CARBOHYDRATES

Carbohydrates may be defined as polyhydroxy aldehydes or ketones or compounds, which

produce them on hydrolysis.
CLASSIFICATION OF CARBOHYDRATES
1. Monosaccharides: It cannot be further hydrolyzed (e.g., Glucose, fructose, galactose).
2. Disaccharides: On hydrolysis, they yield two monosaccharide units (e.g., maltose, lactose,
sucrose).
3. Oligosaccharides: They contain 3 – 10 monosacchraides units (e.g., Trisaccharides,
tetrasaccharides (raffinose), etc.
4. Polysaccharides: On hydrolysis, they yield more than 10 monosaccharides (starch, glycogen,
cellulose).
Glucose is an aldohexose. It is also known as “grape sugar” because of its presence in ripe grapes.
It is also called dextrose because it causes dextro-rotation of plane-polarized light. All starch in
our diet is converted into glucose before absorption from the intestine.
Fructose is a ketohexose. It is sweeter than glucose. It is found in fruits. It is also known as
laevulose because it causes laevorotation of plane-polarized light.
Galactose is an aldohexose. Galactose and glucose are products of hydrolysis of lactose (found in
milk). It is a constituent of glycolipids of neural tissue.
The disaccharides of importance in humans are maltose (formed during the digestion of starch),
lactose (found in milk) and sucrose (table sugar).
Each disaccharide is made up of two monosaccharides as follows:
MALTOSE - Glucose + Glucose
SUCROSE - Glucose + Fructose
LACTOSE - Glucose + Galactose
Maltose and lactose are reducing sugars, because they have a free aldehyde group. On the other
hand, in the case of sucrose, the aldehyde group of glucose is linked with the keto group of
fructose. Therefore, sucrose is a non-reducing sugar.
Starch is made up of a large number of glucose molecules, which are connected by α-1:4 and α-
1:6 glycosidic linkages. It is insoluble in cold water, but when boiled with water it forms a colloidal
solution.
Dextrins are intermediary products formed during the hydrolysis of starch. Depending upon
the colour formed with iodine, they are called amylodextrin (violet colour), erythrodextrin (red
colour) and achrodextrin (no colour). They are only partially precipitated by full saturation with
ammonium sulphate. Dextrin shows slight reducing action because of a free aldehyde group at the
end of the chain in the large molecule.
 REACTIONS OF CARBOHYDRATES
1. MOLISCH’S TEST: (General test for Carbohydrates)
Principle – Carbohydrates undergo dehydration with strong acids to form furfural or a furfural
derivative, which will give a coloured complex with Phenolic compound.
Reagents – Molisch’s reagent – 1 % Alpha Naphthol in 95% Alcohol & Concentrated Sulphuric acid
Procedure – Take 2ml of the given sample in a clean dry test tube. Add 3 drops of Molisch’s reagent
(1% Alpha-Naphthol in alcohol) and mix well. Incline the test tube slightly and add 2ml of
concentrated Sulphuric acid along the sides of the test tube so as to form a layer of acid below the
given solution.
Observation – A purple ring is seen at the junction of the two liquids
Positive
Inference – It shows the presence of Carbohydrates.

Observation – No purple ring is seen at the junction of the two liquids

Negative
Inference – It shows the absence of Carbohydrates.

2. BENEDICT’S TEST: (Test for Reducing sugar)

Principle – Benedict’s reagent contains Copper sulphate, Sodium carbonate and Sodium Citrate.
Copper sulphate provides cupric ions in solution, Sodium Carbonate provides alkaline medium for
the reaction and Sodium citrate keeps the cupric hydroxide in solution without getting precipitated.
Carbohydrates having a free aldehyde or ketone group tautomerize to form enediol in weak alkaline
medium. The enediol formed are highly reactive which has the property of reducing many ions like
copper, silver, and mercury.
Cupric ions of Cupric hydroxide are then reduced by enediol forms of sugar to form
cuprous hydroxide. Cuprous hydroxide on heating is converted to Cuprous oxide (Red precipitate).
The test is commonly used for the detection of glucose in urine as a screening test for detecting
Diabetes mellitus cases. It is positive for all reducing sugars. It is a semi-quantitative test, which can
be reported as:

Observation Reported as Approximate sugar concentration

No change of colour Nil Nil
Green colour (+) < 0.5 g %
Yellow colour (++) 0.5 – 1.0 g %
Orange colour (+++) 1.0 – 2 g %
Brick red precipitate (++++) Above 2 g %

Procedure – Take 5ml of Benedict’s reagent in a clean dry test tube. Then add 8 drops of the given
sample, mix well and boil for two minutes. Then cool the solution.

