Carbohydrate Analysis Experiments

CHAPTER I
PREFACE
A. Background of Experiment
Our body gets energy from three main classes of food: carbohydrates,
proteins, and fats. Of these classes, carbohydrates are the most important
source of energy and the primary source of calorie. When our body begins to
digesting food, carbohydrates is digested first.
Carbohydrates is the primary source of calories for our body, because 80%
calories that we needed is derived from carbohydrates. One gram of
carbohydrates contains 4.1 calories. (Saktiyono, 2004)
Carbohydrates are actually polyhydroxyaldehydes and polyhydroxyketones,
often but not always with the general formula
where n equals 3 or
more. We refer to polyhydroxyaldehydes as aldoses and polyhydroxyketones

as ketoses. (James and William, 1994)
Carbohydrates are divided into three general classes, depending on the
number of carbohydrate molecules they contain. Carbohydrates that contain
one carbohydrate molecules are classified as monosaccharides. Example of
monosaccharides are glucose and fructose. Carbohydrates that contain two
carbohydrate molecules are classified as disaccharides. Example of
disaccharides are sucrose and maltose. And carbohydrates that contain more
than six carbohydrate molecules are classified as polysaccharides. Example of
polisaccharides are amylum and cellulose.
There are some experiment for carbohydrates that is molisch test, benedict
test, seliwanoff test, and iodine test. The experiment is for presence of
carbohydrates
(monosaccharides,
disaccharides,
polysaccharides)
by
qualitative and quantitative analysis, indicates the presence of reducing sugar,

determining the presence of sugar that contain ketone group, and for presence
starch.
1
B. Stating Problem
1. Are there carbohydrates (monosaccharides, disaccharides, polysaccharides)
qualitively in each experiment materials (glucose, fructose, sucrose, lactose,
maltose, cellulose, amylum) by using molischs test?
2. Are there substance that can reduce alkali in each experiment materials
(glucose, fructose, sucrose, lactose, maltose, cellulose, amylum) and how to
distinguish saccharides (sugar) that can reduce and saccharides (sugar) that
can not reduce by using benedicts test?
3. Are there sugar that contains ketone group in each experiment materials
(glucose, fructose, sucrose, lactose, maltose, cellulose, amylum) by using
seliwanoffs test?
4. Is there polysaccharides (mainly amylum) in each experiment materials and
how to distinguish amylum and lactose by using iodines test?
5. Are there carbohydrates in guava (unripe guava, ripe guava, well-ripe
guava) by using molischs test, benedicts test, seliwanoffs test, and
iodines test?
C. Aims of Experiment
1. Identify the presence of carbohydrates (monosaccharides, disaccharides,
polysaccharides) by using molischs test.
2. Identify the presence of substance that can reduce alkali and can distinguish
saccharides (sugar) that can reduce and saccharides (sugar) that can not
reduce by using benedicts test.
3. Identify the presence of sugar that contains ketone group by using
seliwanoffs test.
4. Identify the presence of polysaccharides (mainly amylum) and can
distinguish amylum and lactose by using iodines test.
5. Identify the presence of carbohydrates in guava (unripe guava, ripe guava,
well-ripe guava) by using molischs test, benedicts test, seliwanoffs test,
and iodines test.
D. Benefits
1. College
student
can
identify
the
presence
of
carbohydrates
(monosaccharides, disaccharides, polysaccharides) by using molischs test.

2. College student can identify the presence of substance that can reduce alkali
and can distinguish saccharides (sugar) that can reduce and saccharides
(sugar) that can not reduce by using benedicts test.
3. College student can identify the presence of sugar that contains ketone
group by using seliwanoffs test.
4. College student can identify the presence of polysaccharides (mainly
amylum) and can distinguish amylum and lactose by using iodines test.
5. College student can identify the presence of carbohydrates in guava (unripe
guava, ripe guava, well-ripe guava) by using molischs test, benedicts test,
seliwanoffs test, and iodines test.
CHAPTER II
LITERATURE REVIEW
Name carbohydrate comes from the fact that most of the compounds of this
class have the empirical formula indicates that the compound is a carbon
"hydrate" and has a ratio of carbon to hydrogen and the oxygen as 1: 2: 1.
Although many carbohydrates generally in line with the empirical formula
, others do not exhibit this comparison and several others also contain
nitrogen, phosphorus, or sulfur. (Tatang, 2015)
Carbohydrates are a source of energy for the body, carbohydrates are widely
distributed
in
nature,
both
in
animal
tissue
and
plant
tissue.
Often water-soluble carbohydrates have a sweet taste and therefore are called
sugars. Another term for carbohydrate is saccharides.
Carbohydrates are actually polyhydroxyaldehydes and polyhydroxyketones,
often but not always with the general formula
where n equals 3 or more.
We refer to polyhydroxyaldehydes as aldoses and polyhydroxyketones as ketoses.

(James and William, 1994)
Aldoses: Possesing aldehyde
as functional group, e.g.
glyceraldehyde, glucose.
Ketoses:
Posessing
keto
as
functional
group,
e.g.
dihydroxyacetone, fructose. (Shivaraja, 2008)
Figure 1. Aldoses and Ketoses (Source: Tatang, 2015 )
Carbohydrates, depending on the number of carbohydrate molecules they

contain, they are:
1. Monosaccharides
2. Disaccharides
3. Polysaccharides
Monosaccharides
Monosaccharides are the simplest of carbohydrates and they cannot be
further hydrolyzed into smaller units. The most common monosaccharides of
biological importance are glucose, fructose, galactose, anda mannose. The
monosaccharides are classified into different categories, based on the functional
group and the number of carbon atoms. (Shivaraja, 2008)
Monosaccharides, or simple sugars, consist of a single polyhydroxy
aldehyde or ketone unit. The most abundant monosaccharide in nature is the sixcarbon sugar D-glucose, sometimes referred to as dextrose. Monosaccharides of
more than four carbons tend to have cyclic structures. (David and Michael)
Figure 2. Representative monosaccharides. Two common hexoxes (Source: David and

