Laboratory Activity No. 2 - Analysis of Carbohdrates - 1
Laboratory Activity No. 2 - Analysis of Carbohdrates - 1
Laboratory Activity No. 2 - Analysis of Carbohdrates - 1
SY 2021-2022
First Semester
BIOCHEMISTRY
Analysis of Carbohydrates
Laboratory Activity No. 2
I. LEARNING OBJECTIVES
1. Identify the chemical responses, reaction and properties that characterized a specific
kind of carbohydrates; and
2. Gain insight of the importance of carbohydrates in the series of biochemical reactions
A carbohydrate is an organic compound with the general formula Cn(H2O)n, that is,
consists only of carbon, hydrogen and oxygen. Carbohydrates make up the bulk of organic
substances on earth and perform numerous roles in living things. The carbohydrates
(saccharides) are divided into four chemical groups: monosaccharides, disaccharides,
oligosaccharides and polysaccharides. Polysaccharides serve for the storage of energy (e.g.,
starch in plants and glycogen in animals) and as structural components (e.g., cellulose in plants
and chitin in arthropods). Structural polysaccharides are frequently found in combination with
proteins (glycoproteins or mucoproteins) or lipids (lipopolysaccharides). The 5-carbon
monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD and NAD) and
the backbone of the genetic molecule known as RNA. The related deoxyribose is a component of
DNA. Saccharides and their derivatives include many other important biomolecules that play key
roles in the immune system, fertilization, preventing pathogenesis, blood clotting and
development.
1) Molisch’s Test
Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds
containing carbohydrates in a combined form, based on the dehydration of the carbohydrate by
sulfuric acid to produce an aldehyde (either furfural or a derivative), which then condenses with
the phenolic structure resulting in a red or purple-colored compound.
A. Fehlings Test
Fehling’s Solution (deep blue colored) is used to determine the presence of reducing
sugars and aldehydes.
C. Barfoed’s Test
Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so
that it can only be reduced by monosaccharides but not less powerful reducing sugars.
Disaccharides may also react with this reagent, but the reaction is much slower when
compared to monosaccharides.
3) Seliwanoff’s Test
Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are
distinguished from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone
group, it is a ketose and if it contains an aldehyde group, it is an aldose. This test is based on the
fact that, when heated, ketoses are more rapidly dehydrated than aldoses.
4) Iodine Test
Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an
aqueous solution of potassium iodide) reacts with starch producing a blue-black color.
IV. OBSERVATION
Write your observations from the Experiments/Analysis seen
TESTS ON OBSERVATION
CARBOHYDRATES
Molisch’s Test At the interface between the acid and the maltose reagent in this
test, I noticed a purple coloring that appeared as a ring, suggesting
the presence of carbohydrate. When the test method was performed
with the protein glycine, the surface began to turn green. Therefore,
glycine cannot be classified as a carbohydrate; giving a negative
result.
Fehling's Test In this test, I've come to the conclusion that a precipitate of copper
(1) oxide that displays various hues, as shown in the experiment,
signified a positive outcome.
Benedict’s Test The glycine, glucose, and maltose were tested, and were pronounced
as non-sugar, reducing sugar, and reducing sugar respectively.
Heating a mixture of Benedict’s qualitative reagent and a reducing
sugar such as glucose in a base medium, leads to the reduction of
cupric ions in Benedict’s qualitative reagent to cupric ions; with the
formation of copper (1) oxide seen as a yellow to red precipitate in
the mixture.
Barfoed’s Test In this test, a formation of red or yellow to orange precipitation for
reducing monosaccharides yields a positive result.
Iodine Test In this test, I observed that after applying iodine on the killed/dead
leaf, a blue stain appeared, indicating the presence of starch.
V. QUESTIONS/RESEARCH WORK
Molisch’s Test
1. Write the reaction step(s) involved in this test?
● Measure 2ml. of Maltose, a carbohydrate, into the test tube.
● Add 3 drops of Molisch reagent.
● Swirl to mix; and
● Run 2ml. of concentrated H2SO4 slowly along the inner walls of the test tube (Maltose +
Molisch reagent + Concentrated H2SO4).
2. Give an example of a protein structure that would give positive test with Molisch’s Reagent
Fehling's Test
1. Write the reaction(s) involved in Fehling’s Test.
Copper ions are reduced from the oxidation state of +2 down to +1, which causes copper
(1) oxide to form and precipitate.Positive results in the Fehling test indicate presence of glucose,
fructose and lactose or presence of reducing sugar in the sample. The negative result of the
Fehling test indicates the presence of non-reducing sugars such as sucrose, starch.
Fehling's solution is alkaline. Mixing cupric ions with an alkaline solution would
result in formation of cupric hydroxide of very low water solubility. By adding tartrate
ions, the cupric ions will form a complex with the cupric ions which will keep them
dissolved in the alkaline solution; forming a deep blue complex.
3. Some disaccharides such as maltose are reducing agents, whereas others, such as sucrose
are not. Explain briefly by including the structures of the sugars.
Disaccharides such as maltose have an aldehyde group in its open chain form. That's why
this reaction can also be used to test for aldehydes. Sucrose on the other hand, the ring of the
glucose cannot open due to the glycosidic bond. Therefore, it is not a reducing sugar. But, it can
be splitted in acidic conditions. This way, all disaccharides can be splitted into monosaccharides;
giving a positive result.
Barfoed’s Test
1. Write the reaction(s) involved in the Barfoed’s Test.
2. When you test starch with Barfoed’s reagent, what would be the answer, positive or
negative? Explain your answer by giving reasons and structures.
Testing a starch with Barfoed’s reagent would yield a negative result. As mentioned, the
Barfoed's reagent is used to detect monosaccharides— starch is a polysaccharide consisting of
glucose monomers which are joined together with the help of 1,4 linkages. Hence, the result.
Seliwanoff’s Test
1. Write the reaction(s) involved in Seliwanoff’s Test.
When heated, ketoses are more rapidly dehydrated than aldoses, which explains why
aldoses only form a yellow to orange precipitate. Whereas keto dehydrates in the presence of
concentrated acids to yield furfural derivatives, which reacts with resorcinol in salivanos reagent
to yield a cherry-red complex; seen as a positive result.
Resorcinol works best for quick determination of keto-sugars because it produces a red
precipitate when combined with acid.
On treatment with concentrated/strong acid, ketoses are dehydrated more rapidly to give
furfural derivatives and on condensation than aldoses. Thus, I can say that strong acids are best
used for easy identification of keto-sugars and aldose-sugars, along with resorcinol.
4. What is the result of testing sucrose with Seliwanoff’s reagent? Explain your answers by
giving reasons and structures.
Seliwanoff's reagent would produce a positive result when used to test a sucrose. This is
due to the fact that sucrose, a disaccharide made of fructose and glucose, has a ketone group in
fructose, which interacts with resorcinol to produce a dark red (cherry) color.
Iodine Test
1. Explain the reaction between iodine solution and polysaccharides by giving the structures
of related compounds.
Polysaccharides can trap iodine in the center of their coiled and helical structures,
resulting in a blue-black color complex.
2. Each polysaccharide tested gives different color results with the iodine test. Explain the
reason briefly.
The long chains are needed to make the reaction in an iodine test. Polysaccharides have
long carbon chains. Hence, the longer the chains, the darker the color complex it produces;
situated as “different color” results.