Observation – Green /Yellow /Orange / or Brick red color precipitate is formed*.

Positive
Inference – It shows the presence of reducing sugars
* (Write the color that you see)
Observation – No colored precipitate is formed.
Negative
Inference – It shows the absence of reducing sugars

3. BARFOED’S TEST: (Test for Monosaccharides)

Principle – In acidic medium monosaccharides tautomerize to form enediols which reduce cupric
ions to cuprous hydroxide which on heating is converted to Cuprous oxide (Red precipitate).
Procedure - Take 2 ml of Barfoed’s reagent. Add 2 ml of the given sample and boil for 30 seconds.
Allow the test tube to cool
Reagents – Copper acetate in 1% Glacial acetic acid
Observation – Red precipitate is formed at the bottom of test tube.
Positive
Inference – It shows the Presence of Monosaccharides.

Observation – No red precipitate is formed at the bottom of test tube.

Negative
Inference – It shows the absence of Monosaccharides.

4. SELIWANOFF’S TEST:(Specific test for Ketohexose)

Principle – Ketoses form furfural with lower concentrations of Hydrochloric acid. This reacts with
Resorcinol to form red coloured complex.
Reagents – Resorcinol and Concentrated Hydrochloric acid
Procedure – Take 3 ml of Seliwanoff’s reagent. Add 0.5 ml of the given solution. Boil for 1 minute.
Allow it to cool in the rack. Avoid prolonged boiling.
Observation –Cherry red colored complex is obtained.
Positive
Inference – It shows the presence of ketohexose (Fructose)

Observation – No Cherry red colored complex is obtained.

Negative
Inference – It shows the absence of ketohexose

5. FOULGER’S TEST: (Specific test for Ketohexose)

Principle – Ketoses undergo dehydration with concentrated H2SO4 to form furfural. This furfural
condenses with Urea in the presence of Stannous chloride to form a blue coloured complex.
Reagents – Urea, Stannous chloride, concentrated Sulphuric acid
Procedure – Take 3 ml of Foulger’s reagent. Add 0.5 ml of given solution. Boil vigorously for 2
minutes. Allow it to cool.
Observation –Blue color is formed.
Positive
Inference – It shows the presence of Ketohexose (Fructose)

Observation – No blue color is formed.

Negative
Inference – It shows the absence of Ketohexose

6. OSAZONE TEST:
Principle – The reaction involves the carbonyl group (aldehyde or keto) and the adjacent carbon. One
molecule of sugar reacts with one molecule of phenyl hydrazine to form phenyl hydrazone which
reacts with 2 more phenyl hydrazine molecules to form the osazones.
Reagents – Phenyl hydrazine hydrochloride, Sodium acetate, Glacial acetic acid
Procedure – To 5ml of given solution in a clear test tube, add a spatula full of phenyl hydrazine
sodium acetate mixture and shake it to dissolve. Heat it in a boiling water bath for 20 minutes. Allow
to cool spontaneously in the test tube stand. Examine a small amount of the crystals under
microscope.
Observation – A bright yellow precipitate is formed. Microscopic examination
Positive shows needle shaped/haystack shaped crystals.
Inference – It shows the presence of Glucosazone/ Fructosazone

Observation – A bright yellow precipitate is formed. Microscopic examination

Positive shows powder puff shaped crystals.
Inference – It shows the presence of Lactosazone

Observation – A bright yellow precipitate is formed. Microscopic examination

Positive
shows petal shaped crystals.
 Inference – It shows the presence of Maltosazone

GLUCOSAZONE/ LACTOSAZONE
FRUCTOSAZONE

MALTOSAZONE

7. ACID HYDROLYSIS OF SUCROSE (Specific test for Sucrose)

Principle – Sucrose will not answer for Benedicts test as it is a non-reducing sugar, after hydrolysis
by concentrated HCl sucrose is converted into glucose and fructose which are reducing
monosaccharides, which answers this.
Procedure – Take 5ml of the given solution and add 5 drops of concentrated Hydrochloric acid and
boil for 2 minutes. Cool under tap water. Add 20% Sodium carbonate drop wise until there is no
effervescence to neutralise the acid.
Observation – No effervescence is seen.
Inference – Concentrated Hydrochloric acid is completely neutralised.

