BIOCHEMISTRY AND CLINICAL PATHOLOGY

PRACTICAL LAB MANUAL

LAB MANUAL BIOCHEMISTRY AND CLINICAL

PATHOLOGY
D.PHARM Ist YEAR

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LIST OF EXPERIMENTS

S. No. EXPERIMENTS
1 Introduction to Biochemistry and Clinical Pathology Laboratory

2 To identify given sample of carbohydrate by qualitative tests.

3 To identify given sample of carbohydrate by qualitative tests.

4 To identify given sample of carbohydrate by qualitative tests.

5 To identify given sample of carbohydrate by qualitative tests.

6 To identify given sample of carbohydrate by qualitative tests.

7 To identify given sample of carbohydrate by qualitative tests.

8 To identify given sample of amino acid by qualitative tests.

9 To identify given sample of amino acid by qualitative tests.

10 To identify given sample of amino acid by qualitative tests.

11 To identify given sample of amino acid by qualitative tests.

12 To identify given sample of amino acid by qualitative tests.

13 To identify given sample of protein by qualitative tests.

14 To identify given sample of protein by qualitative tests.

15 To identify given sample of protein by qualitative tests.

16 To isolate casein from milk and its confirmation by chemical tests.

17 To study physical and chemical properties of fats and oils.

18 To conduct test on cholesterol to verify physical properties and chemical tests.

19 To detect normal constituents in given sample of urine by qualitative tests.

20 To detect abnormal constituents in given sample of urine by qualitative test.

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21 To detect abnormal constituents in given sample of urine by qualitative test.

22 To detect abnormal constituents in given sample of urine by qualitative test.

23 To estimate quantity of glucose in given sample of urine.

24 To estimate quantity of creatitine in given sample of urine.

25 To estimate quantity of urea nitrogen in given sample of urine.

26 To estimate quantity of to%I cholesterol in given sample of blood plasma.

27 To estimate quantity of serum alkaline phophatase in given sample of serum.

28 To estimate quantity of calcium in give sample of serum.

29 To estimate quantity of glucose in given sample of serum.

30 To study microscopic examination of sputum.

31 To study microscopic examination of faeces.

32 To visit a hospital to study methods of injecting drugs.

33 To visit to a pathology laboratory to study methods of withdrawal of blood.

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EXPERIMENT NO -1

OBJECT: Introduction to Biochemistry and Clinical Pathology laboratory.

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EXPERIMENT NO -2

OBJECT: To To identify given sample of carbohydrate (sample No. 1)

REFERENCE
1. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 22.

REQUIRMENTS
Chemical required: - Molish reagent, Benedict’s reagent

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel.

THEORY
Sugar that is capable of acting as a reducing agent is known as reducing sugar because it has a
free aldehyde group or a free ketone group. These free groups are oxidized to carboxylic acid. In
order for oxidation to occur, the cyclic form must first ring-open to give the reactive aldehyde.
Thus any sugar that contains a hemi acetal will be a reducing sugar.
Benedict’s test: the reagent that is used in the Benedict ’s test is known as Benedict’s reagent. It is a
clear blue solution containing copper (II) sulphate & sodium tartrate. Copper sulphate provides the cu²+
ions and sodium tartarate provides the required alkaline environment. In the test free aldehyde or keto
group in the reducing sugar reduce the alkaline cupric oxide to red coloured cuprous oxide.

Fehling’s test: It is a test that is done so as to differentiate between reducing and non-
reducing sugar. The test was developed by German chemist Hermann van Fehling in
1849. Fehling’s solution is a preparation of two separate solution, known as Fehling’s
solution A and Fehling’s solution B. Fehling’s solution A is an aqueous solution of
copper sulphate (II), which is deep blue. Fehling’s solution B is a colourless solution of
aqueous potassium sodium tartarate (also known as rochere salt), made in strong alkali,
commonly with sodium hydroxide.
The reducing sugar when reacts with Fehling’s solution gives yellow or brownish
red colour precipitation.

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Figure 1

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PROCEDURE
For Benedict’s test:
(1) Take a sample in a test tube.
(2) Add few amount of benedict’s solution. Then the mixture is shaken well and placed in boiling
water for a while.
(3) If the sample is reducing sugar a brick red precipitate will be formed otherwise the mixture
would remain clear blue.