Michael )
Dissacharides
The aldehyde or ketone group on the anomeric carbon atom of one
monosaccharide can react with the hydroxyl group of a second monosaccharide to
5
form dissacharide. The covalent bond formed is called a glycosidic bond. (David
and Nigel, 2011)
Maltose or malt sugar occurs in germinating seeds and also formed during
digestion of starch by enzymes or by dilute acids. It is formed by the union of two
glucose units linked by , 1-4 glycosidic linkage. Maltose is a reducing
disaccharide, since it contains one free aldehyde group in its molecular structure.
It can be hydrolyzed either by acid or by the enzyme maltase of the intestinal juice.
(Shivaraja, 2008)
Lactose is present in the milk (milksugar). Lactose is formed by one
molecule of galactose and one molecule of glucose linked by , 1-4 glycosidic
linkage. It is a reducing disaccharide, since it contains one free aldehyde group in
its molecular structure. It can be hydrolyzed either by acid or by the enzyme
lactase of the intestinal juice. (Shivaraja, 2008)
Sucrose is the sweetening agent. Present in the sugar cane. Sucrose is
formed by one molecule of glucose and one molecule of fructose linked by , 12 glycosidic linkage. Sucrose is a non-redusing disaccharide, since the reducing
groups of glucose (aldehyde group) and fructose (keto group) are involved in the
formation of glycosidic bon. It can be hydrolyzed either by acod or by the enzyme
sucrase (invertase) of the intestinal juice. (Shivaraja, 2008)
Figure 3. Stucture of common disaccharides (Source: David and Nigel, 2011)
Polysaccharides
They are the macromolecular polymers of monosaccharide units linked by
glycosidic linkage with high molecular weight. Based on the type of
monosaccharide units present, the polysaccharides are further classified into
homoplysaccharides, and heteropolysaccharides. (Shivaraja, 2008)
Presence of carbohydrates can be confirmed qualitvely by several test. Due
to the presence of different numbers of sugar units specific carbohydrates exhibit
typical colour reactions that form the basis for their identification. In this kit the
following tests will be perforned for the qualitative analysis of carbohydrates:
Molischs Test
Molischs test is a general test for all carbohydrates, incluiding
monosaccharides, dissacharides and polysaccharides. The reagent contains
concentrated H2SO4, which hydrolyzes glycolisidic bonds present in a
disaccharide or a polysaccharide to yield monosaccharides, which in the presence
of an acid get dehydrated to form furfural and its derivates. In the case of hexoses,
hydroxymethyl fulfural, which is formed by the action of conc. H2SO4 on these
sugars, is very reactive, and it condenses with -naphthol to give a purple or violet
colored product. (Nigam, 2008)
Figure 4. Molischs reaction
Benedicts Test
This test is based on ther reducing properties of sugars which posses a keto
or an aldehyde group. The principle of this test is the same as that of Fehlings
test. However, in Benedicts test, the reduction reaction is carried out in weak
alkaline conditions in the presence of sodium carbonate, whereas the reduction
reaction in Fehlings test is brought about in strong alkaline conditions by using
KOH. The reagent used for Benedicts test contains
in an alkaline
citrate solution. Sugars with free aldehyde or ketone groups can reduce cupric ions
to insoluble yellow or red colored cuprous oxide, and in turn get oxidizes to sugar
acids. Sodium citrate present in the reagent acts as a chelating agent for metal
ions. (Nigam, 2008)
Figure 5. Benedicts reaction
In this test, a reducing sugar can be evidenced by the formation of red brick
precipitates. It depends on the concentration of reducing sugar contained in each
test solution. In the reducing sugars contained aldehyde group and OH lactol. OH
lactol OH group is bound to the C atom decisive first carbohydrates as reducing
sugars or not.
Seliwanoffs Test
Seliwanoffs test is used to distinguish between ketoses and aldoses. If a
sugar contains a ketone group it is called ketose and if it contains an aldehyde
group then it is called aldose. Here ketoses undergo dehydration to give furfural
derivates, which then condens with resorcinol to form a red-colored complex.

Prolonged heating can hydrolyze disaccharides, which may yield ketose
monosaccharides, eventually giving a colour. For instance, Seliwanoffs test is
responded positively by sucrose as it gets hydrolyzed to give glucose and fructose
due to the action of conc. HCl and NaOH present in the reagent.
Figure 6. Seliwanoffs reaction
Iodines Test
Iodine forms colored complexes with polysaccharides. The color of the complex
depends upon the three dimensional structure of the polysaccharide. Starch is a
coiled structure which turns blue when bound to Iodine whereas glycogen, which
is a branched molecule, turns red-violet.
CHAPTER III
DESIGN OF EXPERIMENT
A. Tools and Materials
1. Molisch Test
Tools :
Reaction tube
Pippete drops
Reaction tubes rack
Measure glass
2. Benedict Test
Tools :
Reaction tube
Pippete drops
Reaction tubes rack
Measure glass
Waterbath
Leg three
Burner methylated
Lighter
3. Seliwanoff Test
Tools :
Reaction tube
Pippete drops
Reaction tubes rack
Measure glass
Waterbath
Leg three
Burner methylated
Lighter
Stopwatch
4. Iodine Test
Tools :
Reaction tube
Pippete drops
Reaction tubes rack
Measure glass
Waterbath
Materials
H2SO4
Molisch reagent
1% Glucose solution
1% Fructose solution
1% Lactose solution
1% Sucrose solution
1% Maltose solution
1% Cellulose solution
1% Amylum solution
Materials
Benedict reagent
1% Glucose solution
1% Lactose solution
1% Sucrose solution
1% Maltose solution
1% Amylum solution
Materials
Seliwanoff reagent
1% Glucose solution
1% Lactose solution
1% Sucrose solution
1% Maltose solution
1% Amylum solution
Materials
Iodine solution
1% Amylum solution
NaOH 6 N
HCL 6 N
10
Leg three
Aquadest
Burner methylated
Lighter
5. Carbohydrates Experiment in Fruit (Guava)
Tools :
Materials
Reaction tube
Molisch reagent
Pippete drops
Benedict reagent
Reaction tubes rack
Seliwanoff reagent
Measure glass
Iodine solution
Waterbath
H2SO4
Leg three
NaOH 6 N
Burner methylated
HCl 6 N
Lighter
Aquadest
Stopwatch
Fruit (Guava) solution
B. Procedure
1. Molisch Test
1) Prepare all kinds of carbohydrates into a solution with a concentration
of 1%.
2) Put 2 ml 1% carbohydrates solution into different reaction tube.
3) Add 2-3 drops of molisch reagent, shake slowly about 5 second.
4) Oblique reaction tube, add 1 ml (20 drops) H2SO4 through the wall of
reaction tube.
5) Stand the reaction tube, observe there are any red-purple ring on
border of the two solution.
2. Benedict Test
of 1%.
2) Put 2 ml benedict reagent into reaction tube.
3) Add 5 drops of 1% glucose solution, then heat it in waterbath about 5
second, let it cool, and compare and write the colour changing.
4) Do this experiment toward the other 1% carbohydrates solution.
(1% Fructose solution, 1% Lactose solution, 1% Sucrose solution, 1%
Maltose solution, 1% Selulose solution, 1% Amylum solution)
3. Seliwanoff Test
of 1%.
2) Put 1 ml seliwanoff reagent into reaction tube.
3) Add 2 drops of 1% amylum solution, at the time heat it in waterbath
until formed colour. Then, write velocity of formed colour.
4) Do this experiment toward the other 1% carbohydrates solution.
11
(1% Fructose solution, 1% Lactose solution, 1% Sucrose solution, 1%

