CHEM 132.

2 – BIOCHEMISTRY (LABORATORY)
LABORATORY REPORT

Name _John Allan T. Pasana_______ Date Submitted: _01/21/21__

Lab Schedule: MTh @ 10:00-12:00 Date Performed: _01/21/21__

Exercise No. 3
Carbohydrates
I. OBJECTIVES:
 Use chemical tests to identify some physical and chemical characteristics of typical
carbohydrates.
 Differentiate between monosaccharides, disaccharides and polysaccharides using the
above tests.
 Identify an unknown carbohydrate.

II. RESULTS AND DISCUSSION:

A. STRUCTURAL FORMULAS FOR CARBOHYDRATES
STRUCTURES
MONOSACCHARIDE FISCHER HAWORTH CHAIR
Glucose

Galactose

Fructose
 STRUCTURES
DISACCHARIDE
HAWORTH CHAIR

Lactose

Maltose

Sucrose

POLYSACCHARIDE STRUCTURE

Amylose

Amylopectin

Discussion:

For the monosaccharides, even if they have the same chemical formula which is C 6H12O6,
they differ structurally and stereochemically. This makes them different molecules despite
sharing the same atoms in the same proportions, and they are all isomers of one another, or
isomeric monosaccharides. For the disaccharides, they are formed by the condensation of two
 monosaccharide molecules. To be more precise glucose and galactose form lactose, two
glucose molecules form maltose and sucrose or table sugar is composed of the monomers
glucose and fructose. For the polysaccharides, amylopectin molecules are huge, branched
polymers of glucose, and in contrast to amylose, amylopectin is branched. Amylose, on the
other hand, consists of linear, helical chains of alpha-D-glucose monomers linked together
through alpha (1-4) glycosidic bonds.

B. BENEDICT’S TEST FOR REDUCING SUGARS

Compound Color Reducing Sugar (Yes/No)

Approaching Brick
Glucose Yes
Red
Fructose Brick Red Yes
Sucrose Blue No
Maltose Orange Yes
Bluish Green with a No
Starch
hint of Yellow

Discussion:
For the Benedict’s test, these 5 known sugars were tested to see if they are reducing sugars.
Glucose, fructose and maltose tested positive which means that they are reducing sugar, especially
Fructose since it possesses a darker shade of red color which indicates that it has a large amount
of it. On the other hand, sucrose tested negative which meant that it is a non-reducing sugar and
therefore did not react to the Benedict’s reagent itself. Starch had a bluish green color with a hint
of yellow in it and this means that it is a non-reducing sugar since it reacts poorly to the reagent
due to the presence of a small number of reducing sugar moieties.

Unknown Solution Color Reducing Sugar (Yes/No)

A Blue No
Bluish Green with a
B No
hint of Yellow
 C Brick Red Yes
D Orange Yes
Approaching Brick Yes
E
Red

Discussion:

For the Benedict’s test of these 5 unknown solutions, only solutions C, D, and E have tested
positive which meant that they are reducing sugars. However, solution A has tested negatively
which indicates that it is a non-reducing sugar. The same goes for solution B since it did not
completely react with the reagent itself. These 5 unknown solutions will be identified later on
the last table.

C. BARFOED’S TEST FOR MONOSACCHARIDES

Compound Color Monosaccharide (Yes/No)

Reddish with a little
Glucose Yes
speck of blue
Fructose Brick Red Yes
Sucrose Blue No
Maltose Blue No
Starch Blue No

Discussion:

The Barfoed’s test is somewhat similar to the Benedict’s test, but this one is usually used to
distinguish monosaccharides from disaccharides. After the experiment, it was found out that
only glucose and fructose tested positive which meant that they are monosaccharides. On the
contrary, sucrose maintained its blue color which meant that it is not a monosaccharide. This is
true since sucrose is a disaccharide formed by the condensation reaction of glucose and
fructose molecules. Maltose tested negatively as well since its blue color indicates that it is not
a monosaccharide. Just like sucrose, maltose is a disaccharide and is formed by the
condensation reaction of two glucose molecules. Finally, starch also had a blue color and this
basically means that it is not a monossacharide just like sucrose and maltose. Unlike sucrose
and maltose, starch is not a disaccharide. Hence, it is a polysaccharide which means that it is a
large molecule made of many smaller monosaccharides.