Procedure – (BENEDICT’S TEST AFTER ACID HYDROLYSIS) – Take 5ml of Benedict’s

reagent in a clean dry test tube. Then add 8 drops of the acid hydrolysed sucrose solution, mix well
and boil exactly for two minutes. Then cool the solution.
Observation – Brick red color precipitate is formed.
Inference – It shows the presence of reducing sugars in the acid hydrolysed sucrose solution.

Procedure – (SELIWANOFF’S TEST AFTER ACID HYDROLYSIS) - Take 3 ml of Seliwanoff’s

reagent. Add 0.5 ml of the acid hydrolysed sucrose solution. Boil for 1 minute. Allow it to cool in the
rack.
Positive Observation – Cherry red colored complex is obtained.
 Inference – It shows the presence of ketohexose in the acid hydrolysed sucrose
solution.

8. IODINE TEST: (General test for Polysaccharides)

Principle – The large polysaccharide molecule adsorbs the smaller iodine molecules on their surface
to form an ill-defined colored complex. On cooling, the complex is reformed, and the color reappears.
Procedure – Take 2 ml of given solution in a test tube and add a drop of N/50 iodine. Record your
observation.
Observation – A blue color is seen.
Positive
Inference – It shows the presence of Polysaccharides

9. HYDROLYSIS OF STARCH:
Procedure – Take 3ml of given solution and add 2 drops of concentrated Hydrochloric acid and boil
for 2 minutes. Add 40% Sodium hydroxide till effervescence ceases to neutralise it. With the
already neutralized hydrolysate perform Benedicts test.
Observation – Green /Yellow /Orange / or Brick red color precipitate is formed
at the bottom of test tube.*
Positive
Inference – It shows the presence of reducing sugars. Hydrochloric acid has
hydrolysed starch into glucose to give positive result to Benedict’s reagent
* (Write the color that you see)

RESULT:
1. Reactions of Glucose were carried out and it is inferred that Glucose is a Monosaccharide, a
reducing sugar and an aldohexose.
2. Reactions of Fructose were carried out and it is inferred that Fructose is a Monosaccharide, a
reducing sugar and a ketohexose.
3. Reactions of Lactose were carried out and it is inferred that Lactose is a Disaccharide and a
reducing sugar.
4. Reactions of Maltose were carried out and it is inferred that Maltose is a Disaccharide and a
reducing sugar.
5. Reactions of Sucrose were carried out and it is inferred that Sucrose is a Disaccharide and a
non-reducing sugar.
6. Reactions of Starch were carried out and it is inferred that Starch is a polysaccharide and is a
non-reducing sugar
 IMPORTANT CARBOHYDRATE QUESTION AND ANSWERS
1) How will you classify carbohydrates?
Triose -
Glyceraldehyde

Tetrose - Erythrose

Number of carbons

Pentose - Ribose

Monosaccharides Hexose - Glucose

Aldoses -
Glyceraldehyde,
Glucose
Functional groups
Ketoses - Dihydroxy
acetone phosphate,
Fructose
Lactose - Glu + Gal

Number of
Maltose - Glu + Glu
saccharides
Disaccharides

Oligosaccharides (2 - Sucrose - Glu + Fru

10 monosaccharide
units covalently
linked) Trehalose - Glu + Glu

Starch, Glycogen,
Homopolysaccharide
Polysaccharides Cellulose, Inulin,
s
(more than Dextran
monosaccharide 10 Glycosaminoglycans-
units) Hetropolysaccharide
Mucopolysaccharide
s
s
2) Name the general test for carbohydrates. What is its principle?
Molisch’s Test - Carbohydrates undergo dehydration with strong acids (concentrated
sulphuric acid) to form furfural derivatives which then condense with phenol (alpha-naphthol)
to form colored complex. The reaction takes place between the two layers which is seen as a
ring.
3) What is a furfural?
Furfurals are dehydrated products of carbohydrates.
4) What are the components of Benedict’s reagent? What are their uses?
Benedict’s reagent contains: Copper suphate – gives cupric ions, Sodium carbonate-
gives an alkaline medium and Sodium citrate- acts as a chelating agent

5) Why is Benedict’s Test a semiquantitative test?