Maltose solution, 1% Selulose solution, 1% Amylum solution)
4. Iodine Test
1) Prepare 3 reaction tube. Then put 3 ml 1% amylum at each tube.
2) Add 2 drops of water in first tube. Add 2 drops of HCl in second tube.
Add 2 drops of NaOH in third tube. Shake all tube. Then observe the
colour changing.
3) Heat the tube that has coloured liquid, and let it cool. Then observe
the alteration.
4) Do this experiment toward 1% Selulose solution.
1) For molisch test, benedict test, and seliwanoff test the method is same
with previous experiment.
2) For iodine :
a) Put 1 drops of unripe, ripe, and well ripe guava into petri cup
b) Add 1 drop iodine solution to each petri cup. Then write the
colour changing.
C. Work Flow
1. Molisch Test
1% Glucose
1% Fructose
- put 2 ml 1% glucose into

reaction tube
- add 2-3 drops of molisch reagent
- shake it slowly about 5 second
- tilt the reaction tube
- add 1ml (20 drops) H2SO4
through the wall of tube reaction
- stand the reaction tube
- observed, are there any redpurple ring on border of the two
solution
- put 2 ml 1% fructose into

reaction tube
solution
Color Change
Color Change
12
1% Lactose
- put 2 ml 1% lactose into reaction
tube
solution
Color Change
1% Sucrose
- put 2 ml 1% sucrose into reaction
tube
solution
Color Change
1% Maltose
1% Selulose
- put 2 ml 1% maltose into

reaction tube
solution
- put 2 ml 1% cellulose into

reaction tube
solution
Color Change
Color Change
13
1% Amylum
- put 2 ml 1% amylum into reaction tube
- add 1ml (20 drops) H2SO4 through the wall of tube reaction
- observed, are there any red- purple ring on border of the two
solution
Color Change
2. Benedict Test
2 ml benedict reagent
- put into reaction tube

- add 5 drops of 1% glucose
solution
- heat it in waterbath about 5
minutes
- let it cool
- compare and write the color
changing

- add 5 drops of 1% fructose
solution
minutes
- let it cool
changing
Color Change
Color Change

- add 5 drops of 1% lactose
solution
minutes
- let it cool
changing

- add 5 drops of 1% sucrose
solution
minutes
- let it cool
changing
Color Change
Color Change
14

- add 5 drops of 1% maltose
solution
minutes
- let it cool
changing

- add 5 drops of 1% cellulose
solution
minutes
- let it cool
changing
Color Change
Color Change
- add 5 drops of 1% amylum
solution
minutes
- let it cool
changing
Color Change
3. Seliwanoff Test
1 ml seliwanoff reagent

- add 2 drops of 1% glucose
solution at the time heat it in
waterbath until formed color
- write velocity of formed
color

- add 2 drops of 1% fructose
color
Color Change
Color Change
15
- add 2 drops of 1% lactose
color
Color Change
- add 2 drops of 1% sucrose
color
Color Change

- add 2 drops of 1% maltose
color

- add 2 drops of 1% cellulose
color
Color Change
Color Change
- add 2 drops of 1% amylum
color
Color Change
16
4. Iodine Test
1% Amylum
- put 3 ml 1% amylum
into reaction tube
- add 2 drops of aquadest
- add 5 drops of iodine
reagent
- shake the tube
- observe color changing
- heat the colored liquid
- reobserve the alteration
Color Change
1% Amylum
into reaction tube
- add 2 drops of NaOH
reagent
- shake the tube
Color Change
1% Amylum
into reaction tube
- add 2 drops of HCl
reagent
- shake the tube
Color Change
1% Cellulose
- put 3 ml 1% cellulose
into reaction tube
- add 2 drops of NaOH
reagent
- shake the tube
Color Change
1% Cellulose
into reaction tube
- add 2 drops of aquadest
reagent
- shake the tube
- heat the colored liquid
- reobserve the alteration
Color Change
1% Cellulose
into reaction tube
- add 2 drops of HCl
reagent
- shake the tube
Color Change
17

a) Carbohydrates experiment with molisch reagent in guava
Unripe guava solution
Ripe guava solution
- put 2 ml unripe guava solution

into reaction tube
solution
Color Change
- put 2 ml ripe guava solution into

reaction tube
solution
Color Change
Well-ripe guava solution

- put 2 ml well-ripe guava solution
into reaction tube
solution
Color Change
18
b) Carbohydrates experiment with benedict reagent in guava


- add 5 drops of unripe guava
solution
minutes
- let it cool
changing

- add 5 drops of ripe guava
solution
minutes
- let it cool
changing
Color Change
Color Change
- add 5 drops of well-ripe
guava solution
minutes
- let it cool
changing
Color Change
c) Carbohydrates experiment with seliwanoff reagent in guava

