Title: Identification of Reducing and Non-reducing Sugars (Carbohydrate Test)

Objective: The objective of this experiment is to use the Fehling’s test, the
Benedict’s test and the Tollen’s test to distinguish between reducing and non-
reducing sugars and also use the Iodine test to identify starch.

Materials Required:

1. Tollen’s Reagent (already prepared)

2. Fehling’s Solution (already prepared)
3. Benedict’s Solution (already prepared)
4. Barfoed solution(already prepared)
5. Iodine Solution (already prepared)
6. Boiling water bath
7. Test tube and holder
8. Pipette and pipette pump/dropper
9. Carbohydrates to be tested (as supplied)

A. Tollen’s Test

Principle: Silver nitrate in an excess of ammonia, complexes to form Ag(NH3)2+

which is reduced by reducing sugars to silver. The silver deposits on the walls of
the test tube and indicates a positive test for this reaction.

Procedure:

1. To 3mLs of silver nitrate solution, add one

drop of aqueous sodium hydroxide.
2. Add dilute ammonia dropwise till the brown
precipitate of silver oxide just dissolves.
3. Add 0.1 gms of the carbohydrate and warm
in a beaker of boiling water for 5 minutes.
4. Record your observations.
Sample +
Silver Nitrate (1 spatula) +
dil NH3 (0.5 ml) +
dil. NaOH (0.5ml)
HEAT
(SILVER MIRROR DEPOSIT)
 Sample +
Fehling I (0.5 ml) +
Fehling II (0.5ml)
HEAT
B. Fehling’s Test (BRICK RED PPT)
Principle: The solution is made by mixing copper (II) sulphate with an alkaline
solution of a salt of tartaric acid which contains a deep blue complexed copper
(II) ion. The copper (II) ion is reduced by reducing agents to copper (I) oxide,
which forms a red precipitate.

Procedure:

1. Mix equal volumes of Fehling’s I

solution to Fehling’s II solution in a
test tube.

2. Add 0.1gms of the carbohydrate and

warm the mixture in a beaker of
boiling water for a few minutes.

3. Record your observations.

C. Benedict’s Test

Principle: The copper (II) sulphate in the Benedict’s solution is reduced to form
colored precipitates of cuprous oxide. The colour of the precipitate depends on the
concentration of the reducing sugar being tested.

Procedure:
1. To 5mLs of Benedict’s
solution, add 0.1gms of the
carbohydrate.
2. Warm in a beaker of beaker of
boiling water for a few minutes.
3. Record your observations.

Sample +
Benedict Sol. (0.5ml)
HEAT
(BRICK RED PPT)
 Sample +
Water (2 ml) +
Iodine sol. (2 drops)
(BLUE BLACK COLOR)
D. Iodine test for starch:
Principle: Starch can be separated in two fractions, amylose and amylopectins.
Amylose forms a colloidal dispersion in hot water whereas amylopectin is
completely insoluble. The structure of amylose consists of a long polymer of
glucose units connected by an alpha-acetal linkage and due to this amylose
usually forms a spiral like coiled spring. The iodine test is used to distinguish
starch from monosaccharides, disaccharides, and other polysaccharides. Because of
its unique coiled geometric configuration, it reacts with iodine to produce a blue-
black color and tests positive. A yellowish-brown color indicates that the test is
negative.

Procedure:

1. Dissolve starch (0.1g) in water (2ml) by

heating at 60  C on a water bath.
2. A viscous translucent jelly will form on
cooling.
3. Add Iodine solution (1drop) to form a
deep blue-black color.

E. Barfoed’s test

Principle: Barfoed’s test used copper (II) ions in a slightly acidic medium,
reducing monosaccharides are oxidized by the copper ion in solution to form a
carboxylic acid and a reddish precipitate of copper (I) oxide within three minutes.
Reducing disaccharides undergo the same
reaction, but do so at a slower rate. The
nonreducing sugars give negative result.

Procedure

1. Pour 1.0 ml of dilute solution of

carbohydrate in a test tube.
2. Add 1.0 ml of reagent
3. Heat the test tube in a
beaker of boiling water.

Sample +
Barfoed Sol. (0.5ml)
HEAT
(BRICK RED PPT)
Remarks:
If red ppt is formed within 2 minutes, a monosaccharide is present. The ppt is
due to the formation of copper (I) oxide.