Benedict’s Test is a semiquantitative test because an approximate sugar content in
the urine can be reported as follows depending on the different colours of precipitate
obtained
 Observation Reported as Approximate sugar concentration
No change of colour Nil Nil
Green colour (+) < 0.5 g %
Yellow colour (++) 0.5 – 1.0 g %
Orange colour (+++) 1.0 – 2 g %
Brick red precipitate (++++) Above 2 g %

6) Why is sucrose a non-reducing sugar?

Sucrose is a non-reducing sugar because the two monosaccharide units (Glucose and
Fructose) are held together by a glycosidic linkage between C-1 of α Glucose and C-2 of beta
Fructose. Since all the reducing groups (functional groups) are involved in glycosidic bond
formation, Sucrose is a non-reducing sugar.
7) Name another test to demonstrate the reducing property of carbohydrates.
Fehling’s Test
8) What are the advantages of Benedict’s test over Fehling’s Test?
Benedict’s Test is more specific than Fehling’s Test because strong alkali in Fehling’s
reagent may reduce uric acid, creatinine and other compounds which are normal
constituents of urine.
9) Name the test which helps to differentiate monosaccharides from reducing
disaccharides.
Barfoed’s Test
10) Name the tests which distinguish glucose from fructose?
Seliwanoff’s Test and Foulger’s Test
11) Why does Glucose, Fructose and Mannose form the same osazone?
Glucose, Fructose and Mannose form the same osazone because they differ only at the first
two carbon atoms which are masked by the attachment of two phenyl hydrazine molecules.
12) Why does Sucrose not form osazones?
Sucrose cannot form osazones because sucrose does not contain a free aldehyde or ketonic
group. It is not a reducing sugar. Only reducing sugars when treated with phenyl hydrazine
hydrochloride in the presence of acetate buffer form characteristic yellow coloured crystals.
13) What is the confirmatory test for sucrose?
Acid hydrolysis of Sucrose followed by Benedict’s Test and Seliwanoff’s Test
14) Name the general test to detect polysaccharides.
Iodine Test
15) Which test is employed in the clinical laboratory to detect glucose in urine?
Benedict’s Test
16) Name some non carbohydrates with reducing property.
Vitamin C, Homogentisic acid, Salicylates, and glucuronides
17) What is the tests done to detect Pentose?
Bial’s Test
18) What is the tests done to detect Galactose?
Mucic acid test
 AMINO ACIDS & PROTEINS
Proteins are macromolecular polypeptides—i.e., very large molecules (macromolecules)
composed of many peptide-bonded amino acids. They are large molecules with definite size, shape
& charge. Proteins acquire charges depending upon the pH of the medium in which it is present. At
the isoelectric pH the net charges will be nil, the solubility will be minimum and the proteins will be
precipitated. If the pH of the medium is acidic to isoelectric pH, the proteins acquire +ve charge.
The protein will acquire negative charge when the pH is alkali to isoelectric.
A colloid is a system in which the particles have a diameter in the range of one millimicron to 200
millimicrons. All proteins form colloidal solution. The colloidal solutions are classified as
Suspensoids & Emulsoids.
.