- add 2 drops of unripe guava
color

- add 2 drops of ripe guava
color
Color Change
Color Change
19
- add 2 drops of well-ripe
guava solution at the time
heat it in waterbath until
formed color
color
Color Change
d) Carbohydrates experiment with iodine solution in guava
Unripe guava solution
- put 1 drops unripe guava
solution into petri cup
- add 1 drops iodine
solution
- observe and write the
color changing
Color Change
Ripe guava solution

- put 1 drops ripe guava
- add 1 drops iodine
solution
- observe and write the
color changing
Color Change
Well-ripe guava solution

- put 1 drops well-ripe guava
- add 1 drops iodine solution
- observe and write the color
changing
Color Change
20
CHAPTER VI
RESULT OF EXPERIMENT
A. Data of Experiment
Tabel 1. Observation Result of Molisch Test
No
Carbohydrates
Solution
Treatment
1.
1% Glucose
solution
2 ml 1% Glucose
solution +
Molisch Reagent
+ H2SO4
2.
1% Fructose
solution
2 ml 1% Fructose
solution +
Molisch Reagent
+ H2SO4
3.
1% Lactose
solution
2 ml 1% Lactose
solution +
Molisch Reagent
+ H2SO4
4.
1% Sucrose
solution
2 ml 1% Sucrose
solution +
Molisch Reagent
+ H2SO4
5.
1% Maltose
solution
2 ml 1% Maltose
solution +
Molisch Reagent
+ H2SO4
6.
1% Cellulose
solution
2 ml 1%
Cellulose solution
+ Molisch
Reagent + H2SO4
7.
1% Amylum
solution
2 ml 1% Amylum
solution +
Molisch Reagent
+ H2SO4
Observation Result
Before
Glucose = colorless
Molisch = dark-brown
Glucose+Molisch=
colorless and there are
dark-brown precipitates
Fructose = colorless
Fructose+Molisch=
Lactose = colorless
Lactose+Molisch=
Sucrose = colorless
Glucose+Molisch=
Maltose = colorless
Maltose+Molisch=
Cellulosee = white cloudy
Glucose+Molisch= turbid
and there are dark-brown
precipitates
Amylum = white cloudy
Glucose+Molisch= turbid
and there are dark-brown
precipitates
After
Ring(thickness):
+
Turbidity(color):
++
Ring(thickness):
+++
Turbidity(color):
+
Ring(thickness):
++
Turbidity(color):
++
Ring(thickness):
++++
Turbidity(color):
++++
Ring(thickness):
+++
Turbidity(color):
+++
Ring(thickness):
++++
Turbidity(color):
+++++
Ring(thickness):
++
Turbidity(color):
+++
21
Tabel 2. Observation Result of Benedict Test
No
Carbohydrates
Solution
Treatment
Observation Result
Before
After
Solutions color =
formed two color
layers
(top=colorless,
bottom-blue)
Precipitates = redthin precipitates
(+)
Solutions color =
Light blue
Precipitates =
Brick red
precipitates (+++)
Solutions color =
Blue
Precipitates = redthin precipitates
(+)
Solutions color =
Blue
Precipitates =
No precipitates
Solutions color =
formed three
layers (top=tosca,
bottom= blue)
Precipitates =
(++)
Solutions color =
Blue
Precipitates =
No precipitates
Solutions color =
Blue
Precipitates =
No precipitates
1.
1% Glucose
solution
2 ml Benedict
reagent + 5 drops
1% Glucose
solution
Benedict + Glucose=
Blue
After heated = formed
two layers
(top=colorless,
bottom=blue), no
precipitates
2.
1% Fructose
solution
2 ml Benedict
reagent + 5 drops
1% Fructose
solution
3.
1% Lactose
solution
2 ml Benedict
reagent + 5 drops
1% Lactose
solution
Benedict + Glucose=
Blue
After heated = Brick
red, formed
precipitates
Benedict + Glucose=
Blue
After heated =
Greenish blue
4.
1% Sucrose
solution
5.
1% Maltose
solution
2 ml Benedict
reagent + 5 drops
1% Sucrose
solution
2 ml Benedict
reagent + 5 drops
1% Maltose
solution
6.
1% Cellulose
solution
7.
1% Amylum
solution
2 ml Benedict
reagent + 5 drops
1% Cellulose
solution
2 ml Benedict
reagent + 5 drops
1% Amylum
solution
Benedict + Glucose=
Blue
After heated = Blue
Benedict + Glucose=
Blue
After heated = formed
three layers (top=red,
middle=greenish blue
bottom= dark blue)
Benedict + Glucose=
Blue
After heated = Blue,
no precipitates
Benedict + Glucose=
Blue
After heated = Blue,
no precipitates
22
Tabel 3. Observation Result of Seliwanoff Test
No
Carbohydrates
Solution
1.
1% Glucose
solution
2.
1% Fructose
solution
3.
1% Lactose
solution
4.
1% Sucrose
solution
5.
1% Maltose
solution
6.
1% Cellulose
solution
7.
1% Amylum
solution
Treatment
1 ml Seliwanoff
reagent + 2 drops
1% Glucose
solution
heated
1 ml Seliwanoff
reagent + 2 drops
1% Fructose
solution
heated
1 ml Seliwanoff
reagent + 2 drops
1% Lactose
solution
heated
1 ml Seliwanoff
reagent + 2 drops
1% Sucrose
solution
heated
1 ml Seliwanoff
reagent + 2 drops
1% Maltose
solution
heated
1 ml Seliwanoff
reagent + 2 drops
1% Cellulose
solution
heated
1 ml Seliwanoff
reagent + 2 drops
1% Amylum
solution
heated
Observation Result
Before
After
Time
Seliwanoff reagent
= colorless
Glucose = colorless
Pale
yellow
(++)
21.33
minutes
Seliwanoff reagent
= colorless
Fructose =
colorless
