University of Tunku Abdul Rahman (Kampar Campus)

Faculty of Engineering and Science

Bachelor of Science (Hons) Biotechnology

Year 1 Semester 1 (Group 1)

UESB 1112 Laboratory 1A (II) Cell Biology

Lecturer: Dr. Han Kwai Hin

Student’s Name: Cheah Hong Leong (08AIB03788)

Partner’s Name: Lim Zhe Choon (08AIB02258)

Experiment No. 6, and 7

Title: Indicative Tests on Macromolecules and Food

samples.

Date: 24 June 2008

Title: Indicative Tests on Macromolecules and Food Samples

Objective: - Describe and carry out the Benedict’s test that indicates the presence of

reducing sugars specifically.

- Describe and carry out the Iodine Test that indicates the presence of starch

specifically.

- Describe and carry out the hydrolysis process on the non-reducing disaccharide

and polysaccharide.

- Describe and carry out the Biuret test that indicates the presence of proteins.

- Describe and carry out the paper test that indicates the presence of lipids.

- Identify the macromolecules presence in food samples given by carry out

indicative tests on the food samples.

Introduction:

Carbohydrates

Glucose is an aldose which has aldehyde group as functional group. Fructose is a

ketose which has ketone group as functional group. All monosaccharides are reducing

sugars due to the presence of free aldehyde group or ketone group.

The Benedict’s reagent is a solution that contains copper (II) sulphate and

alkaline solution, and the copper (II) ions are reduced by the aldehyde or ketone group

to form copper (I) oxide precipitate. Besides, the yellow, green, and orange color of

precipitate also indicates the presence of reducing sugar.

Sucrose is the disaccharide formed from the enzymatic dehydration reaction

between glucose and fructose. Lactose is the disaccharide formed from the

dehydration reaction between the glucose and galactose.

Lactose is reducing sugar due to the presence of aldehyde or ketone group in one

of the monomers that can gives the positive result on the Benedict’s test. Sucrose is

non-reducing sugar, due to the absence of the aldehyde or ketone group after the

formation of the glycosidic linkage. Thus, sucrose gives negative result on the

Benedict’s test.

Starch is a polysaccharide of glucose and thus cannot give positive result on the

Benedict’s test, since all polysaccharides do not have any reducing ability. However,

starch gives positive result on the Iodine test. Starch forms the dark blue color of

solution with iodine solution which is the result from the complex ion formed

between the amylose and the iodine molecules.

Both disaccharides and polysaccharides can be hydrolyzed into their constituents

with addition of water and requiring specific enzyme that catalyze the hydrolysis

reaction. In the laboratory, these compound sugars can be hydrolyzed in the test tube

by addition of concentrated acid and heating process.

Proteins

Proteins consist of one or more polypeptides coiled or folded into specific three

dimensional conformations. The functional group of amino acids are the carboxyl

group (-COOH) and the amino group (-NH2). The carboxyl group and amino group of

the adjacent amino acids can be linked together to form a peptide linkage through

dehydration reaction.

The Biuret reagent is consisting of mixture of the copper (II) sulphate and an

alkaline solution, used to detect the presence of peptide linkages. A solution that

contains proteins will give violet coloration to the Biuret reagent.

Lipids

The lipid is a group of macromolecules that are insoluble in water. The major

kinds of lipids in biological world include fats, phospholipids, and steroids.

The fats or triacylglycerols are made up of glycerol (an alcohol) and fatty acids

(a monocarboxylic acid) which are bonded together by ester linkages.

The lipids are greasy and would turn the area of dry lipids on a brown paper into

semi-transparent.

Materials and Methods:

Part 1: Chemical Tests on Carbohydrates and Food Samples

Investigation 1: Benedict’s Test on Monosaccharides, Disaccharides, Polysaccharide

And Food Samples

Material and Apparatus:

- Benedict’s reagent

- Distilled water

- 1% solution of glucose

- 1% solution of fructose

- 1% solution of lactose

- 1% solution of sucrose

- 1% solution of starch

- Apple juice

- Potatoes ( solution after grinded with distilled water )

- Soft drink

- Onions (solution after grinded with distilled water)

- Instant noodles ( solution after grinded with distilled water )

- Soya Bean milk

- Test tubes

- Test tube rack

- Test tube holder

- Boiling water bath ( prepared )

Methods:

1. 6 test tubes were prepared.

2. 1ml of distilled water was placed into a test tube.

3. 1ml of 1% solution of glucose, fructose, sucrose, lactose, and starch were placed

into the other 5 test tubes, respectively.

4. 5ml of Benedict’s solution was added into each of the test tubes.