For Fehling’s Test:

(1) Fehling’s solution A and B should add in equal amount and then sample will be added to it.
(2) Then the mixture is shaked well and placed in boiling water for a while.
(3) If a sample is reducing sugar a brick red precipitate will be formed, otherwise the mixture would
remain clear blue.
Result and observation
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of carbohydrate is ______________.

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EXPERIMENT NO -3

OBJECT: To To identify given sample of carbohydrate (sample No. 2)

REFERENCE
2. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 25.

REQUIRMENTS
Chemical required: Molish reagent, Benedict’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY
Sugar that is capable of acting as a reducing agent is known as reducing sugar because it has a
free aldehyde group or a free ketone group. These free groups are oxidized to carboxylic acid. In
order for oxidation to occur, the cyclic form must first ring-open to give the reactive aldehyde.
Thus any sugar that contains a hemi acetal will be a reducing sugar.
Benedict’s test: the reagent that is used in the Benedict’s test is known as Benedict’s reagent. It is a
clear blue solution containing copper (II) sulphate & sodium tartrate. Copper sulphate provides the cu²+
ions and sodium tartarate provides the required alkaline environment. In the test free aldehyde or keto
group in the reducing sugar reduce the alkaline cupric oxide to red coloured cuprous oxide.

Fehling’s test: It is a test that is done so as to differentiate between reducing and non-
reducing sugar. The test was developed by German chemist Hermann van Fehling in 1849.
Fehling’s solution is a preparation of two separate solution, known as Fehling’s solution A and
Fehling’s solution B. Fehling’s solution A is an aqueous solution of copper sulphate (II), which
is deep blue. Fehling’s solution B is a colourless solution of aqueous potassium sodium tartarate
(also known as rochere salt), made in strong alkali, commonly with sodium hydroxide.

The reducing sugar when reacts with Fehling’s solution gives yellow or brownish red colour
precipitation.

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Figure 2

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PROCEDURE
For Benedict’s test:
(1) Take a sample in a test tube.
(2) Add few amount of benedict’s solution. Then the mixture is shaken well and placed in boiling
water for a while.
(3) If the sample is reducing sugar a brick red precipitate will be formed otherwise the mixture
would remain clear blue.

For Fehling’s Test:

(1) Fehling’s solution A and B should add in equal amount and then sample will be added to it.
(2) Then the mixture is shaked well and placed in boiling water for a while.
(3) If a sample is reducing sugar a brick red precipitate will be formed, otherwise the mixture would
remain clear blue.
Result and observation
SAMPLE REAGENT ADDED INFERENCE RESULT

RESULT
The given unknown sample of carbohydrate is ______________.

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EXPERIMENT NO -4

OBJECT: To To identify given sample of carbohydrate (sample No. 3)

REFERENCE
1. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 25.

REQUIRMENTS
Chemical required: Molish reagent, Benedict’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY
Sugar that is capable of acting as a reducing agent is known as reducing sugar because it has a
free aldehyde group or a free ketone group. These free groups are oxidized to carboxylic acid. In
order for oxidation to occur, the cyclic form must first ring-open to give the reactive aldehyde.
Thus any sugar that contains a hemi acetal will be a reducing sugar.
Benedict’s test: the reagent that is used in the Benedict’s test is known as Benedict’s reagent. It is a
clear blue solution containing copper (II) sulphate & sodium tartrate. Copper sulphate provides the cu²+
ions and sodium tartarate provides the required alkaline environment. In the test free aldehyde or keto
group in the reducing sugar reduce the alkaline cupric oxide to red coloured cuprous oxide.

Fehling’s test: It is a test that is done so as to differentiate between reducing and non-
reducing sugar. The test was developed by German chemist Hermann van Fehling in 1849.
Fehling’s solution is a preparation of two separate solution, known as Fehling’s solution A and
Fehling’s solution B. Fehling’s solution A is an aqueous solution of copper sulphate (II), which
is deep blue. Fehling’s solution B is a colourless solution of aqueous potassium sodium tartarate
(also known as rochere salt), made in strong alkali, commonly with sodium hydroxide.

The reducing sugar when reacts with Fehling’s solution gives yellow or brownish red
colour precipitation.

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Figure 3

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PROCEDURE
For Benedict’s test:
(1) Take a sample in a test tube.
(2) Add few amount of benedict’s solution. Then the mixture is shaken well and placed in boiling
water for a while.
(3) If the sample is reducing sugar a brick red precipitate will be formed otherwise the mixture
would remain clear blue.