Orange
(+++)
6.22
minutes
Seliwanoff reagent
= colorless
Lactose = colorless
Pale
yellow
(+++)
22.52
minutes
Seliwanoff reagent
= colorless
Sucrose = colorless
Orange
(++)
9.59
minutes
Seliwanoff reagent
= colorless
Maltose = colorless
Pale
yellow
(++++)
15.30
minutes
Seliwanoff reagent
= colorless
Celullose = turbid
Pale
yellow
(+++++)
22.55
minutes
Seliwanoff reagent
= colorless
Amylum = turbid
Pale
yellow
(+)
24.45
minutes
23
Tabel 4. Observation Result of Iodine Test
No
Carbohydrates
Solution
1.
1% Amylum
solution
2.
1% Amylum
solution
3.
1% Amylum
solution
4.
1% Cellulose
solution
5.
1% Cellulose
solution
6.
1% Cellulose
solution
Treatment
3 ml Amylum
solution + 2 drops
Aquadest + 5 drops
iodine reagent
Colored liquid
heated
3 ml Amylum
solution + 2 drops
HCl + 5 drops
iodine reagent
heated
3 ml Amylum
solution + 2 drops
NaOH + 5 drops
iodine reagent
heated
3 ml Cellulose
solution + 2 drops
Aquadest + 5 drops
iodine reagent
Colored liquid
heated
3 ml Cellulose
solution + 2 drops
HCl + 5 drops
iodine reagent
heated
3 ml Amylum
solution + 2 drops
NaOH + 5 drops
iodine reagent
heated
Observation Result
Before
After
Amylum = turbid
Amylum + Aquadest =
turbid
Amylum + Aquadest +
Iodine = Blackish green
(+++)
Amylum = turbid
Amylum + HCl = turbid
Amylum + HCl +
Iodine = Blackish green
(++)
Amylum = turbid (+)
Amylum + NaOH =
turbid (++)
Amylum + NaOH +
Iodine = turbid (+++)
Cellulose = colorless
Cellulose + Aquadest =
colorless
Cellulose + Aquadest +
Iodine = Blackish
purple (+++)
Cellulose + HCl =
feculent white
Cellulose + HCl +
Iodine = Blackish
purple (++)
Amylum + NaOH =
colorless
Amylum + NaOH +
Iodine = colorless
Blackish blue
Blackish blue
Colorless
Blackish blue
Blackish blue
Colorless
24
Tabel 5. Observation Result of Carbohydrates Experiment in Guava
No
Observation Result
Procedure
Before
1.
Molisch
experiment
Unripe guava
Ripe guava
Well-ripe guava
2.
Benedict
experiment
Unripe guava
Ripe guava
Well-ripe guava
3.
Seliwanoff
experiment
Unripe guava
Ripe guava
Well-ripe guava
4.
Iodine
experiment
Unripe guava
Ripe guava
Well-ripe guava
Unripe guava =
Light green
Ripe guava =
Turbid green (+)
Well-ripe guava =
Turbid green (++)
Unripe guava =
Light green
Ripe guava =
turbid green (+)
Well-ripe guava =
turbid green (++)
Unripe guava =
Light green
Ripe guava =
turbid green (+)
Well-ripe guava =
turbid green (++)
Unripe guava =
Light green
Ripe guava =
turbid green (+)
Well-ripe guava =
turbid green (++)
After
Unripe guava =
Solution color: top = turbid yellow (++),
bottom = purple (+)
Ring : Thick red (+)
Ripe guava =
Solution color: top = turbid yellow (+),
bottom = purple (++)
Ring : Red (+)
Well-ripe guava =
Solution color: top = red, middle =
turbid yellow (+), bottom = reddish
white
Ring : Thick red (+++)
Unripe guava =
Solution color : dark red (+++)
Precipitates : (+)
Ripe guava =
Solution color : dark red (++)
Precipitates : (++)
Well-ripe guava =
Solution color : dark red (+)
Precipitates : (+++)
Unripe guava =
Pink (+++)
Ripe guava =
Pink (++)
Well-ripe guava =
Pink (+)
Unripe guava =
Yellow (++)
Ripe guava =
Yellow (+)
Well-ripe guava =
Yellow (+++)
25
B. Data Analyzing
1. Molischs test
In molisch test there are seven experiment materials, there are 1% Glucose
solution,1% Fructose solution, 1% Lactose solution, 1% Sucrose solution, 1%
Maltose solution, 1% Selulose solution, 1% Amylum solution.
First material is Glucose. 2 ml 1% glucose solution added molisch reagent
show solution color is clear and there are dark-brown precipitates. Then the
solution added H2SO4 there are purplish-black ring (++) with thickness (+).
Second material is Fructose. 2 ml 1% fructose solution added molisch
reagent show solution color is clear and there are dark-brown precipitates.
Then the solution added H2SO4 there are purplish-black ring (+) with
thickness (+++).
Third material is Lactose. 2 ml 1% lactose solution added molisch reagent
solution added H2SO4 there are purplish-black ring (++) with thickness (++).
Fourth material is Sucrose. 2 ml 1% sucrose solution added molisch reagent
solution added H2SO4 there are purplish-black ring (++++) with thickness
(++++).
Fifth material is Maltose. 2 ml 1% maltose solution added molisch reagent
solution added H2SO4 there are purplish-black ring (+++) with thickness
(+++).
Sixth material is Cellulose. 2 ml 1% cellulose solution added molisch
reagent show solution color is turbid and there are dark-brown precipitates.
Then the solution added H2SO4 there are purplish-black ring (+++++) with
thickness (++++).
26
Seventh material is Amylum. 2 ml 1% amylum solution added molisch