5. The test tubes were then placed in a boiling water bath prepared in the laboratory

for about 2 to 3 minutes.

6. The test tubes were taken out from the boiling water bath.

7. The color and precipitate formation of the solutions were observed.

8. The test was repeated with different kinds of solution of food samples (apple

juice, potato, soft drink, onion, instant noodles, and Soya bean milk).

9. The color and precipitate formation of the solutions were observed.

Investigation 2: Iodine Test on Starch and Food samples

Materials and Apparatus:

- Iodine reagent

- Distilled water

- 1% solution of starch

- Apple juice

- Potatoes ( solution after grinded with distilled water )

- Soft drink

- Onions (solution after grinded with distilled water)

- Instant noodles ( solution after grinded with distilled water )

- Soya Bean milk

- Test tubes

- Test tube rack

Methods:

1. About 1ml of distilled water was placed into a test tube.

2. About 1ml of 1% solution of starch was placed into another test tube.

3. A few drops of iodine reagent were added into the two test tubes.

4. The colors of the solutions were observed.

5. The iodine test was repeated with different solution of apple juice, potatoes, soft

drink, onions, instant noodles, and Soya Bean milk.

6. The colors of the solutions were observed.

Investigation 3: Hydrolysis of Sucrose and Starch

Materials and Apparatus:

- Benedict’s reagent

- Iodine reagent

- 1% solution of starch

- 1% solution of sucrose

- 2N of hydrochloric acid (HCl) solution

- Large test tubes

- Test tubes

- Pipettes

- Test tube rack

- Beaker

- Test tube holder

- Boiling water bath ( prepared )

Methods:

1. Eight test tubes were labeled from 1 to 8.

2. These test tubes were lined up in accordance to their numbers in a test tube rack.

3. Two large test tubes were labeled with sucrose and starch respectively.

4. 6ml of 1% solution of starch was pipette into the large test tube labeled starch.

5. 3ml of 2N of hydrochloric acid (HCl) solution was pipette into the large test tube

containing starch solution.

6. 5ml of 1% solution of sucrose was pipette into the large test tube labeled sucrose.

7. 1ml of 2N of hydrochloric acid (HCl) solution was pipette into the large test tube

containing sucrose solution.

8. Each of the large test tubes was swirled gently so that the contents were mixed.

9. 1ml of solution from the large test tube labeled sucrose was drawn and put into

test tube 1.

10. 1ml of solution from the large test tube labeled starch was drawn and put into test

tube 3.

11. An additional 1ml of solution from the large test tube labeled starch was drawn

and put into test tube 4.

12. The large test tubes were placed in the boiling water bath prepared in the

laboratory.

13. After 2 to 3 minutes, the large test tube labeled sucrose was taken out from the

boiling water bath; 1ml of solution from the large test tube was drawn and put into

test tube 2.

14. After 5 minutes, 1ml of solution from the large test tube was drawn and put into

test tube 5.

15. Another 1ml of solution from the large test tube (labeled starch) was drawn and

put into test tube 6.

16. After another 10 minutes, solution from the large test tube (labeled starch) was

drawn and put into test tube 7 and 8 with 1ml for each test tube.

17. Benedict’s reagent was added into the test tube 1, 2, 3, 5, and 7 with 5 ml for each

test tube.

18. These test tubes were placed in the boiling water bath for 5 minutes.

19. 3 drops of iodine reagent were added into test tube 4, 6, and 8.

20. The test tube in the boiling water bath were removed and placed in test tube rack

for them to cool.

21. The colors of the solutions and formation of precipitate in the test tubes were

observed.

Part 2: Biuret Test on Proteins and Food Samples

Material and Apparatus:

- Concentrated potassium hydroxide (KOH) solution

- 0.5% of copper(II) sulphate (CuSO4) solution

- Distilled water

- 1% of egg albumin

- Apple juice

- Potatoes ( solution after grinded with distilled water )

- Soft drink

- Onions (solution after grinded with distilled water)

- Instant noodles ( solution after grinded with distilled water )

- Soya Bean milk

- Test tubes

Methods:

1. 3ml of distilled water was placed into a test tube.

2. 3ml of 1% egg albumin was placed into another test tube.

3. Concentrated potassium hydroxide (KOH) solution was added into both of the test

tubes with equal volume.

4. The mixtures were swirled gently.

5. 1ml of 0.5% of copper (II) sulphate (CuSO4) solution was added into the mixtures

of both test tubes, slowly.

6. The mixtures were swirled gently.

7. The colors of the solutions in the test tubes were observed after 2 minutes.

8. The Biuret test was repeated with different solutions of apple juice, potatoes, soft

drink, onions, instant noodles, and Soya Bean milk.