1. Suspensoid: The stability of colloidal solution in suspensoid type is due to the charges on the
particles which prevent particles from coagulation. If the charges are neutralized, protein get
precipitated.
2. Emulsoid: In emulsoid type, the stability of solution is due to charges and the hydration (shell of
solvent around the particle). So in this type of protein, it gets precipitated by neutralisation of the
charge and removal of the hydration (by concentrated salt solution). Most of the proteins form
emulsoid type of solution.
Reactions of amino acids and proteins can be studied under two broad headings:
A. Precipitation Reactions of Proteins – Salting out, Isoelectric precipitation, Precipitation by
Organic Solvents, Precipitation by Heavy Metal Ions & Precipitation by Alkaloidal Reagents

B. Color Reactions of amino acids - Proteins undergo certain reactions producing coloured
compounds/complexes, which are characteristic of the side chains (functional groups) of the amino
acids they contain.
 REACTIONS OF PROTEINS

1. PRECIPITATION BY HEAVY METAL IONS

a) By Lead acetate:
Principle – In alkaline medium provided by the salt of any metal, proteins become negatively
charged and exist as anions. The positively charged metal ions neutralise the negatively charged
proteins to form a precipitate of lead proteinate.
Procedure – To 2ml of protein solution, add 5-10 drops of 10% lead acetate solution drop by drop.
Mix well.
Observation – A white precipitate is obtained.
Inference – It shows the presence of Proteins.

b) By Mercuric chloride:
Principle – In alkaline medium provided by the salt of any metal, proteins become negatively charged
and exist as anions. The positively charged metal ions neutralise the negatively charged proteins to
form a precipitate of mercuric proteinate.
Procedure – To 2 ml of protein solution add 5 drops of 2% mercuric chloride. Mix well.
Observation – A white precipitate is formed.
Inference – It shows the presence of Proteins.

2. PRECIPITATION BY ALKALOIDAL ANIONIC REAGENTS:

a) By Sulphosalicylic acid:
Principle – Alkaloidal reagents are negatively charged complexes. Proteins acquire a positive charge
in acidic medium and are neutralised by the negatively charged alkaloidal reagent to form a
precipitate of protein salicylate.
Procedure – To 2ml of protein solution add 2-3 drops of freshly prepared 3% Sulphosalicylic acid.
Observation – A White precipitate is formed.
Inference – It shows the presence of Proteins

b) By Picric acid:
Principle – Picric acid being an alkaloidal reagent is a negatively charged complex and it neutralises
the positively charged protein to form a precipitate of protein picrate.
Procedure – To 2-3 ml of protein solution, add 2-3 drops of Picric acid reagent. Mix well.
Observation – A yellow precipitate is formed.
Inference – It shows the presence of Proteins
c) By Tannic acid:
Principle – Alkaloidal reagents are negatively charged complex. Protein acquires positive charge in
acidic medium and are neutralised by negatively charged alkaloidal medium to form a precipitate of
protein tannate.
Procedure – To 2ml of protein solution, add 2-3 drops of Tannic acid reagent. Mix well.
Observation – A light brown precipitate is formed.
Inference – It indicates the presence of proteins.
 3. PRECIPITATION BY CONCENTRATED SALT SOLUTION:
a) Precipitation by half saturation with ammonium sulphate:
Principle – Ammonium sulphate solution precipitates protein by removing shell of hydration (salting
out). As albumin has a greater degree shell of hydration; it cannot be precipitated by half saturation
with ammonium sulphate.

Procedure – To 3ml of protein solution add 3ml of saturated solution of ammonium sulphate and mix
well. Allow to stand for 5 minutes. Filter it. To the filtrate add 3ml of 40% Sodium hydroxide and 2-
3 drops of Copper sulphate and mix well.
Observation – Filtrate gives violet color
Inference – Violet color is obtained on Biuret test as proteins are not precipitated by half saturation
with ammonium sulphate.

b) Precipitation by full saturation with ammonium sulphate:

Principle – Ammonium sulphate solution precipitates protein by removing shell of hydration (salting
out). As albumin has a greater degree shell of hydration; it is precipitated by full saturation with
ammonium sulphate.
Procedure – Saturate 3ml of protein solution with solid ammonium sulphate. Mix well till a few
undissolved crystals settle down. Filter it. To the filtrate add 3ml of 40% Sodium hydroxide and 2- 3
drops of Copper sulphate and mix well.
Observation – Filtrate gives no violet color
Inference – Violet color is not obtained as proteins are fully precipitated by full saturation with
ammonium sulphate.