reagent show solution color is turbid and there are dark-brown precipitates.
Then the solution added H2SO4 there are purplish-black ring (++) with
thickness (+++).
This experiment showed that seven experiment materials 1% Glucose
Maltose solution, 1% Selulose solution, 1% Amylum solution are containing or
presence of carbohydrate, but there are different thickness of purplish-black
ring in each experiment materials, that is show carbohydrates content in each
experiment materials is different. In this experiment is show that carbohydrate
content in sucrose is more than others. Sucrose is disaccharides that composed
of glucose and fructose. So thats why sucrose can condense directly with
molisch reagent without hydrolysis and the others must go through a stage of
dehydration.
2. Benedicts test
In benedict test there are seven experiment materials, there are 1% Glucose
Maltose solution, 1% Selulose solution, 1% Amylum solution.
First material is Glucose. 2 ml benedict reagent added 5 drops of 1%
glucose solution show the solution color is blue. Then the solution heated
solution color formed two layers (top=colorless, bottom=blue), and no
precipitates. When the solution is cool the color is not change but are there
thin-red precipitates (+).
Second material is Fructose. 2 ml benedict reagent added 5 drops of 1%
fructose solution show the solution color is blue. Then the solution heated
solution color changes into brick red and formed precipitates. When the
solution is cool solution color changes into light blue and formed brick red
precipitates (+++).
27
Third material is Lactose. 2 ml benedict reagent added 5 drops of 1% lactose

solution show the solution color is blue. Then the solution heated solution color
changes into greenish blue. When the solution is cool solution color changes
into blue and formed thin-red precipitates (+).
Fourth material is Sucrose. 2 ml benedict reagent added 5 drops of 1%
sucrose solution show the solution color is blue. Then the solution heated
solution color is not change. When the solution is cool the color is not change
and there is no precipitates.
Fifth material is Maltose. 2 ml benedict reagent added 5 drops of 1%
maltose solution show the solution color is blue. Then the solution heated
solution
color
formed
three
layers
(top=red,
middle=greenish-blue,
bottom=dark-blue). When the solution is cool formed three color layers

(top=tosca, bottom=blue) and there is red precipitates.
Sixth material is Cellulose. 2 ml benedict reagent added 5 drops of 1%
cellulose solution show the solution color is blue. Then the solution heated
solution color is not change and there no precipitates. When the solution is cool
the color is not change and there is no precipitates.
Seventh material is Amylum. 2 ml benedict reagent added 5 drops of 1%
amylum solution show the solution color is blue. Then the solution heated
solution color is not change and there no precipitates. When the solution is cool
the color is not change and there is no precipitates.
Positive test result is indicated by fructose, glucose, maltose, and lactose is
characterized by the deposition of red brick on a test benedict, whereas the
carbohydrate type of sucrose and starch showed a negative result is marked in
blue. Even aldoses or ketoses are in the form of cyclic, but this form are in
equilibrium with small amounts of aldehydes or ketones open chain, so the
group aldehyde or ketone can reduce a wide range of reducing agents,
therefore, carbohydrate which showed a positive reaction called reducing
sugars. Sucrose, although composed of glucose and fructose, but both anomeric
28
carbon atoms bound to each other, so that on each monosaccharide units no

longer have an aldehyde group or a ketone to mutarotation be open chain, it
causes no sucrose can reduce reagent benedict. In starch, even if there are
open-chain glucose at the end of the polymer chain, but its concentration is
very small, so that the color of the reaction did not appear by sight. (Hamdan,
2007)
3. Seliwanoffs test
In seliwanoff test there are seven experiment materials, there are 1%
Glucose solution,1% Fructose solution, 1% Lactose solution, 1% Sucrose
solution, 1% Maltose solution, 1% Selulose solution, 1% Amylum solution.
First material is Glucose. 1 ml seliwanoff reagent added 2 drops of 1%
glucose solution. Then the solution heated, at 21 minutes 33 second solution
color changes into pale yellow (++).
Second material is Fructose. 1 ml seliwanoff reagent added 2 drops of 1%
fructose solution. Then the solution heated, at 6 minutes 22 second solution
color changes into orange (+++).
Third material is Lactose. 1 ml seliwanoff reagent added 2 drops of 1%
lactose solution. Then the solution heated, at 22 minutes 52 second solution
color changes into pale yellow (+++).
Fourth material is Sucrose. 1 ml seliwanoff reagent added 2 drops of 1%
sucrose solution. Then the solution heated, at 9 minutes 59 second solution
color changes into orange (++).
Fifth material is Maltose. 1 ml seliwanoff reagent added 2 drops of 1%
maltose solution. Then the solution heated, at 15 minutes 30 second solution
color changes into pale yellow (++++).
29
Sixth material is Cellulose. 1 ml seliwanoff reagent added 2 drops of 1%

cellulose solution. Then the solution heated, at 22 minutes 55 second solution
color changes into pale yellow (++++).
Seventh material is Amylum. 1 ml seliwanoff reagent added 2 drops of 1%
amylum solution. Then the solution heated, at 24 minutes 45 second solution
color changes into pale yellow (+).
Selliwanoff test positive only on karbohidarat containing monosaccharides
with number 6 C atoms called hexoses and contains a ketone group. This
experiment showed that sucrose and fructose are containing ketose, that is
showed by solution color is changing into orange. While, amylum, glucose,
lactose, cellulose, and maltose are indicates negative result, because they are
not containing ketose.
4. Iodines test
In seliwanoff test there are two experiment materials, there are 1% Selulose
solution and 1% Amylum solution with three different treatment for each
materials.
First. 3 ml of 1% amylum solution added 2 drops of aquadest added 5 drops
of iodine reagent show solution color is blackish green (+++). Then the
solution heated solution color changes into blackish blue.
Second. 3 ml of 1% amylum solution added 2 drops of HCl added 5 drops
of iodine reagent show solution color is blackish green (++). Then the solution
heated solution color changes into blackish blue.
Third. 3 ml of 1% amylum solution added 2 drops of NaOH added 5 drops
of iodine reagent show solution color is feculent white
(+++). Then the
solution heated solution color changes into colorless.
30
Fourth. 3 ml of 1% cellulose solution added 2 drops of aquadest added 5

