Course Code: BIO 024

Student’s Guide #1

Lesson title: CARBOHYDRATES Materials:

Lesson Objectives: by the end of this
home experiment, you should be able to
… 
1. Know the different test for
detection of reducing and non-
reducing sugar.
2. Understand the principle
behind the general and specific
test for
carbohydrates and sugars.
Pen and paper
References:
Bathan, G. I., Crisostomo, A. C., Daya, M. L., De Guia, R. M., Farrow, F. L., Gabona,
M. G., . . . Ysrael, M. C. (2017). Laboratory Manual in General Biochemistry. (2nd, Ed.)
Quezon: C & E Publishing.
 Belen Espino-Cabatit, P. P. (1988). BIOCHEMISTRY. In P. P. Belen Espino-Cabatit,
BIOCHEMISTRY (p. 851). Manila, Philippines: UST PRESS.
 Josue, P. B., & Ocenar, PhD, M. M. (2014). Biochemistry Laboratory Manual and
workbook for Health Sciences . Quezon : C & E Publishing Inc. .

 Stoker, H. S. (2013). General, organic and biological chemistry (6 ed.). Retrieved 8

September, 2020

PRE-LABORATORY CONCEPTS

Sugar like sucrose, is a

carbohydrate that can be found in fruits
and vegetables. All green plants
manufacture sugar through
photosynthesis. Photosynthesis is a
process by which plants synthesize
carbohydrates from carbon dioxide and
water using sunlight as the energy
source. Once photosynthesis produces
sugar, plants have the unique ability to
change sugar into starch, and then turn
starch into various forms of sugar for
storage. This diversity makes a wide
variety of fruits and vegetables as an
indispensable food group. (Josue &
Ocenar, PhD, 2014)

All carbohydrates are made up of

one or more molecules of sugars. The
family of sugars includes:

Carbohydrates can be divided into four major classes:

1. Monosaccharides are sugars with only one molecule. They cannot be hydrolyzed into simpler carbohydrate molecules.
They are simple sugars and further classified according to the length of the carbon chain present, as triose (C3H6O3), tetroses
(C4H8O4), pentoses (C5H10O5) and hexoses (C6H12O6). Examples like:
 Glucose (dextrose or blood sugar)
 Fructose (levulose or fruit sugar)
 Galactose (occurs only in milk)

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 Course Code: BIO 024
Student’s Guide #1

2. Disaccharides are two monosaccharides linked together: Hence, yield two monosaccharide when hydrolyzed.
 Sucrose (table sugar) = glucose + fructose
 Lactose (milk sugar) = glucose + galactose
 Maltose (malt sugar) = glucose + glucose

3. Oligosaccharides yield two to ten monosaccharide molecules which be of only one,

two, or more kinds.
4. Polysaccharides yield many monosaccharide molecules upon hydrolysis, and these
molecules are typically of only one kind. Examples are starch, glycogen, cellulose
which are all polymers of glucose units.

TEST FOR CARBOHYDRATES and SUGARS

Aside from iodine test, Molisch test is also a general test for the presence of carbohydrates, and the presence of any monosaccharides
gives positive result. Since the test solution is acidic, disaccharides and polysaccharides hydrolyze to produce monosaccharides which
gives positive purple color. Molisch reagent is a mixture of α-naphthol and ethanol. (Josue & Ocenar, PhD, 2014)

It is a group test for all carbohydrates, whether free or in combined form. Despite its limitations, it is routinely used to detect the
presence of carbohydrates. This test is named after Hans Molisch, an Austrian botanist.

By principle, the reaction is based on the fact that concentrated H 2SO4 (dehydrating agent) catalysis the dehydration of sugars to form
furfural (from pentoses) or hydroxymethyl furfural (from hexoses).These furfurals then condense with sulfonated alpha-naphthol to
give a purple or violet colored ring at the junction. Polysaccharides and glycoproteins also give a positive reaction. In the event of the
carbohydrate being a poly- or disaccharide, the acid first hydrolyses it into component monosaccharides, which then get dehydrated to
form furfural or its derivatives. (Belen Espino-Cabatit, 1988).

