Carbohydrate Experiment

8/18/2019 Carbohydrate Experiment

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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.


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B. Stating Problem

1. Are there carbohydrates (monosaccharides, disaccharides, polysaccharides)

qualitively in each experiment materials (glucose, fructose, sucrose, lactose,

maltose, cellulose, amylum) by using molisch’s 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 benedict’s test?

3. Are there sugar that contains ketone group in each experiment materials

(glucose, fructose, sucrose, lactose, maltose, cellulose, amylum) by using

seliwanoff’s test?

4. Is there polysaccharides (mainly amylum) in each experiment materials and

how to distinguish amylum and lactose by using iodine’s test?

5. Are there carbohydrates in guava (unripe guava, ripe guava, well-ripe

guava) by using molisch’s test, benedict’s test, seliwanoff’s test, and

iodine’s test?

C. Aims of Experiment

1.

Identify the presence of carbohydrates (monosaccharides, disaccharides,

polysaccharides) by using molisch’s 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 benedict’s test.

3.

Identify the presence of sugar that contains ketone group by using

seliwanoff’s test.

4. Identify the presence of polysaccharides (mainly amylum) and can

distinguish amylum and lactose by using iodine’s test.

5. Identify the presence of carbohydrates in guava (unripe guava, ripe guava,

well-ripe guava) by using molisch’s test, benedict’s test, seliwanoff’s test,

and iodine’s test.


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D. Benefits

1. College student can identify the presence of carbohydrates

(monosaccharides, disaccharides, polysaccharides) by using molisch’s 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 benedict’s test.

3.

College student can identify the presence of sugar that contains ketone

group by using seliwanoff’s test.

4. College student can identify the presence of polysaccharides (mainly

amylum) and can distinguish amylum and lactose by using iodine’s test.

5. College student can identify the presence of carbohydrates in guava (unripe

guava, ripe guava, well-ripe guava) by using molisch’s test, benedict’s test,

seliwanoff’s test, and iodine’s test.


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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 )


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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 six-

carbon 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


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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 α, 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 β, 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 α β, 1 -2 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)


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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:

Molisch’s Test

Molisch’s 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 α-naphthol to give a purple or violet

colored product. (Nigam, 2008)

Figure 4. Molisch’s reaction


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Benedict’s 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 Fehling’s

test. However, in Benedict’s test, the reduction reaction is carried out in weak

alkaline conditions in the presence of sodium carbonate, whereas the reduction

reaction in Fehling’s test is brought about in strong alkaline conditions by using

KOH. The reagent used for Benedict’s 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. Benedict’s 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.

Seliwanoff’s Test

Seliwanoff’s 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


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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, Seliwanoff’s 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. Seliwanoff’s reaction

Iodine’s 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.


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CHAPTER III

DESIGN OF EXPERIMENT

A. Tools and Materials

1. Molisch TestTools : Materials

Reaction tube H2SO4

Pippete drops Molisch reagent

Reaction tube’s rack 1% Glucose solution

Measure glass 1% Fructose solution

1% Lactose solution

1% Sucrose solution

1% Maltose solution

1% Cellulose solution

1% Amylum solution

2. Benedict Test

Tools : Materials

Reaction tube Benedict reagent

Pippete drops 1% Glucose solution

Reaction tube’s rack 1% Fructose solution

Measure glass 1% Lactose solution

Waterbath 1% Sucrose solution

Leg three

1% Maltose solution Burner methylated 1% Cellulose solution

Lighter 1% Amylum solution

3. Seliwanoff TestTools : Materials

Reaction tube Seliwanoff reagent

Pippete drops 1% Glucose solution

Reaction tube’s rack 1% Fructose solution

Measure glass 1% Lactose solution

Waterbath 1% Sucrose solution

Leg three

1% Maltose solution Burner methylated 1% Cellulose solution

Lighter 1% Amylum solution

Stopwatch

4. Iodine TestTools : Materials

Reaction tube Iodine solution

Pippete drops 1% Amylum solution

Reaction tube’s rack 1% Cellulose solution

Measure glass NaOH 6 N

Waterbath

HCL 6 N


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Leg three Aquadest

Burner methylated

Lighter5. Carbohydrates Experiment in Fruit (Guava)

Tools : Materials

Reaction tube Molisch reagent

Pippete drops Benedict reagent

Reaction tube’s 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 ofreaction 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.