drops of iodine reagent show solution color is blackish purple (+++). Then the
solution heated solution color changes into blackish blue.
Fifth. 3 ml of 1% cellulose solution added 2 drops of HCl added 5 drops of
iodine reagent show solution color is blackish puple (++). Then the solution
heated solution color changes into blackish blue.
Sixth. 3 ml of 1% amylum solution added 2 drops of NaOH added 5 drops
of iodine reagent show solution color is colorless. Then the solution heated
solution color is colorless.
Iodine test aims to identify polysaccharides. Polysaccharides generally form
helical chains (circular), which can bind with iodine. This experiment show
that amylum and cellulose can react with aquadest and HCl, that is showed by
color changing in the solutions. While, amylum and cellulose does not react
with NaOH.
NaOH is inhibit a reaction between starch with iodine. This is because the
iodine reacts with a base so did not experience a reaction with the starch. This
situation occurs because the existing NaOH solution first reacts with iodine to
form compounds NaI and Naoi, so the test with the addition of NaOH have
been no changes in the starch solution. (Anonym, 2011)
5. Carbohydrate experiment in fruit (guava)
Molischs Test
Unripe guava. 2 ml 1% unripe guava solution added molisch reagent show
solution color is light green. Then the solution added H2SO4 solution color on
the top is turbid yellow (++), on the bottom is purple (+) and there is red ring
with thickness (+).
Ripe guava. 2 ml 1% ripe guava solution added molisch reagent show
solution color is turbid green (+). Then the solution added H2SO4 solution
31
color on the top is turbid yellow (+), on the bottom is purple (++) and there is
red ring (+).
Well-ripe guava. 2 ml 1% well-ripe guava solution added molisch reagent
show solution color is turbid green (++). Then the solution added H2SO4
solution color on the top is red, the middle is turbid yellow (+), on the bottom
is reddish white and there is red ring with thickness (+++).
Benedicts Test
Unripe guava. 2 ml benedict reagent added 5 drops of unripe guava solution
show the solution color is light green. Then after treatment the solution color
changes into dark red (+++) and there are precipitates (+).
Ripe guava. 2 ml benedict reagent added 5 drops of ripe guava solution
show the solution color is turbid green. Then after treatment the solution color
changes into dark red (++) and there are precipitates (++).
Well-ripe guava. 2 ml benedict reagent added 5 drops of well-ripe guava
solution show the solution color is turbid green. Then after treatment the
solution color changes into dark red (+) and there are precipitates (+++).
Seliwanoffs Test
Unripe guava. 1 ml seliwanoff reagent added 2 drops of unripe guava
solution show solution color is light green. Then the solution heated solution
color changes into pink (+++).
Ripe guava. 1 ml seliwanoff reagent added 2 drops of ripe guava solution
show solution color is turbid green (+). Then the solution heated solution color
changes into pink (++).
Well-ripe guava. 1 ml seliwanoff reagent added 2 drops of well-ripe guava
solution show solution color is turbid green (++). Then the solution heated
solution color changes into pink (+).
32
Iodines Test
Unripe guava. 3 ml of unripe guava solution (light green) added 1 drops of
iodine reagent then the solution color changes into yellow (+).
Ripe guava. 3 ml of ripe guava solution (turbid green) added 1 drops of
iodine reagent then the solution color changes into pale yellow.
Well-ripe guava. 3 ml of well-ripe guava solution (turbid green) added 1
drops of iodine reagent then the solution color changes into yellow (++).
In molisch experiment show that unripe, ripe, and well-ripe guava solution
are containing carbohydrates but the content of carbohydrates is different in
each solution. In benedict experiment show that unripe, ripe, and well-ripe
guava solution have reducing sugar, and the reducing sugar in each solution is
fructose. In seliwanoff experiment show that unripe, ripe, and well-ripe guava
solution have ketone group in their solution because they are containing of
fructose. In iodine experiment show that unripe, ripe, and well-ripe guava
solution show negative result.
C. Discussion Of Questions
Molischs Test
1. Why the red-purple ring is formed in the material containing
carbohydrates?
Red-purple ring formed caused by dehydration of concentrated sulfuric
acid (H2SO4) to carbohydrates.
2. Is it the same the color intensity of the red-puple ring on the

experiment materials used in this experiment? Explain!
No, because the color intensity of the red-purple ring on the experiment
materials it depends on the amount of carbohydrates content.
33
Benedicts Test
1. What the color of the precipitates formed? Why?
Brick red precipitates. Alkaline copper solution which will be reduced by
having a sugar-free aldehyde or ketone group to form a colored
cuprooksida.
2. In benedicts test why sucrose not include a reducing sugar?
Sucrose, although composed of glucose and fructose, but both anomeric
carbon atoms bound to each other, so that on each monosaccharide units
no longer have an aldehyde group or a ketone to mutarotaion be open
chain, it causes no sucrose can reduce reagent benedict. In starch, even if
there are open-chain glucose at the end of the polymer chain, but its
concentration is very small, so that the color of the reaction did not appear
by sight. (Hamdan, 2007)
Seliwanoffs Test
1. What group of carbohydrates that give a positive reaction to
seliwanoff's test? Why?
Ketose. Seliwanoff's test positive only on a carbohydrate-containing
monosaccharides with number 6 C atoms called hexoses and contains a
ketone group.
2. Can seliwanoff's test can be used to distinguish sucrose from fructose?
Yes, it can. Because in seliwanoffs test sucrose hydrolyzed into glucose
and fructose.
Iodines Test
1.
Why there is color changing after heating?

Because the heating serves to accelerate the reaction. Then after heating
the reaction will appear, then there was a color changing.
2. Which substances except amylum that gives color with iodine?