Unknown Solution Color Monosaccharide (Yes/No)

A Blue No
B Blue No
Bluish with a hint of
C Yes
Red
D Blue No
Bluish with a hint of Yes
E
Red

Discussion:

For the Barfoed’s test of these 5 unknown solutions, the only solutions that tested
positively is solutions C and E. Therefore, it can be assumed that these two solutions are
monosaccharides. On the other hand, solutions A, B and D tested negatively in this test which
basically means that they are not monosaccharides. It may be safe to assume that 2 solutions
are disaccharides and one is a polysaccharide. In the last table, these 5 unknown solutions will
be identified to bring about clarity and enlightenment.

D. SELIWANOFF’S TEST FOR KETOHEXOSES

Compound Color Ketohexose/Aldohexose

Approaching Clear
Glucose Aldohexose
Color
 Fructose Dark Red Ketohexose
Sucrose Dark Red Ketohexose
Approaching Clear
Maltose Aldohexose
Color
Approaching Clear Aldohexose
Starch
Color

Discussion:

Seliwanoff’s test is used to differentiate between sugars that have a ketone group (ketose)
and those that have an aldehyde group (aldose). For the result of the 5 known sugars, only
fructose and sucrose resulted in a dark red color. The formation of this dark red or cherry red-
colored complex indicates a positive result which basically means that they contain ketoses.
The absence of such color or the appearance of the color after a period of time indicates a
negative result which means that glucose, maltose and starch don’t have ketoses in them. Thus,
they are classified as aldohexose.

Unknown Solution Color Ketohexose/Aldohexose

A Dark Red Ketohexose
Pale Yellow

B approaching clearer Aldohexose

color
C Red Ketohexose
Approaching Clear
D Aldohexose
Color
Pale Orange Aldohexose

E approaching clearer

color

Discussion:
 For the Seliwanoff’s test of these 5 unknown solutions, only solutions A and C have
obtained a red-dark red color which indicates that there are ketoses present in them, thus
classifying them as ketohexose. In contrast, solutions B, D and E have corresponding pale
yellow and orange to clear color which indicates that they have tested negatively in this test
and that they do not contain ketones in them. Thus, they are aldohexose. These 5 unknown
solutions will be identified in the last table and match them with the known sugars.

E. IODINE TEST FOR POLYSACCHARIDES

Compound Color Polysaccharide (Yes/No)

Glucose Light Yellow No
Fructose Light Yellow No
Sucrose Light Yellow No
Maltose Light Yellow No
Dark Blue Yes
Starch
approaching Black

Discussion:

Iodine test is used to indicate the presence of polysaccharides. After the experiment, it was
found out that only starch resulted in a dark blue-black color which means that it tested
positively. The appearance of this color confirms the presence of polysaccharide in it. Since
starch is composed of a straight chain sub-unit called amylose, this amylose is responsible for
the reaction with iodine itself. The molecules of iodine become trapped within its helical
structure and form a complex. And this iodine will therefore cause a blue-black coloration with
starch once it detects amylose is present. Contrarily, glucose and fructose tested negatively due
to its light yellow color and the fact that these two are monosaccharides and not
polysaccharides. Similarly, sucrose and maltose were known to be negative in this test due to
its present of light yellow color and the fact that they are disaccharides and not
polysaccharides.
 Unknown Solution Color Polysaccharide (Yes/No)
A Light Yellow No
Dark Blue
B Yes
approaching Black
C Light Yellow No
D Light Yellow No
E Light Yellow No

Discussion:

For the results of the Iodine test of these 5 unknown solutions, it was found out that only
solution B had the color we were looking for which is dark blue-black which indicates that it
tested positively in this test and that it is a polysaccharide. On the other hand, the other four
solutions (A,C,D and E) had a light yellow appearance which meant that they are negative in
this test and therefore, they are not polysaccharides. The last table will be identifying these
unknown solutions and match them to the known sugars.

F. HYDROLYSIS OF DISACCHARIDE AND POLYSACCHARIDE

Direction: Write the color of solution observed on the following tests.
Compounds Benedict’s Test Iodine Test

Sucrose only Blue Yellow

Sucrose + HCl + NaOH Red Orange Yellow

Starch only Blue Deep Blue

Starch + HCl + NaOH Muddy Green Yellow

Discussion:
 Hydrolysis is a chemical reaction in which water is used to break down the bonds of a
particular substance. Four (4) compounds were experimented to see their results for the
Benedict’s and Iodine’s test. After the Benedict’s test was conducted, it was found out that only
the second one (sucrose + HCl + NaOH) tested positively which was evident in its red orange
color which indicates it has large amount of reducing sugars. The fourth one (Starch + HCl +
NaOH) had a muddy green color which meant that it has a bit of a traceable amount of reducing
sugar. The first (sucrose only) and the third (starch only) tested negatively for the benedict’s
test as unveiled in its blue-colored appearance which indicates they don’t have any reducing
sugars at all. For the Iodine test, only the third (starch only) one tested positively as depicted in
its deep blue-black-colored appearance which affirms that it is a polysaccharide. However, the
three (3) remaining solutions tested negatively as they had a yellow-colored appearance.