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(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.

5. Carbohydrates Experiment in Fruit (Guava)

1)

For molisch test, benedict test, and seliwanoff test the method is samewith 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% Fructose

- put 2 ml 1% fructose 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 red-

purple ring on border of the two

solution

Color Change

1% Glucose

- put 2 ml 1% glucose into

reaction tube









solution

Color Change


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1% Selulose

- put 2 ml 1% cellulose into

reaction tube









solution

Color Change

1% Maltose

- put 2 ml 1% maltose into

reaction tube









solution

Color Change

1% Sucrose

- put 2 ml 1% sucrose into reactiontube








purple ring on border of the twosolution

Color Change

1% Lactose

- put 2 ml 1% lactose into reaction

tube









solution

Color Change


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2. Benedict Test

- put into reaction tube

- add 5 drops of 1% sucrose

solution

- heat it in waterbath about 5

minutes

- let it cool

- compare and write the color

changing

Color Change

2 ml benedict reagent


- add 5 drops of 1% lactose

solution


minutes

- let it cool


changing

Color Change



- add 5 drops of 1% fructose

solution


minutes

- let it cool


changing

Color Change



- add 5 drops of 1% glucose

solution


minutes

- let it cool


changing

Color Change


-

put 2 ml 1% amylum into reaction tube- add 2-3 drops of molisch reagent



- add 1ml (±20 drops) H2SO4 through the wall of tube reaction


- observed, are there any red- purple ring on border of the two

solution

1% Amylum

Color Change


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3. Seliwanoff Test


- add 2 drops of 1% fructose

solution at the time heat it in

waterbath until formed color

- write velocity of formed

color

1 ml seliwanoff reagent

Color Change


- add 2 drops of 1% glucose




color


Color Change


- add 5 drops of 1% amylum

solution


minutes- let it cool


changing

Color Change



- add 5 drops of 1% cellulose

solution


minutes

- let it cool


changing

Color Change



- add 5 drops of 1% maltose

solution


minutes

- let it cool


changing

Color Change



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- add 2 drops of 1% amylum




color


Color Change


- add 2 drops of 1% cellulose




color


Color Change


- add 2 drops of 1% maltose




color


Color Change


- add 2 drops of 1% sucrose




color


Color Change


- add 2 drops of 1% lactose




color


Color Chan e


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4. Iodine Test

- put 3 ml 1% cellulose

into reaction tube

- add 2 drops of HCl

- add 5 drops of iodine

reagent

- shake the tube

- observe color changing

1% Cellulose

Color Change


into reaction tube- add 2 drops of aquadest


reagent

- shake the tube


- heat the colored liquid

- reobserve the alteration

1% Cellulose

Color Change


into reaction tube

- add 2 drops of NaOH

- add 5 drops of iodinereagent

- shake the tube


1% Cellulose

Color Change

- put 3 ml 1% amylum

into reaction tube

- add 2 drops of NaOH

- add 5 drops of iodinereagent

- shake the tube


1% Amylum

Color Change


into reaction tube

- add 2 drops of HCl


reagent

- shake the tube


1% Amylum

Color Change


into reaction tube

- add 2 drops of aquadest


reagent

- shake the tube


- heat the colored liquid

- reobserve the alteration

1% Amylum

Color Change


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5. Carbohydrates Experiment in Fruit (Guava)

a) Carbohydrates experiment with molisch reagent in guava

- put 2 ml well-ripe guava solution

into reaction tube









solution

Color Change

Well-ripe guava solution

- put 2 ml ripe guava solution into

reaction tube









solution

Color Change

Ripe guava solution

- put 2 ml unripe guava solution

into reaction tube









solution

Color Change

Unripe guava solution


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d)

