Carbohydrate Experiment

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 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 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 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, 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)

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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

7

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

8

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.

9

CHAPTER III DESIGN OF EXPERIMENT A. Tools and Materials 1. Molisch Test Tools :  Reaction tube  Pippete drops  Reaction tube’s rack  Measure glass

2. Benedict Test Tools :  Reaction tube  Pippete drops  Reaction tube’s rack  Measure glass  Waterbath  Leg three  Burner methylated  Lighter 3. Seliwanoff Test Tools :  Reaction tube  Pippete drops  Reaction tube’s rack  Measure glass  Waterbath  Leg three  Burner methylated  Lighter  Stopwatch 4. Iodine Test Tools :  Reaction tube  Pippete drops  Reaction tube’s 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% Fructose solution  1% Lactose solution  1% Sucrose solution  1% Maltose solution  1% Cellulose solution  1% Amylum solution Materials  Seliwanoff reagent  1% Glucose solution  1% Fructose solution  1% Lactose solution  1% Sucrose solution  1% Maltose solution  1% Cellulose solution  1% Amylum solution

Materials  Iodine solution  1% Amylum solution  1% Cellulose solution  NaOH 6 N  HCL 6 N

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 Leg three  Aquadest  Burner methylated  Lighter 5. 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 of reaction tube. 5) Stand the reaction tube, observe there are any red-purple ring on border of the two solution. 2. Benedict Test 1) Prepare all kinds of carbohydrates into a solution with a concentration 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 1) Prepare all kinds of carbohydrates into a solution with a concentration 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. 5. Carbohydrates Experiment in Fruit (Guava) 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 - 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

Color Change

Color Change

12

1% Lactose - put 2 ml 1% lactose 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 Color Change

1% Sucrose - put 2 ml 1% sucrose 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 Color Change

1% Maltose

1% Selulose

- put 2 ml 1% maltose 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% cellulose 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

Color Change

Color Change

13

1% Amylum - put 2 ml 1% amylum 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 2. Benedict Test 2 ml benedict reagent

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

- put into reaction tube - add 5 drops of 1% fructose solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing

Color Change

Color Change

2 ml benedict reagent

2 ml benedict reagent

- put into reaction tube - add 5 drops of 1% lactose solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing

- 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

Color Change

14

2 ml benedict reagent

2 ml benedict reagent

- put into reaction tube - add 5 drops of 1% maltose solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing

- put into reaction tube - add 5 drops of 1% cellulose solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing

Color Change

Color Change

2 ml benedict reagent - put into reaction tube - add 5 drops of 1% amylum solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing Color Change 3. Seliwanoff Test 1 ml seliwanoff reagent

1 ml seliwanoff reagent

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

- put into reaction tube - add 2 drops of 1% fructose solution at the time heat it in waterbath until formed color - write velocity of formed color

Color Change

Color Change

15

1 ml seliwanoff reagent - put into reaction tube - add 2 drops of 1% lactose solution at the time heat it in waterbath until formed color - write velocity of formed color Color Change

1 ml seliwanoff reagent - put into reaction tube - add 2 drops of 1% sucrose solution at the time heat it in waterbath until formed color - write velocity of formed color Color Change

1 ml seliwanoff reagent

1 ml seliwanoff reagent

- put into reaction tube - add 2 drops of 1% maltose solution at the time heat it in waterbath until formed color - write velocity of formed color

- put into reaction tube - add 2 drops of 1% cellulose solution at the time heat it in waterbath until formed color - write velocity of formed color

Color Change

Color Change

1 ml seliwanoff reagent - put into reaction tube - add 2 drops of 1% amylum solution at the time heat it in waterbath until formed color - write velocity of formed color Color Change

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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 - put 3 ml 1% amylum into reaction tube - add 2 drops of NaOH - add 5 drops of iodine reagent - shake the tube - observe color changing Color Change

1% Amylum - put 3 ml 1% amylum into reaction tube - add 2 drops of HCl - add 5 drops of iodine reagent - shake the tube - observe color changing Color Change