(Mono – Reducing – Glucose, Galactose) (Disac – Reducing – Maltose, Lactose)

(Polysac – Non Red – Starch) (Disac – Non reducing – Sucrose)
Report Writing:
1. Principle
2. Materials required
3. Procedure
4. Observation/Justification
5. Reactions
6. Result
7. Precautions
8.Figure
Include in your report, the equations for every experiment ( Tollen’s, Fehling’s,
Benedicts, Barfoed’s and Iodine test)
Video link for carbohydrate
https://www.youtube.com/watch?v=TDFbtEwbmz0 ......carb
https://www.youtube.com/watch?v=QacQmS3aaTI.....carb,prot,fat
https://www.youtube.com/watch?v=Ewe7i1D9lSQ ……..carb
https://www.youtube.com/watch?v=yQfMqvOxPrc …….barfoed test…imp
https://www.youtube.com/watch?v=ojhdTFmkY1c ……carb qualitative
test/20…..imp
Title: Qualitative test for Lipid. (Lipid Test)

Objective: The object of this experiment is to use the solubility test & Sudan
red test for the qualitative identification of lipids.

Materials Required:

1. Polar solvent
2. Non-polar solvent
3. Sudan Red
4. Test tube and holder
5. Pipette and pipette pump/dropper
6. Water
7. Lipids to be tested (as supplied)

Procedure:
A. Solubility Test
Principle: Lipids are insoluble in polar solvent and soluble in non-polar solvent
(Benzene, Ether, Chloroform).
Procedure:
1. Take 1 ml water in one test tube
and 1 ml of non-polar solvent in
another test tube.
2. Add 1 ml of sample (to be tested)
to both test tubes.
3. Mix the contents and wait for 1
minute.
4. Record your observations
(soluble or insoluble).
B. Sudan Red Test
Principle: Sudan Red is a lipid soluble dye. When Sudan red is added to a mixture
of lipid and water, the dye will move into the lipid layer, coloring it red.

Procedure:
1. To a mixture of water and lipid, add one drop of Sudan red dye.
2. Mix the contents and wait for 1minute.
3. Record your observations (which layer does the dye move to?).

C. Emulsification Test:

Principle: The emulsion test is a method to determine the presence of lipids using

wet chemistry. The procedure is for the sample to be suspended in ethanol,
allowing lipids present to dissolve (lipids are soluble in alcohols). The liquid
(alcohol with dissolved fat) is then decanted into water. Emulsifying agent bile
salt, soap water etc.
Emulsification:

Oil or liquid fat becomes finely divided and is dispersed in water when shaken
with water to form emulsification. Emulsification is permanent and complete in the
presence of emulsifying agent. The important emulsifying agents are bile salts,
proteins, soaps, mono- and diglycerides. Emulsification is important in the
processes of fat digestion in the intestine. Emulsifying agents lower surface
tension of the liquid.

Procedure:

Take 2 clean and dry test tubes, in one test tube added 2 ml water and in other 2ml
dilute bile salt solution. Now to each tube added 2 drops of mustard oil and shaken
vigorously for about one minute. Allow the tubes to stands for two minutes and
note that the water, oil is broken in small pieces and floats on the surface; where as
in the bile salt solution, the oil can be seen in minute droplets suspended in the
liquid (permanent emulsification).

3. What is the significance of the emulsification of fat?

Ans: Emulsification is the process of breaking down the fat into smaller blood
cells which makes it easy for enzymes to function and digest food. Fat
emulsification helps digest fats into fatty acids and glycerol that are easily
absorbed by the small intestine.

Report Writing:
Submit a laboratory report on this experiment one week from the date of the
experiment.
Include the following in your report:
1. Title of the Experiment
2. objective
3. Principle
4. Materials Required
5. Procedure
6. Observation
7. Reactions
8. Figure
9. result
10. Precautions

https://www.youtube.com/watch?v=QacQmS3aaTI.....carb,prot,fat sudan
https://www.youtube.com/watch?v=l2QOi9mZoFc …….....fat solubility
,translucent
https://www.youtube.com/watch?v=d3YCFwhPkYM ……..fat
solubility,translucent imp

https://www.youtube.com/watch?v=Boo1o2soTbA&t=18s
……..EMULSIFICATION
https://www.youtube.com/watch?v=IgMopgqnWqo …….SOLUBILITY good

Title: Qualitative test for protein.