Carbohydrates experiment with iodine solution in guava

- put 1 drops well-ripe guava

solution into petri cup

- add 1 drops iodine solution

- observe and write the color

changing

Color Change

Well-ripe guava solution

- put 1 drops ripe guava


- add 1 drops iodine

solution

- observe and write the

color changing

Color Change

Ripe guava solution

- put 1 drops unripe guava


- add 1 drops iodine

solution

- observe and write the

color changing

Color Change

Unripe guava solution


- add 2 drops of well-ripe

guava solution at the time

heat it in waterbath until

formed color


color

Color Change



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CHAPTER VI

RESULT OF EXPERIMENT

A. Data of Experiment

Tabel 1. Observation Result of Molisch Test

No Carbohydrates

SolutionTreatment

Observation Result

Before After

1. 1% Glucose

solution

2 ml 1% Glucose

solution +

Molisch Reagent

+ H2SO4

Glucose = colorlessMolisch = dark-brown

Glucose+Molisch=colorless and there are

dark-brown precipitates

Ring(thickness):

+

Turbidity(color):

++

2. 1% Fructose

solution

2 ml 1% Fructose

solution +

Molisch Reagent

+ H2SO4

Fructose = colorlessMolisch = dark-brown

Fructose+Molisch=

colorless and there are


Ring(thickness):

+++

Turbidity(color):

+

3. 1% Lactose

solution

2 ml 1% Lactose

solution +

Molisch Reagent

+ H2SO4

Lactose = colorless

Molisch = dark-brownLactose+Molisch=

colorless and there aredark-brown precipitates

Ring(thickness):

++

Turbidity(color):

++

4. 1% Sucrose

solution

2 ml 1% Sucrose

solution +

Molisch Reagent

+ H2SO4

Sucrose = colorlessMolisch = dark-brown

Glucose+Molisch=



Ring(thickness):

++++

Turbidity(color):

++++

5. 1% Maltose

solution

2 ml 1% Maltose

solution +

Molisch Reagent

+ H2SO4

Maltose = colorlessMolisch = dark-brown

Maltose+Molisch=



Ring(thickness):

+++

Turbidity(color):

+++

6. 1% Cellulose

solution

2 ml 1%

Cellulose solution

+ Molisch

Reagent + H2SO4

Cellulosee = white cloudyMolisch = dark-brown

Glucose+Molisch= turbid

and there are dark-brown

precipitates

Ring(thickness):

++++

Turbidity(color):

+++++

7. 1% Amylum

solution

2 ml 1% Amylum

solution +

Molisch Reagent

+ H2SO4

Amylum = white cloudyMolisch = dark-brown

Glucose+Molisch= turbid

and there are dark-brown

precipitates

Ring(thickness):

++

Turbidity(color):

+++


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Tabel 2. Observation Result of Benedict Test

NoCarbohydrates

SolutionTreatment

Observation Result

Before After

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

Solutions color =

formed two color

layers

(top=colorless,

bottom-blue)

Precipitates = red-

thin precipitates

(+)

2. 1% Fructose

solution

2 ml Benedict

reagent + 5 drops

1% Fructose

solution

Benedict + Glucose=

Blue

After heated = Brick

red, formed

precipitates

Solutions color =

Light blue

Precipitates =

Brick red

precipitates (+++)

3. 1% Lactose

solution

2 ml Benedict

reagent + 5 drops

1% Lactose

solution

Benedict + Glucose=

Blue

After heated =

Greenish blue

Solutions color =

Blue

Precipitates = red-

thin precipitates

(+)4. 1% Sucrose

solution

2 ml Benedict

reagent + 5 drops

1% Sucrose

solution

Benedict + Glucose=

Blue

After heated = Blue

Solutions color =

Blue

Precipitates =

No precipitates

5. 1% Maltose

solution

2 ml Benedict

reagent + 5 drops

1% Maltose

solution

Benedict + Glucose=

Blue

After heated = formed

three layers (top=red,

middle=greenish blue

bottom= dark blue)

Solutions color =

formed three

layers (top=tosca,

bottom= blue)