Anthrone’s test is another general test for carbohydrates. Its principle is same as that for molisch’s test except that the furfurals and
hydroxy -methyl furfurals give condensation products with anthrone that are bluish green in colour.

REDUCING and NON-REDUCING SUGAR

Sugars can be classified as either reducing or non-reducing, based on their ability to reduce copper (II) ions to copper (I)
ions during the Benedict’s test.

Reducing sugar is a carbohydrate that reacts with a mild oxidizing agent to

form an aldonic acid (refer to figure above or to lecture discussion on reactions
of monosaccharide). The carbohydrate reduces the oxidizing agent. A reducing
agent donates electrons during the redox reaction and is itself oxidized.

One of the common oxidizing agents used to test for the presence of reducing
sugar is benedict’s solution. All common monosaccharides are reducing
sugars. Examples are glucose, fructose, maltose, and lactose. All
oligosaccharides except sucrose are reducing sugars.

Sucrose, unlike maltose, cellobiose, and lactose, is a nonreducing sugar.

In sucrose, the hemiacetal center (anomeric carbon atom) of each monosaccharide is involved in the glycosidic linkage. The result is a
molecule that contains two acetal centers. Sucrose, in the solid state and in solution, exists in only one form—there are no α and β
isomers, and an open-chain form is not possible. The glycosidic linkage in sucrose is very different from that in maltose, cellobiose,
and lactose. The linkages in the latter three compounds can be characterized as

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 Course Code: BIO 024
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“head-to-tail” linkages—that is, the front end (carbon 1) of one monosaccharide is linked to the back end (carbon 4) of the other
monosaccharide. Sucrose has a “head-to-head” glycosidic linkage; the front ends of the two monosaccharides (carbon 1 for glucose
and carbon 2 for fructose) are linked. (Stoker, 2013)

Sucrase, the enzyme needed to break the α, β(1 : 2) linkage in sucrose, is present in the human body. Hence sucrose is an easily
digested substance. Sucrose hydrolysis (digestion) produces an equimolar mixture of glucose and fructose called invert sugar.

TEST for REDUCING and NON-REDUCING SUGAR:

Benedict’s solution is a clear blue solution of sodium and copper salts. In the presence of simple sugars, the blue solution turns green,
yellow, and brick red, depending on the amount of sugar present. Any carbohydrate that can exist in a solution, even to a small extent,
has a reducing ability because they are able to form a free aldehyde or ketone group. The Benedict’s reagent contains Cu 2+ and sodium
carbonate. This solution, being alkaline in nature, can oxidize the aldehyde group to the carboxylic acid group as Cu 2+ is reduced to
Cu1+ (Cu2O) in the form of brick-red precipitate. The formation of this colored precipitate indicates the presence of a reducing sugar.

glucose + 2Cu(OH)2---------------> Cu2O + 2H2O + glucose

(oxidized cupric ions: blue) (reduced cuprous ions: red)

Non-reducing sugars do not contain an aldehyde group. The Benedict’s test determines the presence of non-reducing sugars. It is often
conducted on a food sample which tested negative for reducing sugar. If reducing sugar is present, a heavier precipitate is often
observed when the test for non-reducing sugar is conducted. The Fehling’s test is an alternative to the Benedict’s test. However, it is
less sensitive and requires that the reagents Fehling’s solutions A and B be kept separate until the experiment is carried out.

Fehling’s test, a test to detect reducing and non-reducing sugar. Sugar which contain a free aldehyde or ketone group reduce alkaline
solution of copper salts to cuprous oxide. This test can be used to screen for glucose in urine, thus detecting diabetes. Fehling’s
Solution: Prepared by combining two separate solutions, known as Fehling’s A and B.