For Fehling’s Test:

(1) Fehling’s solution A and B should add in equal amount and then sample will be added to it.
(2) Then the mixture is shaked well and placed in boiling water for a while.
(3) If a sample is reducing sugar a brick red precipitate will be formed, otherwise the mixture would
remain clear blue.
Result and observation
SAMPLE REAGENT ADDED INFERENCE RESULT

RESULT
The given unknown sample of carbohydrate is ______________.

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EXPERIMENT NO -5

OBJECT: To To identify given sample of carbohydrate (sample No. 4)

REFERENCE
1. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 25.

REQUIRMENTS
Chemical required: Molish reagent, Benedict’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY
Sugar that is capable of acting as a reducing agent is known as reducing sugar because it has a
free aldehyde group or a free ketone group. These free groups are oxidized to carboxylic acid. In
order for oxidation to occur, the cyclic form must first ring-open to give the reactive aldehyde.
Thus any sugar that contains a hemi acetal will be a reducing sugar.
Benedict’s test: the reagent that is used in the Benedict’s test is known as Benedict’s reagent. It is a
clear blue solution containing copper (II) sulphate & sodium tartrate. Copper sulphate provides the cu²+
ions and sodium tartarate provides the required alkaline environment. In the test free aldehyde or keto
group in the reducing sugar reduce the alkaline cupric oxide to red coloured cuprous oxide.

Fehling’s test: It is a test that is done so as to differentiate between reducing and non-
reducing sugar. The test was developed by German chemist Hermann van Fehling in 1849.
Fehling’s solution is a preparation of two separate solution, known as Fehling’s solution A and
Fehling’s solution B. Fehling’s solution A is an aqueous solution of copper sulphate (II), which
is deep blue. Fehling’s solution B is a colourless solution of aqueous potassium sodium tartarate
(also known as rochere salt), made in strong alkali, commonly with sodium hydroxide.

The reducing sugar when reacts with Fehling’s solution gives yellow or brownish red colour
precipitation.

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Figure 4

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PROCEDURE
For Benedict’s test:
(1) Take a sample in a test tube.
(2) Add few amount of benedict’s solution. Then the mixture is shaken well and placed in boiling
water for a while.
(3) If the sample is reducing sugar a brick red precipitate will be formed otherwise the mixture
would remain clear blue.

For Fehling’s Test:

(1) Fehling’s solution A and B should add in equal amount and then sample will be added to it.
(2) Then the mixture is shaked well and placed in boiling water for a while.
(3) If a sample is reducing sugar a brick red precipitate will be formed, otherwise the mixture would
remain clear blue.
Result and observation
SAMPLE REAGENT ADDED INFERENCE RESULT

RESULT
The given unknown sample of carbohydrate is ______________.

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EXPERIMENT NO -6

OBJECT: To To identify given sample of carbohydrate (sample No. 5)

REFERENCE
1. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 25.

REQUIRMENTS
Chemical required: Molish reagent, Benedict’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY
Sugar that is capable of acting as a reducing agent is known as reducing sugar because it has a
free aldehyde group or a free ketone group. These free groups are oxidized to carboxylic acid. In
order for oxidation to occur, the cyclic form must first ring-open to give the reactive aldehyde.
Thus any sugar that contains a hemi acetal will be a reducing sugar.
Benedict’s test: the reagent that is used in the Benedict ’s test is known as Benedict’s reagent. It is a
clear blue solution containing copper (II) sulphate & sodium tartrate. Copper sulphate provides the cu²+
ions and sodium tartarate provides the required alkaline environment. In the test free aldehyde or keto
group in the reducing sugar reduce the alkaline cupric oxide to red coloured cuprous oxide.

Fehling’s test: It is a test that is done so as to differentiate between reducing and non-
reducing sugar. The test was developed by German chemist Hermann van Fehling in 1849.
Fehling’s solution is a preparation of two separate solution, known as Fehling’s solution A and
Fehling’s solution B. Fehling’s solution A is an aqueous solution of copper sulphate (II), which
is deep blue. Fehling’s solution B is a colourless solution of aqueous potassium sodium tartarate
(also known as rochere salt), made in strong alkali, commonly with sodium hydroxide.
The reducing sugar when reacts with Fehling’s solution gives yellow or brownish red
colour precipitation.