Precipitates =

(++)6. 1% Cellulose

solution

2 ml Benedict

reagent + 5 drops

1% Cellulose

solution

Benedict + Glucose=

Blue

After heated = Blue,

no precipitates

Solutions color =

Blue

Precipitates =

No precipitates

7. 1% Amylum

solution

2 ml Benedict

reagent + 5 drops

1% Amylum

solution

Benedict + Glucose=

Blue

After heated = Blue,

no precipitates

Solutions color =

Blue

Precipitates =

No precipitates


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Tabel 3. Observation Result of Seliwanoff Test

NoCarbohydrates

SolutionTreatment

Observation Result

Before After Time

1. 1% Glucose

solution

1 ml Seliwanoff

reagent + 2 drops

1% Glucose

solution

heated

Seliwanoff reagent

= colorless

Glucose = colorless

Pale

yellow

(++)

21.33minutes

2. 1% Fructose

solution

1 ml Seliwanoff

reagent + 2 drops

1% Fructose

solution

heated

Seliwanoff reagent

= colorless

Fructose =

colorless

Orange

(+++)

6.22minutes

3. 1% Lactose

solution

1 ml Seliwanoff

reagent + 2 drops

1% Lactose

solution

heated

Seliwanoff reagent

= colorless

Lactose = colorless

Pale

yellow

(+++)

22.52minutes

4. 1% Sucrose

solution

1 ml Seliwanoff

reagent + 2 drops

1% Sucrosesolution

heated

Seliwanoff reagent

= colorless

Sucrose = colorless

Orange

(++)

9.59minutes

5. 1% Maltose

solution

1 ml Seliwanoff

reagent + 2 drops

1% Maltose

solution

heated

Seliwanoff reagent

= colorless

Maltose = colorless

Pale

yellow

(++++)

15.30minutes

6. 1% Cellulose

solution

1 ml Seliwanoff

reagent + 2 drops

1% Cellulosesolution

heated

Seliwanoff reagent

= colorless

Celullose = turbid

Pale

yellow

(+++++)

22.55minutes

7. 1% Amylum

solution

1 ml Seliwanoff

reagent + 2 drops

1% Amylum

solution

heated

Seliwanoff reagent

= colorless

Amylum = turbid

Pale

yellow

(+)

24.45minutes


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Tabel 4. Observation Result of Iodine Test

No Carbohydrates

SolutionTreatment

Observation Result

Before After

1. 1% Amylum

solution

3 ml Amylum

solution + 2 drops

Aquadest + 5 drops

iodine reagent

Colored liquid

heated

Amylum = turbid

Amylum + Aquadest =

turbid

Amylum + Aquadest +

Iodine = Blackish green

(+++)

Blackish blue

2. 1% Amylum

solution

3 ml Amylum

solution + 2 drops

HCl + 5 drops

iodine reagent

heated

Amylum = turbid

Amylum + HCl = turbid

Amylum + HCl +

Iodine = Blackish green

(++)

Blackish blue

3. 1% Amylum

solution

3 ml Amylum

solution + 2 drops

NaOH + 5 drops

iodine reagent

heated

Amylum = turbid (+)

Amylum + NaOH =

turbid (++)

Amylum + NaOH +

Iodine = turbid (+++)

Colorless

4. 1% Cellulose

solution

3 ml Cellulose

solution + 2 dropsAquadest + 5 drops

iodine reagent

Colored liquid

heated

Cellulose = colorless

Cellulose + Aquadest =colorless

Cellulose + Aquadest +

Iodine = Blackish

purple (+++)

Blackish blue

5. 1% Cellulose

solution

3 ml Cellulose

solution + 2 drops

HCl + 5 drops

iodine reagent

heated


Cellulose + HCl =

feculent white

Cellulose + HCl +

Iodine = Blackish

purple (++)

Blackish blue

6. 1% Cellulose

solution

3 ml Amylum

solution + 2 drops

NaOH + 5 drops

iodine reagent

heated


Amylum + NaOH =

colorless

Amylum + NaOH +

Iodine = colorless

Colorless


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Tabel 5. Observation Result of Carbohydrates Experiment in Guava