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 Course Code: BIO 024
Student’s Guide #1

Fehling’s A is an aqueous solution of copper (II) sulphate, which is deep blue. Fehling’s B is a colorless solution of
aqueous potassium sodium tartrate (aka Rochelle Salt) made strong alkali, commonly with sodium hydroxide. The copper
(II) complex in Fehling’s solution is an oxidizing agent and the reactive reagent in the test. The deep blue active ingredient in
Fehling’s solution in the bis(tartrate) complex of Cu 2+. The two solutions are not mixed until just before using, because the tartrate
itself would tend to reduce the copper in time. When a sugar solution containing a free aldehyde or ketone group is boiled with
Fehling’s solution, the characteristic result in a brick-red precipitate of Cu2O.

Rochelle salt acts as chelating agent in this reaction:

CuSO4 + 2KOH ------------ Cu(OH)2 + K2SO4 2Cu(OH)2 + Reducing Sugar-------------------2Cu2O + Aldonic acid

ALDONIC ACID
• Acid group on top
• uses weak oxidizing agent

Table 1. Common oxidizing agents used to test for the presence of a reducing sugar
Oxidizing Composition Color of Color reaction Oxidizing agent Reducing
reagent solution with a reducing agent
sugar
Benedict’s Copper sulfate Deep blue Brick-red Cu2+ Reducing
solution in alkaline precipitate Cu2+ + e→Cu+ sugar oxidized to
Cu2O(s)
citrate carboxylate
Fehling’s Copper sulfate Deep blue Brick-red Cu2+ Reducing
solution in alkaline precipitate Cu2+ + e→Cu+ sugar oxidized to
Cu2O(s)
tartrate carboxylate
Tollen’s Silver nitrate colorless Silver mirror Ag+ Reducing sugar
reagent in aqueous forms Ag(s) Ag+ + e→ Ag(s) oxidized
ammonia to carboxylate
Source: Chemistry tutorial: Reducing and Non-reducing sugars. www.ausetute.com .au/redsugar.htm-I

OTHER SPEFICIFIC TEST FOR SUGARS:

BASED ON THE FURFURAL FORMATION AND ITS DERIVATIVES

1. SELIWANOFF’S TESTS for Keto Sugar

Principle: This test is a timed colour reaction specific for ketohexoses. Thus, it
is used to distinguish aldoses from ketoses. In the presence of HCl ketohexoses
undergo dehydration to yield 4-hydroxy methyl furfural more rapidly than
aldohexoses. Further these furfural derivatives condense with resorcinol to
form a red coloured complex. This test is named after Theodor Seliwanoff.

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 Course Code: BIO 024
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2. BIAL’S ORCINOL: Test for Pentoses

Principle: This test is specific for pentoses and the compounds
containing pentoses and thus useful for the determination of pentose
sugars. Reaction is due to the formation of furfural in the acid
medium which condenses with orcinol in the presence of ferric ions
to give a blue green coloured complex.

BASED ON OXIDATION:

1. MUCIC ACID TEST

Principle: Aldohexoses are converted to their corresponding dicarbixylic
acids in the presence of strong oxidizing agent HNO3This test is highly
specific for galactose which is either independently present in solutions or
obtained by the hydrolysis of lactose. Galactose is converted to Saccharic
acid on heating with HNO3. Mucic acid (galactaric acid, a dicarboxylic
acid) which is formed from galactose due to the oxidation of both
aldehyde & primary alcoholic group at C1&C6. It is the only Saccharic
acid
which is insoluble in cold water and thus helps in the identification of galactose. Result: The presence of insoluble crystals in
the bottom of the tube indicates the presence of mucic acid.