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Figure 5

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PROCEDURE
For Benedict’s test:
(1) Take a sample in a test tube.
(2) Add few amount of benedict’s solution. Then the mixture is shaken well and placed in boiling
water for a while.
(3) If the sample is reducing sugar a brick red precipitate will be formed otherwise the mixture
would remain clear blue.

For Fehling’s Test:

(1) Fehling’s solution A and B should add in equal amount and then sample will be added to it.
(2) Then the mixture is shaked well and placed in boiling water for a while.
(3) If a sample is reducing sugar a brick red precipitate will be formed, otherwise the mixture would
remain clear blue.

Result and observation

SAMPLE REAGENT ADDED INFERENCE RESULT

RESULT
The given unknown sample of carbohydrate is ______________.

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EXPERIMENT NO -7

OBJECT: To To identify given sample of carbohydrate (sample No. 6)

REFERENCE
1. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 25.

REQUIRMENTS
Chemical required: Molish reagent, Benedict’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY
Sugar that is capable of acting as a reducing agent is known as reducing sugar because it has a
free aldehyde group or a free ketone group. These free groups are oxidized to carboxylic acid. In
order for oxidation to occur, the cyclic form must first ring-open to give the reactive aldehyde.
Thus any sugar that contains a hemi acetal will be a reducing sugar.
Benedict’s test: the reagent that is used in the Benedict’s test is known as Benedict’s reagent. It is a
clear blue solution containing copper (II) sulphate & sodium tartrate. Copper sulphate provides the cu²+
ions and sodium tartarate provides the required alkaline environment. In the test free aldehyde or keto
group in the reducing sugar reduce the alkaline cupric oxide to red coloured cuprous oxide.

Fehling’s test: It is a test that is done so as to differentiate between reducing and non-
reducing sugar. The test was developed by German chemist Hermann van Fehling in 1849.
Fehling’s solution is a preparation of two separate solution, known as Fehling’s solution A and
Fehling’s solution B. Fehling’s solution A is an aqueous solution of copper sulphate (II), which
is deep blue. Fehling’s solution B is a colourless solution of aqueous potassium sodium tartarate
(also known as rochere salt), made in strong alkali, commonly with sodium hydroxide.

The reducing sugar when reacts with Fehling’s solution gives yellow or brownish red colour
precipitation.

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Figure 6

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PROCEDURE
For Benedict’s test:
(1) Take a sample in a test tube.
(2) Add few amount of benedict’s solution. Then the mixture is shaken well and placed in boiling
water for a while.
(3) If the sample is reducing sugar a brick red precipitate will be formed otherwise the mixture
would remain clear blue.

For Fehling’s Test:

(1) Fehling’s solution A and B should add in equal amount and then sample will be added to it.
(2) Then the mixture is shaked well and placed in boiling water for a while.
(3) If a sample is reducing sugar a brick red precipitate will be formed, otherwise the mixture would
remain clear blue.
Result and observation
SAMPLE REAGENT ADDED INFERENCE RESULT

RESULT
The given unknown sample of carbohydrate is ______________.

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EXPERIMENT NO -8

OBJECT: To To identify given sample of amino acid (sample No. 1)

REFERENCE
1. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 14.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test

tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines. Amino
acid react with Ninhydrin at pH 4, then reduction product is obtained from Ninhydrin to
yield a blue coloured substance. Amino acid is extremely sensitive to this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These aromatic
group undergoes reaction that have characteristics of benzene and benzene derivatives.
One such reaction is nitration of benzene ring with nitric acid . Activated benzene ring of
amino acid undergoes nitration. The nitration reaction in the presence of activated
benzene ring forms yellow product. This test is application to aromatic amino acid like
Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like glutamic acid.

Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5 drops
of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was observed. 

Xanthoprotic test

 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid was
added and heated over flame for 2 minutes till there was a colour change.

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 It was cooled under running tap water and 40% NaOH was added to make the solution
alkaline in nature.
 The colour change in nitro derivative of aromatic structure was observed.


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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of amino acid is identified as _________________.

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EXPERIMENT NO -9

OBJECT: To To identify given sample of amino acid (sample No. 2)

REFERENCE
Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 14.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test

tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines. Amino
acid react with Ninhydrin at pH 4, then reduction product is obtained from Ninhydrin to
yield a blue coloured substance. Amino acid is extremely sensitive to this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These aromatic
group undergoes reaction that have characteristics of benzene and benzene derivatives.
One such reaction is nitration of benzene ring with nitric acid . Activated benzene ring of
amino acid undergoes nitration. The nitration reaction in the presence of activated
benzene ring forms yellow product. This test is application to aromatic amino acid like
Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like glutamic acid.

Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5 drops
of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was observed.

Xanthoprotic test

 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid was
added and heated over flame for 2 minutes till there was a colour change.
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 It was cooled under running tap water and 40% NaOH was added to make the
solution alkaline in nature.
 The colour change in nitro derivative of aromatic structure was observed.


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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of amino acid is identified as _________________.

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EXPERIMENT NO -10

OBJECT: To To identify given sample of amino acid (sample No. 3)

REFERENCE
Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 14.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test

tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines. Amino
acid react with Ninhydrin at pH 4, then reduction product is obtained from Ninhydrin to
yield a blue coloured substance. Amino acid is extremely sensitive to this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These aromatic
group undergoes reaction that have characteristics of benzene and benzene derivatives.
One such reaction is nitration of benzene ring with nitric acid. Activated benzene ring of
amino acid undergoes nitration. The nitration reaction in the presence of activated
benzene ring forms yellow product. This test is application to aromatic amino acid like
Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like glutamic acid.

Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5 drops
of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was 

.Xanthoprotic test

 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid was
added and heated over flame for 2 minutes till there was a colour change.
 It was cooled under running tap water and 40% NaOH was added to make the solution

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 BIOCHEMISTRY AND CLINICAL PATHOLOGY

alkaline in nature.
 The colour change in nitro derivative of aromatic structure was observed.


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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of amino acid is identified as _________________.

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EXPERIMENT NO -11

OBJECT: To To identify given sample of amino acid (sample No. 4)

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 14.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.
Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test
tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines. Amino
acid react with Ninhydrin at pH 4, then reduction product is obtained from Ninhydrin to
yield a blue coloured substance. Amino acid is extremely sensitive to this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These aromatic
group undergoes reaction that have characteristics of benzene and benzene derivatives.
One such reaction is nitration of benzene ring with nitric acid . Activated benzene ring of
amino acid undergoes nitration. The nitration reaction in the presence of activated
benzene ring forms yellow product. This test is application to aromatic amino acid like
Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like glutamic acid.
Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5 drops
of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was observed. 
Xanthoprotic test
 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid was
added and heated over flame for 2 minutes till there was a colour change.
 It was cooled under running tap water and 40% NaOH was added to make the solution
alkaline in nature.
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 BIOCHEMISTRY AND CLINICAL PATHOLOGY

 The colour change in nitro derivative of aromatic structure was observed.



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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of amino acid is identified as _________________.

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EXPERIMENT NO -12

OBJECT: To To identify given sample of amino acid (sample No. 5)

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 14.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test

tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines. Amino
acid react with Ninhydrin at pH 4, then reduction product is obtained from Ninhydrin to
yield a blue coloured substance. Amino acid is extremely sensitive to this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These aromatic
group undergoes reaction that have characteristics of benzene and benzene derivatives.
One such reaction is nitration of benzene ring with nitric acid . Activated benzene ring of
amino acid undergoes nitration. The nitration reaction in the presence of activated
benzene ring forms yellow product. This test is application to aromatic amino acid like
Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like glutamic acid.
Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5 drops
of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was observed. 

Xanthoprotic test
 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid was
added and heated over flame for 2 minutes till there was a colour change.
 It was cooled under running tap water and 40% NaOH was added to make the solution
alkaline in nature.
 The colour change in nitro derivative of aromatic structure was observed.
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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of amino acid is identified as _________________.

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EXPERIMENT NO -13

OBJECT: To isolate casein from milk and its confirmation by chemical tests.

REFERENCE
2. Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 7.

REQUIRMENTS
Chemical required: Neumann’s solution,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

Principle
Neumann’s test
On heating with sodium hydroxide, casein is digested and phosphorous is released as
inorganic phosphate. Ammonium molybdate reacts with phosphorous in presence of
nitric acid to form a canary yellow precipitate.
Half saturation test
Neutral salt precipitate proteins by neutralization of charges on protein dehydration.
Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5
drops of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was observed. 

Xanthoprotic test

 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid
was added and heated over flame for 2 minutes till there was a colour change.
 It was cooled under running tap water and 40% NaOH was added to make the
solution alkaline in nature.
 The colour change in nitro derivative of aromatic structure was observed.

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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample is identified as _________________.