4. HEAT COAGULATION TEST:

Principle – Heat coagulable protein undergoes denaturation on heating. This denatured protein
solution is a suspension which gets precipitated at isoelectric pH by adding 1% acetic acid. (Acetic
acid addition also excludes the phosphate interference with coagulation reaction). The upper portion
of the test tube is heated because lower half acts as a control.
Procedure – i) Fill ¾ of the test tube with the protein solution. Hold the test tube at its bottom, incline
it slightly and heat the upper portion of the solution.
ii) If coagulum is obtained then add few drops of 1% acetic acid.
Observation – i) A white coagulum is formed. ii)The coagulum is precipitated.
Inference – It indicates the presence of proteins.
NOTE:
Albumin – Heat coagulable; Precipitated by full saturation.
Casein & Gelatin - Non heat coagulable; Precipitated by half saturation
Peptone - Non Heat coagulable; not precipitated even by full saturation. Peptone
is highly hydrated, and the size of the molecule is so small and hence not precipitated even by
full saturation test

5. BIURET TEST: (Group test for proteins)

Principle – This is a group test for proteins. Protein in serum reacts with Biuret reagent to form
a purple-coloured complex the purple colour is due to coordination complex between cupric
ions and nitrogen atoms of the peptide bond (CO- NH). All proteins and peptides possessing at
least two peptide bonds answer Biuret test.
Procedure – To 2ml of given solution add 2ml of 5% Sodium hydroxide solution and mix well.
Then add 3 to 4 drops of 1% copper sulphate.
Observation – Violet color is obtained.
Inference – It shows the presence of Proteins.

6. XANTHOPROTEIC TEST: (Specific test for aromatic amino acids)

Principle – Nitration of benzene ring gives yellow colour which turns into orange colour in
alkaline medium due to formation of sodium salt of nitrobenzene.
Procedure – i) To 3ml of given solution add 1ml of concentrated Nitric acid. Boil for a minute
and then cool it under tap water. Divide the solution into 2 parts. ii) To one part add 1 ml of
40% Sodium hydroxide and use the other part as control.
Observation – Yellow color intensifies and orange color is obtained.
Inference – It shows the presence of aromatic amino acids.

7. COLE’S MERCURIC NITRITE TEST: (Test for Phenolic group)

Principle – Mercury forms complex with phenolic hydroxyl group of tyrosine to form mercuric
phenolate, which in the presence of sodium hydroxide form red coloured sodium salt of
mercuric phenolate.
Procedure – i) To 1ml of given solution add 1ml of 10% Mercuric sulphate in Sulphuric acid.
Boil for 1 minute. Cool ii) Add 3 drops of Sodium nitrite.
Observation – A red precipitate is obtained.
Inference – It shows the presence of Phenylalanine and Tyrosine.

8. ALDEHYDE TEST: (Test for Tryptophan)

Principle – The indole group of Tryptophan forms purple colour with formaldehyde in the
presence of Mercuric sulphate in acidic medium.
Procedure – To 2ml of given solution, 2 drops of 1/500 formalin and 1 drop of 10% Mercuric
sulphate in 10% Sulphuric acid is added and mixed well. Then 2ml of concentrated Sulphuric
acid is added along the sides of the test tube.
 Observation – A deep violet color ring is slowly developed at the junction of two fluids.
Inference – It shows the presence of Tryptophan.

9. SAKAGUCHI’S TEST: (Test for Arginine)

Principle – Guanidinium group of Arginine gives red colour with the reagents.
Procedure – To 3ml of given solution, add 5 drops of 20% sodium hydroxide, 4 drops of 1%
alpha naphthol. Mix well and add 10 drops of freshly prepared bromine water.
Observation – A bright red color is obtained.
Inference – It shows the presence of Arginine.

10. SULPHUR TEST: (Test for Sulphur containing amino acid)

Principle – Sulphur group combines with Sodium hydroxide to form Sodium sulphide which
reacts with Lead acetate to form black Lead sulphide precipitate.
Procedure – i) To 2ml of given solution add 2ml of 40% Sodium hydroxide, mix well, boil for
a minute.
ii) Then add 5 drops of Lead acetate.
Observation – A brownish black solution is obtained.
Inference – It shows the presence of Sulphur containing amino acids – Cysteine and Cystine.