Ex. reaction of formation of aldaric acid (or dicarboxylic acidic sugar )

ALDARIC ACID
• acid groups both on top and
bottom
• uses strong oxidizing agent

2. OSAZONE TEST OR PHENYLHYDRAZINE TEST OR KOWARSKY TEST

Principle: An organic compound phenylhydrazine reacts with carbonyl carbon (or more specifically C1 and C2) of sugar to
form the compound called osazones or phenylhydrazone. These osazone crystals have yellow colour characteristics shapes
and melting point, definite time of formation and relative solubility in hot water. All reducing sugars form osazone with
excess of phenylhydrazine (C6H5NHNH2) when kept at boiling temperature. This property is attributed to the presence of
aldehyde or ketone group in their molecules. A microscopic examination and comparison of the formed osazones with
reference standards may lead to the identification of the reducing sugar. Needle-shaped yellow osazone crystals will be observed
for glucose and fructose, whereas lactosazone shows mushroom shaped and maltose produces flower-shaped crystals.
 Course Code: BIO 024
Student’s Guide #1

3. BARFOED’S TEST:
Principle: This test is performed to distinguish between a reducing mono- and
disaccharide. Monosaccharides are more reactive (easily oxidized) reducing agents
than disaccharides and thus react in about 1-2 min while the reducing disaccharides
take 7-12 min to get hydrolysed in the acidic solution and then react. Thus, the
difference in reducing property can be detected. Thin brick red precipitates, at the
bottom or sides of the tube indicates the presence of a reducing monosaccharide.

GUIDE QUESTIONS: Based on your readings, please answer the following queries.

1. Which of the following saccharides is a reducing and nonreducing sugar?

a. Glucose - Ans: Reducing
b. Galactose - Ans: Reducing
c. Fructose - Ans: Reducing
d. Ribose - Ans: Reducing
e. Sucrose - Ans: Non-reducing
f. Maltose - Ans: Reducing
g. Lactose - Ans: Reducing

2. Identify the name of each of the structures of sugar and identify as reducing or nonreducing sugar. One of the structures may
be not identified from the usual structures of disaccharides.

Answer: Reducing Answer: Reducing

Answer: Reducing Answer: Reducing

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 Course Code: BIO 024
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3. Which test can be used to differentiate the following pairs of carbohydrates? Explain why and why other tests ar not possible.
a. Fructose and galactose
- Mucic Acid test and Seliwanoff’s test. The Mucic acid test is a test used to distinguish Lactose and
Galactose. Since fructose is a ketohexose, it will not be converted to an aldaric acid or dicarboxylic acid,
or acidic sugar. On the other hand, Seliwanoff’s test is used to distinguish aldoses from ketoses.
Ketohexose, such as Fructose, undergoes dehydration to form a cherry-red condensation product. Ketoses
react more quickly than aldoses and thus the reaction time is a means of measurement or detection.
Ketoses react within a minute of heating while it takes longer than that for aldoses.
- It cannot be differentiated either with Benedict’s test since both are reducing sugars or barfoed’s test since
they are both monosaccharides reducing sugar and with Phenylhydrazine test since both are reducing
sugars capable of forming osazone crystals. Bial’s orcinol test is also impossible since both are hexoses
and the test is only for furanose or pentose sugar.

b. Ribose and glucose

- Bial’s Test. This test is for pentose sugars or sugars with furanose ring. A positive result of this test will
give a green or olive color. This distinguishes it from other sugars. On the other hand, it gives out a muddy
brown color if it is a hexose sugar and there will be no change in color if the sugar is a pyranose.
- Seliwanoff’s test is not possible since both are aldoses. Osazone does not apply since both are reducing
sugars capable of forming osazone crystals. Lastly, barfoed’s test also can’t be used because none of the
sugars are reducing disaccharides.

c. Glucose and maltose

- Barfoed’s Test. This test is used to distinguish monosaccharides from disaccharides and polysaccharides.
This is due to the conditions of lower pH and shorter incubation time. Only monosaccharides can react fast
enough to reduce copper ions. The reagent is similar to Benedict’s except the pH is lower and heating time
is reduced to 2 minutes. It will not take longer than that for monosaccharides to react.
- Seliwanoff’s test cannot be used to differentiate the sugars since maltose also contains glucose units and
one of which is a ketose sugar. Bial’s is not possible because the test is only for aldohexose sugar
galactose. Osazone does not apply since both are reducing sugars or contain reducing sugar capable of
forming osazone crystals.

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