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EXPERIMENT NO -14

OBJECT: To To identify given sample of protein (sample No. 1)

REFERENCE
Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 9.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test

tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines. Amino
acid react with Ninhydrin at pH 4, then reduction product is obtained from Ninhydrin to
yield a blue coloured substance. Amino acid is extremely sensitive to this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These
aromatic group undergoes reaction that have characteristics of benzene and benzene
derivatives. One such reaction is nitration of benzene ring with nitric acid . Activated
benzene ring of amino acid undergoes nitration. The nitration reaction in the presence of
activated benzene ring forms yellow product. This test is application to aromatic amino
acid like Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like glutamic acid.
Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5 drops
of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was observed. Xanthoprotic test
 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid was
added and heated over flame for 2 minutes till there was a colour change.
 It was cooled under running tap water and 40% NaOH was added to make the solution
alkaline in nature.
 The colour change in nitro derivative of aromatic structure was observed.

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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of protein is identified as _________________.

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EXPERIMENT NO -15

OBJECT: To To identify given sample of protein (sample No. 2)

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 9.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test

tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines. Amino
acid react with Ninhydrin at pH 4, then reduction product is obtained from Ninhydrin to
yield a blue coloured substance. Amino acid is extremely sensitive to this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These aromatic
group undergoes reaction that have characteristics of benzene and benzene derivatives.
One such reaction is nitration of benzene ring with nitric acid . Activated benzene ring of
amino acid undergoes nitration. The nitration reaction in the presence of activated
benzene ring forms yellow product. This test is application to aromatic amino acid like
Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like glutamic acid.

Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and 5 drops
of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was observed. 

Xanthoprotic test
 2ml of sample was taken in test tube and equal volume of concentrated Nitric acid was
added and heated over flame for 2 minutes till there was a colour change.
 It was cooled under running tap water and 40% NaOH was added to make the solution
alkaline in nature.

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 The colour change in nitro derivative of aromatic structure was observed.

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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of protein is identified as _________________.

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EXPERIMENT NO -16

OBJECT: To To identify given sample of protein (sample No. 3)

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 9.

REQUIRMENTS
Chemical required: Ninhydrin solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

Principle
Ninhydrin test
Ninhydrin is a chemical used to detect ammonia or primary or secondary amines.
Amino acid react with Ninhydrin at pH 4, then reduction product is obtained from
Ninhydrin to yield a blue coloured substance. Amino acid is extremely sensitive to
this test.
Xanthoprotic test
Some amino acid contains aromatic group that are derivatives of Benzene. These
aromatic group undergoes reaction that have characteristics of benzene and benzene
derivatives. One such reaction is nitration of benzene ring with nitric acid . Activated
benzene ring of amino acid undergoes nitration. The nitration reaction in the presence
of activated benzene ring forms yellow product. This test is application to aromatic
amino acid like Tyrosin , Tryptophan, Phenylalanine, and non- aromatic acid like
glutamic acid.

Procedure:
Ninhydrin test
 1ml of sample (ex: filtered wheat flour or crushed seeds solution) solution and
5 drops of 0.2% Ninhydrin solution in acetone was added.
 It was incubated in water bath for 2 minutes.
 Allow it to cool for some time and blue color complex was

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observed. Xanthoprotic test

 2ml of sample was taken in test tube and equal volume of concentrated Nitric
acid
was added and heated over flame for 2 minutes till there was a colour change.
 It was cooled under running tap water and 40% NaOH was added to make the
solution alkaline in nature.
 The colour change in nitro derivative of aromatic structure was observed.

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OBSERVATION
SAMPLE REAGENTS ADDED INFERENCE RESULT

RESULT
The given unknown sample of protein is identified as _________________.

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EXPERIMENT NO -17

OBJECT: To To study physical and chemical properties of fats and oils

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 23.

REQUIRMENTS
Chemical required: acrolein solution, Million’s reagent,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY
Lipids are an essential component of the cell membrane. The structure is typically made of a
glycerol backbone, 2 fatty acid tails (hydrophobic), and a phosphate group (hydrophilic). As
such, phospholipids are amphipathic. In the cell membrane, phospholipids are arranged in a
bilayer manner, providing cell protection and serving as a barrier to certain molecules. The
hydrophilic part faces outward and the hydrophobic part faces inward. This arrangement helps
monitor which molecules can enter and exit the cell. For example, nonpolar molecules and small
polar molecules, such as oxygen and water, can easily diffuse in and out of the cell. Large, polar
molecules, for example, glucose, cannot pass freely so they need the help of transport proteins.
PROCEDURE
ACROLEIN TEST
It is used to detect the presence of glycerol and fats. When fat is treated strongly in presence of
dehydrating agent, the glycerol portion is dehydrated to form an unsaturated aldehyde.
OBSERVATION
SAMPLE REAGENT ADDED INFERENCE RESULT

RESULT
The sample is identified as ___________________.