Cellulose
34
Carbohydrates Experiment in Fruit

1. Why the well-ripe fruit still found their carbohydrates in the form of
polysaccharides?
At the well-ripe fruit still found their carbohydrates in the form of
polysaccharides, is due to the well-ripe fruit is not found one of the
enzymes that break down polysaccharides into monosaccharides. therefore
carbohydrate is in the form of polysaccharides. Enzymatic hydrolysis of
starch is important in the digestion process.
2. Describe the process of enzymatic hydrolysis of starch!
Amylose hydrolysis by a-amylase occurs in two stages. The first stage is
degraded into maltose and maltotriosa that occurs randomly. This
degradation occurs rapidly followed by decreasing the viscosity quickly.
The second phase is relatively slow with the formation of glucose and
maltose as the final result. As for amylopectin, a-amylase hydrolysis to
produce glucose, maltose and various types of a-limit dextrins which are
oligosaccharides consisting of 4 or more sugar residues which all contain
a-1,6 glycosidic bond. (Suhartono, 1989)
35
CHAPTER V
ENCLOSURE
A. Summary
Carbohydrates are divided into three general classes (monosaccharides,

disaccharides, polysaccharides).
Glucose, fructose, maltose, sucrose, lactose, amylum, and cellulose are

containing carbohydrate.
There are reducing sugar on carbohydrate, they are glucose, fructose,

lactose, and maltose. They can reuce the alkaline atmosphere.
Sucrose and fructose have ketone group in their solution. And the
others(glucose, maltose, sucrose, lactose) have aldose group and both of
them.
Amylum and cellulose are containing polysaccharides.
The result of experiment about guava show that guava containing fructose,
and there are ketone group, that can reduce the alkaline atmosphere.
B. Suggestion
For college student and all residents anyone who using biochemistry lab in
biology departement must take care of materials/tools in biochemistry
laboratory. Like test tube, pippete, meassure glass, etc. After using
materials/tools must cleaned up and return materials/tools in the right place.
36
REFFERENCES
Julianto, Tatang S. 2015. Biokimia: Biomolekul dalam Prespektif Al-Quran.
Yogyakarta: Deepublish
Shankara, Shivaraja. 2008. Laboratory Manual for Practical Biochemistry. New
Delhi: Jaypee Brothers Medical Publisher
Hames, David and Nigel Hooper. 2011. Biochemistry. New York: Garland
Science
Nigam, Arti and Archana Ayyagari. 2008. Lab Manual in Biochemistry:
Immunology and Biotechnology. New Delhi: Tata McGraw-Hill Publishing
Company Limited
37
APENDIX
EXPERIMENT
PICTURE
EXPLANATION
Sucrose and Fructose

(sucrose or fructose +
molisch reagent +
H2SO4)
Molischs Test
Amylum and Glucose

(amylum or glucose +
molisch reagent +
H2SO4)
Lactose, Maltose and

Sellulose
(lactose or maltose or
sellulose + molisch
reagent + H2SO4)
38
Amylum
(amylum in cool
condition after
treatment)
Glucose
(glucose in cool
condition after
treatment)
Fructose
(fructose in cool
condition after
treatment)
Benedicts Test
Lactose
(lactose in cool
condition after
treatment)
Maltose
(malose in cool
condition after
treatment)
Sellulose
(sellulose in cool
condition after
treatment)
Sucrose
(sucrose in cool
condition after
treatment)
39
Amylum
(amylum after
treatment)
Fructose
(fructose after
treatment)
Glucose
(glucose after
treatment)
Seliwanoffs
Test
Lactose
(lactose after
treatment)
Maltose
(malose after
treatment)
Sellulose
(sellulose after
treatment)
Sucrose
(sucrose after
treatment)
40
Amylum
(amylum + aquadest +
iodine reagent)
Amylum
(amylum + HCl +
iodine reagent)
Amylum
(amylum + NaOH +
iodine reagent)
Iodines Test
Sellulose
(sellulose + aquadest +
iodine reagent)
Sellulose
(sellulose + HCl +
iodine reagent)
Sellulose
(sellulose + NaOH +
iodine reagent)
41
Molischs Test
Carbohydrate
Experiment in
Guava
Benedicts Test
Selliwanofs Test
Iodines Test
42

Carbohydrate Analysis Experiments

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CHAPTER I

more. We refer to polyhydroxyaldehydes as aldoses and polyhydroxyketones

qualitative and quantitative analysis, indicates the presence of reducing sugar,

(monosaccharides, disaccharides, polysaccharides) by using molischs test.

where n equals 3 or more.

We refer to polyhydroxyaldehydes as aldoses and polyhydroxyketones as ketoses.

as functional group, e.g.

dihydroxyacetone, fructose. (Shivaraja, 2008)

Figure 1. Aldoses and Ketoses (Source: Tatang, 2015 )

Carbohydrates, depending on the number of carbohydrate molecules they

Figure 2. Representative monosaccharides. Two common hexoxes (Source: David and

Figure 3. Stucture of common disaccharides (Source: David and Nigel, 2011)

Figure 4. Molischs reaction

Figure 5. Benedicts reaction

derivates, which then condens with resorcinol to form a red-colored complex.

Figure 6. Seliwanoffs reaction

(1% Fructose solution, 1% Lactose solution, 1% Sucrose solution, 1%

- put 2 ml 1% glucose into

- put 2 ml 1% fructose into

- put 2 ml 1% maltose into

- put 2 ml 1% cellulose into

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

5. Carbohydrates Experiment in Fruit (Guava)

Ripe guava solution

- put 2 ml unripe guava solution

- put 2 ml ripe guava solution into

Well-ripe guava solution

b) Carbohydrates experiment with benedict reagent in guava

- put into reaction tube

- put into reaction tube

- put into reaction tube

- put into reaction tube

Ripe guava solution

Well-ripe guava solution

Tabel 2. Observation Result of Benedict Test

Tabel 3. Observation Result of Seliwanoff Test

Tabel 4. Observation Result of Iodine Test

Tabel 5. Observation Result of Carbohydrates Experiment in Guava

Seventh material is Amylum. 2 ml 1% amylum solution added molisch

Third material is Lactose. 2 ml benedict reagent added 5 drops of 1% lactose

bottom=dark-blue). When the solution is cool formed three color layers

carbon atoms bound to each other, so that on each monosaccharide units no

Sixth material is Cellulose. 1 ml seliwanoff reagent added 2 drops of 1%

(+++). Then the

solution heated solution color changes into colorless.

Fourth. 3 ml of 1% cellulose solution added 2 drops of aquadest added 5

2. Is it the same the color intensity of the red-puple ring on the

Why there is color changing after heating?

2. Which substances except amylum that gives color with iodine?

Carbohydrates Experiment in Fruit

Carbohydrates are divided into three general classes (monosaccharides,

Glucose, fructose, maltose, sucrose, lactose, amylum, and cellulose are

There are reducing sugar on carbohydrate, they are glucose, fructose,