9. The colors of the solutions were observed.

Part 3: Paper Test on Lipids and Food Samples

Materials and Apparatus:

- Brown paper

- Distilled water

- Vegetable oil

- Apple juice

- Potatoes ( solution after grinded with distilled water )

- Soft drink

- Onions (solution after grinded with distilled water)

- Instant noodles ( solution after grinded with distilled water )

- Soya Bean milk

- Glass rod

Methods:

1. A small square of brown paper was divided into 8 regions.

2. Each region were labeled with “water”, “oil”, “apple juice”, “potatoes”, “soft

drink”, “onions”, “instant noodles”, and “Soya Bean milk”.

3. A tiny drop of water, vegetable oil, apple juice, potatoes (solution), soft drink,

onions (solution), instant noodles (solution), and Soya Bean milk was put onto the

brown paper on respective regions.

4. These tiny drops on brown paper were rubbed gently with glass rod.

5. The brown paper was left so that the spots were allowed to dry.

6. The brown paper was held up to the light.

7. The transparencies of the spots on the brown paper were observed.

Results:

(I) Chemical Tests on Carbohydrates and Food Samples

Table 1: Benedict’s Test on Monosaccharides, Disaccharides, Polysaccharides, and

Food Samples

Samples Observations:

Distilled water Formation of blue color solution

Glucose Formation of brown-yellow color solution and precipitate

Fructose Formation of brick-red color solution and precipitate

Sucrose Formation of blue color solution

Lactose Formation of dark-green color solution and precipitate

Starch Formation of blue color solution

Apple juice Formation of brick-red color solution and precipitate

Potatoes Formation of brown-yellow color solution and precipitate

Soft drink Formation of brick-red color solution and precipitate

Onions Formation of brown-yellow color solution and precipitate

Instant noodles Formation of green color solution and precipitate

Soya Bean milk Formation of green color solution and precipitate

Table 2: Iodine Test on Starch Solution and Food Samples

Samples Observation:

Distilled water The water became light yellow color

Starch Formation of dark blue color solution

Apple juice Formation of light yellow color solution

Potatoes Formation of dark blue color solution

Soft drink Formation of light orange-yellow color solution

Onions Formation of light orange-yellow color solution

Instant noodles Formation of dark blue color solution

Soya Bean milk Formation of yellow-brown color solution

Table 3: Benedict’s Test and Iodine Test on the Hydrolysis of Sucrose and Starch

Observations:

Samples: Sucrose Starch

Test tube

numbers

1 2 3 4 5 6 7 8

Time(minute) 0 2-3 0 0 5 5 15 15

Benedict’s test LB BR LB - LB - BR -

Iodine test - - - DB - LY - LY

*LB – Formation of light blue color solution

BR – Formation of brick-red solution and precipitate

DB – Formation of dark blue color solution

LY – Formation of light yellow color solution

(II) Proteins

Table 4: Biuret Test on Proteins and Food Samples

Samples Observation

Distilled water The water became light blue color

Egg albumin Formation of violet color solution

Apple juice Formation of light orange color solution

Potatoes Formation of dark violet color solution

Soft drink Formation of light blue color solution

Onions Formation of light green color solution

Instant noodles Formation of light blue color solution

Soya Bean milk Formation of violet color solution

(III) Lipids

Table 5: Paper Test on Lipids and Food Samples

Samples Observations

Distilled water No transparency occur on the region

Vegetable oil The region became semitransparent

Apple juice No transparency occur on the region

Potatoes No transparency occur on the region

Soft drink No transparency occur on the region

Onions Semi transparency was only observed on the side of

the spot

Instant noodles No transparency occur on the region

Soya Bean milk No transparency occur on the region

\

Discussion:

Benedict’s Test on Carbohydrates

Glucose, fructose, and lactose are all reducing sugars that have carbonyl group,

which has reducing ability.

Glucose is an aldose that contains free aldehyde group as its functional group. In

an alkaline solution (usually KOH), the copper (II) ions in the Benedict’s reagent are

reduced by the aldehyde group to form copper (I) oxide, a colored precipitate;

whereas the aldehyde group itself is oxidized to form a carboxyl group. Therefore,

glucose shows positive result on the Benedict’s test.

Fructose is the structural isomer of glucose which contains free ketone group

instead of aldehyde group as functional group. Fructose’s ketone group is also a

reducing agent, which can reduce the copper (II) ions in the Benedict’s reagent into

copper (I) oxide precipitate.

Lactose is also a reducing sugar. The formation of lactose involves the reaction

of the aldehyde group of galactose with the hydroxyl group of the glucose. Therefore,

the disaccharide lactose has one free aldehyde group on the glucose subunit. Thus,

lactose shows positive result on the Benedict’s test.