Objective: The object of this experiment is to use the Biuret Test &
Ninhydrin test to identify protein.

Materials Required:
1. Biuret Reagent (already prepared)
2. Ninhydrin Solution (already prepared)
3. Ethanol/Isopropyl alcohol
4. Test tube and holder
5. Pipette and pipette pump
6. Proteins to be tested (as supplied)
Procedure:
A. Biuret Test
Principle: Biuret reagent is a light blue solution, which turns purple when mixed
with a solution containing protein. The purple color is formed when copper ion in
the Biuret reagent reacts with the peptides bond of the polypeptide chains to form
a complex.
Procedure:
1. To 1 ml of sample, add 0.5 ml of Biuret reagent (Alkaline CuSO4) and 0.5 ml
NaOH solution.
2. Mix the contents.
3. Wait for 2 minutes to observe the color.
4. Record your observations.

B. Test by Alcohol: Principle: 5ml of 95% alcohol is added to 1ml of protein

solution and mixed. White ppt forms due to precipitation of protein by alcohol as
a result of dehydration and denaturation.
C. Ninhydrin Test - Principle: Amino acid contains a free amino group which is
readily detected with Ninhydrin reagent. The reagent reacts with free amino groups
to form a purple or violet colored substance. Ninhydrin reagent can also react with
protein but the protein must be heated or digested to hydrolyze the protein into free
amino acids.
Procedure-1:
1. To 1 ml of sample add I drop of Ninhydrin solution
2. Wait for sometime (5-10 min) for the appearance the color.
3. Record your observations.
Procedure-2:
1. Put a drop of sample on a piece of filter paper & draw a circle around the
spot with a soft pencil.
2. Allow the spot to dry and then put a drop of Ninhydrin reagent on the spot.
3. Wait for at least 20 min to observe the color.
4. Record your observations.

Report Writing:
Submit a laboratory report on this experiment one week from the date of the
experiment.
Include the following in your report:
1. Principle
2. Materials Required
3. Procedure
4. Observation
5. Reactions
6. result
7. Precautions
Give reactions for all test (Biuret and Ninhydrin Test)

https://www.youtube.com/watch?v=OsdhNtNNNds&t=11s ….ninhydrin test

tube and biuret
https://www.youtube.com/watch?v=mzoHF3VK9Ek …..ninhydrin
https://www.youtube.com/watch?v=wmhmAESv72E …..ninhydrin first part

https://www.youtube.com/watch?v=QacQmS3aaTI.....carb,prot,fat
https://youtu.be/7Vk3oxm8zxw …..test by alcohol
https://www.youtube.com/watch?v=EyGVlgZoHaQ ....biuret

Principle of Biuret test:

 Biuret test is a general test for compounds having a  peptide  bond. Biuret is a
compound formed by heating urea to 180° C. When biuret is treated with dilute
copper sulfate in alkaline condition, a purple colored compound is formed. This is
the basis of biuret test widely used for identification of proteins and amino acids.

This test is given by compounds containing two or more peptide bond (CO-NH
group). Since all proteins and peptides possessing at least two peptide linkage ie.
tripeptide gives positive biuret test.
 The principle of biuret test is conveniently used to detect the presence of
proteins in biological fluids.
 Alkaline CuSO4 reacts with compounds containing two or more peptide bonds
to give a violet colored product which is due to formation of co-ordination
complex of cupric ions with un-shared electron pairs of peptide nitrogen and O2 of
water.

Biuret reagents:  Copper sulfate (CuSO4)

 Sodium hydroxide (NaOH)
 Sodium potassium tartarate (commonly known as Rochelle salt)

Ninhydrin Test:
Principle:
 This test is a general test and thus given by all amino acids. This test is due to a
reaction between a amino group of free amino acid and ninhydrin. Ninhydrin is a
powerful oxidizing agent and its presence, amino acid undergo oxidative
deamination liberating ammonia, CO2, a corresponding aldehyde and reduced
form of ninhydrin (hydrindantin). The NH3 formed from an amino group reacts
with another molecule of ninhydrin and is reduced product (hydrination) to give a
blue substance diketohydrin (Ruhemanns complex). However, in case of imino
acid like proline and hydroxyproline, a different product having a bright yellow
color is formed. Asparagine, which has a free amide group, reacts to give a brown
colored product.