1% Cellulose - put 3 ml 1% cellulose into reaction tube - add 2 drops of NaOH - add 5 drops of iodine reagent - shake the tube - observe color changing Color Change

1% Cellulose - put 3 ml 1% cellulose 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% Cellulose - 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 Color Change

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5. Carbohydrates Experiment in Fruit (Guava) a) Carbohydrates experiment with molisch reagent in guava Unripe guava solution

Ripe guava solution

- put 2 ml unripe guava solution 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 Color Change

- put 2 ml ripe guava solution 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 Color Change

Well-ripe guava solution - put 2 ml well-ripe guava solution 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 Color Change

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b) Carbohydrates experiment with benedict reagent in guava 2 ml benedict reagent

2 ml benedict reagent

- put into reaction tube - add 5 drops of unripe guava solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing

- put into reaction tube - add 5 drops of ripe guava solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing

Color Change

Color Change

2 ml benedict reagent - put into reaction tube - add 5 drops of well-ripe guava solution - heat it in waterbath about 5 minutes - let it cool - compare and write the color changing Color Change c) Carbohydrates experiment with seliwanoff reagent in guava 1 ml seliwanoff reagent

1 ml seliwanoff reagent

- put into reaction tube - add 2 drops of unripe guava solution at the time heat it in waterbath until formed color - write velocity of formed color

- put into reaction tube - add 2 drops of ripe guava solution at the time heat it in waterbath until formed color - write velocity of formed color

Color Change

Color Change

19

1 ml seliwanoff reagent - put into reaction tube - add 2 drops of well-ripe guava solution at the time heat it in waterbath until formed color - write velocity of formed 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 solution into petri cup - add 1 drops iodine solution - observe and write the color changing Color Change

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

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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 Molisch = dark-brown Fructose+Molisch= colorless and there are dark-brown precipitates Lactose = colorless Molisch = dark-brown Lactose+Molisch= colorless and there are dark-brown precipitates Sucrose = colorless Molisch = dark-brown Glucose+Molisch= colorless and there are dark-brown precipitates Maltose = colorless Molisch = dark-brown Maltose+Molisch= colorless and there are dark-brown precipitates Cellulosee = white cloudy Molisch = dark-brown Glucose+Molisch= turbid and there are dark-brown precipitates Amylum = white cloudy Molisch = dark-brown 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): +++

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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

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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

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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 = colorless Cellulose + HCl = feculent white Cellulose + HCl + Iodine = Blackish purple (++) Cellulose = colorless Amylum + NaOH = colorless Amylum + NaOH + Iodine = colorless

Blackish blue

Blackish blue

Colorless

Blackish blue

Blackish blue

Colorless

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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 (+++)

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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 show solution color is clear and there are dark-brown precipitates. Then the solution added H2SO4 there are purplish-black ring (++) with thickness (++). Fourth material is Sucrose. 2 ml 1% sucrose 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 (++++). Fifth material is Maltose. 2 ml 1% maltose 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 (+++). 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 solution,1% Fructose solution, 1% Lactose solution, 1% Sucrose solution, 1% 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 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 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 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. 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 guava solution 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 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 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 contain a-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

Sucrose and Fructose (sucrose or fructose + molisch reagent + H2SO4)

Molisch’s Test

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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Amylum (amylum in cool condition after treatment)

Glucose (glucose in cool condition after treatment)

Fructose (fructose in cool condition after treatment)

Benedict’s 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)

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Amylum (amylum after treatment)

Fructose (fructose after treatment)

Glucose (glucose after treatment)

Seliwanoff’s Test

Lactose (lactose after treatment)

Maltose (malose after treatment)

Sellulose (sellulose after treatment)

Sucrose (sucrose after treatment)

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Amylum (amylum + aquadest + iodine reagent)

Amylum (amylum + HCl + iodine reagent)

Amylum (amylum + NaOH + iodine reagent) Iodine’s Test Sellulose (sellulose + aquadest + iodine reagent)

Sellulose (sellulose + HCl + iodine reagent)

Sellulose (sellulose + NaOH + iodine reagent)

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Molisch’s Test

Carbohydrate Experiment in Guava

Benedict’s Test

Selliwanof’s Test

Iodine’s Test

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