Sucrose is not a reducing sugar. The formation of sucrose involves the

dehydration reaction between the aldehyde group of glucose and the ketone group of

fructose. Therefore, the sucrose has neither free aldehyde group nor ketone group in

both of its subunits. Thus, sucrose shows negative result on the Benedict’s test.

Starch consists of glucose subunits that joint side by side where their aldehyde

group and hydroxyl group are used to form glycosidic bonds to from a long chain of

starch molecule. Since there are no or little free aldehyde and ketone group in starch

molecule, starch shows negative result on the Benedict’s test.

Any food samples that show positive results on the Benedict’s test (formation of

brick-red, green, yellow, or orange precipitate) indicate that the food contains

reducing sugars.

Iodine Test on Starch

Starch gives positive result on the Iodine test. Starch forms the dark blue color of

solution with iodine solution due to the complex formed between the amylose helix

and the iodine molecules.

Any food samples that show the positive result when added with iodine reagent

can be concluded to contain starch.

Hydrolysis of Sucrose and Starch

When the sucrose and starch are heated with HCl solution, the high temperature

and low pH conditions can catalyze the hydrolysis of sucrose and starch into their

monomers without the presence of the specific enzymes. These conditions are

required to break the glycosidic bonds formed between the monomers so that the free

aldehyde and ketone group are available as reducing agents. Therefore, the

hydrolyzed sucrose and starch can gives positive results on the Benedict’s test. The

hydrolyzed starch is no longer can gives the positive result on the iodine test since the

polymer amylose has been hydrolyzed into its monomers.

The rate of hydrolysis of sucrose is higher than starch. Each sucrose molecule

consists of only one glycosidic bond whereas the each starch molecule consists of

multiple of glycosidic bonds. More heat and the amount of HCl solution are required

to hydrolyze the starch so that the rate of hydrolysis is equal to that of sucrose.

Therefore, under the same amount of HCl solution and temperature, the hydrolysis of

starch is lower than that of the sucrose since more amount of glycosidic bonds have to

be hydrolyzed.

Biuret Test on Proteins

Egg albumin contains huge amount of proteins that functions as storage proteins

that supply the embryo with amino acids. Therefore, egg albumin gives the positive

result on the Biuret test (violet coloration of solution). The Biuret test only indicates

the presence of polypeptides that consist of more than one peptide bonds to gives the

violet coloration of solution.

Any other food samples that give the positive result on the Biuret test also

contain proteins.

Paper Test on Lipids

The vegetable oil is the plant oil which is greasy and can turns the brown paper into

semi-transparent. This is not an accurate and specific test as it’s only a physical test to

indicate the presence of lipids. Any other food samples that show the same semi-

transparent result also can be concluded to contain lipids. However, other organic

molecules such as petroleum can also give the same results. A further chemical test is

needed to be carried out to indicate the presence of lipids more accurately and

specifically.

Conclusion:

1. Glucose, fructose, and lactose show positive results on Benedict’s test.

2. Glucose, fructose, and lactose are reducing sugar.

3. Sucrose and starch solution shows negative result on the Benedict’s test.

4. Sucrose and starch are not reducing.

5. Apple juice and soft drink contains fructose.

6. Potato and onion contains glucose.

7. Instant noodles and Soya bean milk contains lactose.

8. Apple juice, soft drink, potato, onion, instant noodles, and Soya bean milk are all

contain reducing sugars.

9. Starch solution shows positive result on iodine test.

10. Potato and instant noodles contains starch.

11. Apple juice, soft drink, onion, and Soya bean milk does not contain starch.

12. The monomers of sucrose and starch are reducing sugars.

13. Proteins with multiple of peptide bonding shows positive result on Biuret test.

14. Potato and Soya bean milk contains proteins.

15. Apple juice, soft drink, onion, and instant noodles do not contain proteins.

16. Vegetable oil and onion shows positive results on paper test.

17. Vegetable oil and onion contains lipids.

18. Apple juice, potato, soft drink, instant noodles, and Soya bean milk does not

contain lipids.

References:

Neil A. Campbell, Jane B. Reece, Biology, Seventh Edition. Benjamin Cummings,

Pearson, 2005

Gerald Karp, Cell and Molecular Biology: Concept and Experiments. John Wiley &

Sons, 2004

C. J. Cleeg, D. G. Mackean, Advanced Biology: Principles and Application, Second

Edition. John Murray, 2000

Hein, Peisen, Ritchey, Introduction to General, Organic, and Biochemistry in The

Laboratory, Eighth Edition. John Wiley & Sons, 2005