3 0.6 0.4 0 0.5
4 0.8 0.2 0 0.5
5 1.0 0 0 0.5
6 0 0 1.0 0.5
Figure 3
Figure 5
http://www.chem.ucalgary.ca/courses/351/C
arey5th/Ch25/ch25-2-5.html
DISCUSSION
The most important thing in this experiment is
to determine the carbohydrate
(monosaccharide, disaccharide..). We can use
chromatography when determining
carbohydrates, even chromatography is
better. However we used color reactions in
experiment. One of the most important
considerations in the experiment is the
conversion of carbohydrates to furfural. (first
stage) After that, it combines with the
necessary chemicals to give color.
Carbohydrates arent given color section
experiment in reaction with orcinol and
hydrochloric acid. Probably 2 g orcinol and 0.2
ferric chloride per liter of concertrated HCI
condition may not have been made. On the
other hand, lactose and glucose are blue-green
color (Benedicts 10 drop) in reaction with
Benedicts Reagent. Great posibillity they are
not well mixed so they are blue- green. Actually
they had to be red.
REFERENCES
msu.edu/course/lbs/145/luckie/Lab1.html
katedrachemii.sum.edu.pl/uploaded/Carbohy
drates%20reactions_students.pdf