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EXPERIMENT NO -18

OBJECT: To study physical and chemical properties of cholesterol.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 21.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY
Cholesterol is a waxy, fat-like substance that's found in all the cells in your body. Your body
needs some cholesterol to make hormones, vitamin D, and substances that help you digest foods.
Your body makes all the cholesterol it needs. Cholesterol is also found in foods from animal
sources, such as egg yolks, meat, and cheese.

If you have too much cholesterol in your blood, it can combine with other substances in the
blood to form plaque. Plaque sticks to the walls of your arteries. This buildup of plaque is
known as atherosclerosis. It can lead to coronary artery disease, where your coronary arteries
become narrow or even blocked.
PROCEDURE
LIBBERMAN BURCHAD TEST
2 ml of sample is allowed to treated with acetic anhydride then 3 drops od conc. Sulphuric acid
is allowed to added ro it, there is appearance of green colour.

SALKOWSKI TEST
With 2 ml of sample solution add 2 ml of chloroform. Then 2 ml of conc. Sulphuric acid is
allowed to add to the side wall of test tube. Acidic layer shows yellow colour while chloroform
ayer have deep red colouration.

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OBSERVATION
SAMPLE REAGENT ADDED INFERENCE RESULT

RESULT

The sample is identified as ___________________.

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EXPERIMENT NO -19

OBJECT: To detect the normal constituent in given sample of urine by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 27.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY

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EXPERIMENT NO -20

OBJECT: To detect the abnormal constituent in given sample of urine by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemistry and Clinical Pathology”,
Nirali Prakashan, Ed VIst, 2019, pp 29.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test

tubes,test tube holder.

THEORY

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EXPERIMENT NO -21

OBJECT: To detect the abnormal constituent in given sample of urine by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 29.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY

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EXPERIMENT NO -22

OBJECT: To detect the abnormal constituent in given sample of urine by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 29.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY

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EXPERIMENT NO -23

OBJECT: To detect the glucose in given sample of urine by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 45.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY
Principle: Glucose that is capable of acting as a reducing agent is known as reducing
sugar because it has a free aldehyde group or a free ketone group. These free groups are
oxidized to carboxylic acid. In order for oxidation to occur, the cyclic form must first ring-
open to give the reactive aldehyde. Thus any sugar that contains a hemi acetal will be a
reducing sugar.

Benedict’s test: the reagent that is used in the Benedict ’s test is known as Benedict’s
reagent. It is a clear blue solution containing copper (II) sulphate & sodium tartrate. Copper
sulphate provides the cu²+ ions and sodium tartarate provides the required alkaline
environment. In the test free aldehyde or keto group in the reducing sugar reduce the
alkaline cupric oxide to red coloured cuprous oxide.

RESULT
The given sample of urine contains _______________.

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EXPERIMENT NO -24

OBJECT: To detect the ceretine in given sample of urine by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 46.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY

Creatine is estimated by Folin modified method using photoelectric method. It represents the
waste product of creatine metabolism. Its appearance in urine is called creatinuria. Its excretion
increases in fever,starvation or in diabetes.

PROCEDURE

RESULT
The given sample of urine contains_------------------.

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EXPERIMENT NO -25

OBJECT: To detect the urea in given sample of urine by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 45.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY
It is formed mainly in liver by breakdown of amino acids. Its main function is to maintain the
reaction of blood constant. There are so many condition when the urea contain increases in the
body like nephritis, hepato-renal syndrome,uremia,etc.

PROCEDURE

RESULT
The given sample of urine contains_------------------.

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EXPERIMENT-26

OBJECT: To estimate the cholesterol in blood plasma by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 61.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY
Cholesterol is a waxy, fat-like substance that's found in all the cells in your body. Your body
needs some cholesterol to make hormones, vitamin D, and substances that help you digest foods.
Your body makes all the cholesterol it needs. Cholesterol is also found in foods from animal
sources, such as egg yolks, meat, and cheese.