11. PAULY’S TEST: (Test for Histidine)

Principle – This reaction is specific for imidazole group of Histidine. Diazotised Sulphanilic
acid reacts with imidazole group of histidine to give a cherry red colour under alkaline
condition.
Procedure – Take 1 ml of 0.5% Sulphanilic acid in a test tube. Add 1 ml of Sodium Nitrite.
Mix well and 2 ml of given solution. Add 1 ml of 10% of Sodium Carbonate to make it alkaline.
Observation – A deep cherry red color is obtained.
Inference – It shows the presence of Histidine.

12. MOLISCH’S TEST: (Test for Carbohydrates)

Principle – Carbohydrates in the glycoprotein undergo dehydration in the presence of
concentrated sulphuric acid to form furfural which gives purple colour ring with alpha
naphthol.
Procedure – To 2ml of given solution, 3 drops of Molisch’s reagent is added and mixed. The
test tube is inclined, and 2 ml of concentrated Sulphuric acid is added along the sides of the
test tube.
Observation – A purple ring is formed at the junction of the two liquids.
Inference – It shows the presence of Carbohydrates. Albumin forms the ring, Gelatine,
Casein and Peptone have no ring formation

RESULT:
1. The precipitation reactions of proteins are thus verified.
2. The color reactions of amino acids are thus studied.
IMPORTANT PROTEIN QUESTION AND ANSWERS

1) Name the heavy metals which are used to precipitate proteins.

Lead, Cadmium, Mercury etc. Based on this principle raw egg white is used as antidote
for mercury poisoning which binds with mercury and prevents its absorption.
2) Name the alkaloidal reagents which are used to precipitate proteins.
Sulpho salicylic acid, phosphor tungstic acid, trichloroacetic acid.
3) What is the practical significance of heavy metal precipitation?
Based on this principle raw egg white is used as antidote for mercury poisoning which
binds with mercury and prevents its absorption.
4) What is denaturation?
Denaturation is a change in the native character of a protein which is brought about by
various physical and chemical agents. It involves the breaking of all weak bonds. Only
peptide bonds remain intact.
5) Name few denaturing agents?
Denaturation is caused by external stress on the proteins such as solvents, inorganic
salts, exposure to acids or bases or by heat.
6) Is denaturation reversible?
Mostly reversible if the denaturing agent is removed. Denaturation of albumin is
irreversible.
7) What is the clinical significance of heat coagulation test?
Heat coagulation test is done to detect the presence of proteins in urine (proteinuria)
8) What is isoelectric pH?
The pH at which the molecule carries no net charge is known as isoelectric pH
9) What is the principle of Biuret test?
Cupric ions in an alkaline medium, form a violet colour with the peptide bonds present
in proteins.
10) Which amino acid answers Cole’s mercuric nitrite test?
Phenolic hydroxyl group of tyrosine
11) What is the other name for Cole’s mercuric nitrite test?
Modified Millon’s Test
12) What is the red precipitate formed in this reaction?
Sodium salt of mercuric phenolate
13) Name the amino acid with benzene ring .
Phenylalanine
14) Name the amino acid with indole ring.
Tryptophan
15) Which test is specific for indole group containing amino acid?
Aldehyde test
16) Name the sulphur containing amino acids.
Cysteine, Methionine
17) Name the amino acid which answers sulphur test.
 Cystiene
18) Why does Methionine not answer sulphur test?
Methionine doesn’t answer sulphur test due to the presence of thioether bond which
does not allow the release of sulphur in this reaction.
19) Name the test which is answered by the amino acid with imidazole group.
Pauly’s test for histidine
20) Which color reactions are not answered by (a) Casein. (b) Gelatin?
(a) Sulphur test- Because casein contains Methionine. Methionine doesn’t answer
sulphur test due to the presence of thioether bond which does not allow the release of
sulphur in this reaction.
(b) Millon's test is given by proteins containing phenolic amino acids. Gelatin does
not give this test.
21) Why does egg albumin answer Molisch’s test?
Egg albumin is a glycoprotein. So it answers Molisch’s test
*****