G. FERMENTATION TEST

Compound Observations Fermented (Yes/No)

Has a Cloudy

Appearance and
Water No
there are no bubbles

that formed
Has a clear color

and there’s a
Glucose Yes
formation of

bubbles
Has a clear color

Sucrose and there are no No

bubbles formed
Lactose Has a clear color No

and there are no

 bubbles formed
Has a cloudy

appearance and lots

Lactose + Lactaid Yes
of bubbles have

formed
Has a clear

appearance and

Fructose little amount of Yes

bubbles have

formed
Has a clear color No

and there are no

Starch
formation of

bubbles

Discussion:

Some sugars ferment in the presence of yeast, which possess the enzyme zymase. This
experiment will then inform us if the enzymes present in yeast are able to ferment the
carbohydrates. After this certain test was done, it was found out that only glucose and lactose
+ lactaid and fructose were the ones that tested positive for the test. To know whether or not
fermentation took place, one should look out for the formation of bubbles. These three had
some evident bubble formation and that is exactly why it was very obvious that fermentation
took place in these solutions. However, water, sucrose, lactose and starch tested negatively
since they didn’t have any bubble formation and had some clear and some cloudy-like
appearance.

H. TESTING AN UNKNOWN CARBOHYDRATE

Direction: Put (√) mark if the solution shows a positive result while (X) if not.
 Test A B C D E
Benedict’s X X √ √ √
Barfoed’s X X √ X √
Seliwanoff’s √ X √ X X
Iodine X √ X X X
Identification of Sucrose Starch Fructose Maltose Glucose

unknown

Discussion:
For the test results of unknown carbohydrates, solution A had negative results for Benedict’s,
Barfoed’s and Iodine test and a positive result for Seliwanoff’s test. I have identified solution A as
sucrose since sucrose had the same results as solution A for all these tests. Solution B, on the other
hand, had negative test results for Benedict’s, Barfoed’s and Seliwanoff’s test and had a positive
test result of the Iodine Test. That is exactly why I have identified solution B as starch since starch
obtained the same results like solution B did. Moving on, solution C gained positive results for
Benedict’s, Barfoed’s and Seliwanoff’s test while it only had a negative result in the Iodine test.
Due to that, I have made a clear identification of this solution as fructose since fructose had
identical results with solution C. Fourth on the table is solution D. For this solution, it only had a
positive result for the Benedict’s test while it had negative results for Barfoed’s, Seliwanoff’s and
Iodine test. These results are the reasons why I have chosen to identify solution D as maltose
because maltose had parallel results with solution D. The final unknown solution is solution E. For
this, I have identified it as glucose since I have observed that glucose also had positive results for
the Benedict’s and Barfoed’s test while having negative results for the Seliwanoff’s and Iodine test
just like what solution E had. These are the reasons I have considered to identify these unknown
solutions and match them with the known sugars.

III. CONCLUSION:
 Carbohydrates are essential in foods as an energy source (starch is the main source of
human calories), a flavouring (simple sugars are usually sweet) and as a functional ingredient
(sucrose allows ice cream to be soft in the freezer; xanthan gum thickens a low-fat salad
dressing). As with all our approaches to food ingredients/ constituents, we also need to
examine the structure of carbohydrates and then try to elucidate how their structures allow
them to function as they do. Carbohydrates are classified based on size of base carbon chain,
number of sugar units, location of their bonds and stereochemistry. Furthermore, there are
certain tests like Benedict’s test (for reducing sugars), Barfoed’s test (for monosaccharides),
Seliwanoff’s test (for ketohexoses), iodine test (for polysaccharides), hydrolysis of
disaccharides and polysaccharides, and fermentation test which we could perform to
differentiate carbohydrates from each other (either monosaccharide, disaccharide or
polysaccharide). We could also utilize these tests in order for us to identify unknown
carbohydrates and match them with those one which are already known. Carbohydrates, in
general, are important and vital for every being on this planet as without it, we could cease to
exist in this world for we cannot live without energy sources. Thus, let us give importance and
appreciate its essence and learn more about it and what it does for us.