If you have too much cholesterol in your blood, it can combine with other substances in the
blood to form plaque. Plaque sticks to the walls of your arteries. This buildup of plaque is known
as atherosclerosis. It can lead to coronary artery disease, where your coronary arteries become
narrow or even blocked.

PROCEDURE
PREPARATION OF TEST SAMPLE
Take .2 ml of serum and add colouring reagent.mix well and allow to stand for 10 mins.
Obtained the optical density.
PREPARATION OF STANDARD SAMPLE
Take .2 ml of std solution and add colouring reagent.mix well and allow to stand for 10
mins. Obtained the optical density.

RESULT
Cholesterol present in given sample is ________________mg%.

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EXPERIMENT-27

OBJECT: To estimate the serum alkaline phosphate in blood plasma by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 62.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY
The normal alkaline phosphate values varies from person to person. It is highest in childhood
during active bone growth and development observed. Its increasement can cause jaundice,
cirrohsis, hepatitis, etc.

PROCEDURE
PREPARATION OF TEST SAMPLE
Take .2 ml of serum and add alkaline buffer about 1 ml.mix well and allow to stand for 15
mins at room temperature.add 1ml sodium bicarbonate,1 ml potassium ferricynide. Obtained
the optical density.
PREPARATION OF STANDARD SAMPLE
Take .2 ml of standard solution and add alkaline buffer about 1 ml.mix well and allow to
stand for 15 mins at room temperature.add 1ml sodium bicarbonate,1 ml potassium
ferricynide. Obtained the optical density.

RESULT
Alkaline phosphte present in given sample is ________________kAunits

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EXPERIMENT-28

OBJECT: To estimate the calcium in blood plasma by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 66.

REQUIRMENTS
Chemical required: acetic anhydride,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY

Calcium present in the urine is treated with ammonium oxalate to produce a precipitate of
calcium oxalate.It found mostly in increased condition in hyperparathyroidism and stone
formation.

PROCEDURE
Collect 24 hour urine sample,and preserve it with 10 ml HCl solution.In centrifuge tube add 2ml
of urine. To this add 2 ml of water and 4%of ammonium oxalate along with few drops of methyl
blue indicator. Then add 2% of ammonia solution dropwise till red colour changes to yellow
keep to freeze for overnight. Take the supernatant and add 2% of ammonia solution keep it to hot
waterbath and titrate it with 0.01 NKMn04 till pink colour obtained. Note the burette reading.
Repeat it same with the blank.
RESULT
Calcium in given urine is ______________g %.

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EXPERIMENT-29

OBJECT: To estimate the glucose in blood plasma by qualitative test.

REFERENCE
1.Gupta G.D., Sharma S. “Practical Manual of Biochemislinical Pathology”, Nirali
Prakashan, Ed VIst, 2019, pp 66.

REQUIRMENTS
Chemical required: acetic anhydride ,sulphuric acid,chloroform,etc.

Apparatus required: - Beaker, glass rod, measuring cylinder, funnel,test tubes,test tube
holder.

THEORY

Glucose that is capable of acting as a reducing agent is known as reducing sugar because it
has a free aldehyde group or a free ketone group. These free groups are oxidized to
carboxylic acid. In order for oxidation to occur, the cyclic form must first ring-open to give
the reactive aldehyde. Thus any sugar that contains a hemi acetal will be a reducing sugar.

Benedict’s test: the reagent that is used in the Benedict ’s test is known as Benedict’s
reagent. It is a clear blue solution containing copper (II) sulphate & sodium tartrate. Copper
sulphate provides the cu²+ ions and sodium tartarate provides the required alkaline
environment. In the test free aldehyde or keto group in the reducing sugar reduce the
alkaline cupric oxide to red coloured cuprous oxide.

PROCEDURE
For Benedict’s test:
(1) Take a sample in a burette.
(2) Add few amount of benedict’s solution. Then the mixture is shaken well and placed
in boiling water for a while.
(3) If the sample is reducing sugar a brick red precipitate will be formed otherwise the
mixture would remain clear blue. Record the burette reading.

RESULT
Glucose in given serum is ______________g %.

EXPERIMENT-30

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OBJECT: To study microscopic examination of sputum.

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EXPERIMENT-31

OBJECT: To study microscopic examination of faeces.

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EXPERIMENT-32

OBJECT: To visit a hospital for study of injecting drugs.

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EXPERIMENT-33

OBJECT: To visit a pathology for study of methods of withdrawal of bloods.

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