No ProcedureObservation Result

Before After

1. Molisch

experiment

Unripe guava

Ripe guava

Well-ripe guava

Unripe guava =Light green

Ripe guava =

Turbid green (+)

Well-ripe guava =Turbid green (++)

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 = reddishwhiteRing : Thick red (+++)

2. Benedict

experiment

Unripe guava

Ripe guava

Well-ripe guava

Unripe guava =

Light green

Ripe guava =turbid green (+)

Well-ripe guava =turbid green (++)

Unripe guava =

Solution color : dark red (+++)Precipitates : (+)

Ripe guava =

Solution color : dark red (++)Precipitates : (++)

Well-ripe guava =Solution color : dark red (+)Precipitates : (+++)

3. Seliwanoff

experiment

Unripe guava

Ripe guava

Well-ripe guava


Ripe guava =

turbid green (+)

Well-ripe guava =turbid green (++)

Unripe guava =Pink (+++)

Ripe guava =

Pink (++)

Well-ripe guava =Pink (+)

4. Iodine

experiment

Unripe guava Ripe guava

Well-ripe guava


Ripe guava =turbid green (+)

Well-ripe guava =

turbid green (++)

Unripe guava =Yellow (++)

Ripe guava =Yellow (+)

Well-ripe guava =

Yellow (+++)


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B. Data Analyzing

1. Molisch’s 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 (++++).


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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. Benedict’s 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 (+++).


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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 heatedsolution 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


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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. Seliwanoff’s 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 (++++).


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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. Iodine’s 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.


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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)

Molisch’s 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


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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 (+++).

Benedict’s 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 guavasolution show the solution color is turbid green. Then after treatment the

solution color changes into dark red (+) and there are precipitates (+++).

Seliwanoff’s 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 (+).


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Iodine’s 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

Molisch’s 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.


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Benedict’s 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 benedict’s 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)

Seliwanoff’s Test

1.

What group of carbohydrates that give a positive reaction toseliwanoff'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 seliwanoff’s test sucrose hydrolyzed into glucose

and fructose.

Iodine’s 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


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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 containa-1,6 glycosidic bond. (Suhartono, 1989)


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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.


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REFFERENCES

Julianto, Tatang S. 2015. Biokimia: Biomolekul dalam Prespektif Al-Qur’an.

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


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APENDIX

EXPERIMENT PICTURE EXPLANATION

Molisch’s Test

Sucrose and Fructose(sucrose or fructose +

molisch reagent +

H2SO4)

Amylum and Glucose

(amylum or glucose +molisch reagent +

H2SO4)

Lactose, Maltose and

Sellulose(lactose or maltose or

sellulose + molisch

reagent + H2SO4)


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Benedict’s Test

Amylum(amylum in cool

condition after

treatment)

Glucose(glucose in cool

condition after

treatment)

Fructose(fructose in cool

condition after

treatment)

Lactose(lactose in cool

condition aftertreatment)

Maltose(malose in cool

condition after

treatment)

Sellulose

(sellulose in coolcondition after

treatment)

Sucrose(sucrose in cool

condition after

treatment)


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Seliwanoff’s

Test

Amylum(amylum after

treatment)

Fructose

(fructose after

treatment)

Glucose(glucose after

treatment)

Lactose(lactose after

treatment)

Maltose(malose after

treatment)

Sellulose

(sellulose aftertreatment)

Sucrose(sucrose after

treatment)


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Iodine’s Test

Amylum(amylum + aquadest +

iodine reagent)

Amylum

(amylum + HCl +

iodine reagent)

Amylum(amylum + NaOH +

iodine reagent)

Sellulose(sellulose + aquadest +

iodine reagent)

Sellulose(sellulose + HCl +

iodine reagent)

Sellulose(sellulose + NaOH +

iodine reagent)


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Carbohydrate

Experiment in

Guava

Molisch’s Test

Benedict’s Test

Selliwanof’s Test

Iodine’s Test

Carbohydrate Experiment

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