PHARMACEUTICAL ENGINEERING & TECHNOLOGY

Essentials of Biochemistry – B Tech

Batch Experiment
A Identification of carbohydrate sample
B Identification of carbohydrate sample
A Identification of carbohydrate sample
B Identification of carbohydrate sample
A Identification of carbohydrate sample
B Identification of carbohydrate sample
A Identification of protein/amino acid sample
B Identification of protein/amino acid sample
A Identification of protein/amino acid sample
B Identification of protein/amino acid sample
A Identification of protein/amino acid sample
B Identification of protein/amino acid sample
A Identification of unknown carbohydrate/protein/amino acid
sample
B Identification of unknown carbohydrate/protein/amino acid
sample
A Identification of normal/abnormal constituents in urine sample
B Identification of normal/abnormal constituents in urine sample
A Identification of normal/abnormal constituents in urine sample
B Identification of normal/abnormal constituents in urine sample
A Identification of normal/abnormal constituents in urine sample
B Identification of normal/abnormal constituents in urine sample
A Examination (20 Marks)
B Examination (20 Marks)
A Viva Voce (10 Marks)
B Viva Voce (10 Marks)
A File Submission & Compilation
B File Submission & Compilation
Note: 1. Students are required to be present in the lab on time
2. Students must bring all lab essentials (apron, file etc) to the lab.
3. Extra Lab sessions may be scheduled if needed.

Department of Pharmaceutics, IIT (BHU) CY-103 Biochemistry Lab Manual

 BIOCHEMISTRY (CY103) PRACTICAL MANUAL
SCHEME I. GENERAL PROCEDURE FOR THE QUALITATIVE ANALYSIS OF CARBOHYDRATES

Objective: To identify the nature of carbohydrate by carrying out the systematic qualitative analysis

Materials Required: Test tubes, test tube stand, test tube holder, water bath, spatula, and dropper.

Reagents required: Molisch’s reagent, Iodine solution, Barfoed’s reagent, Fehling’s reagent, Benedict’s
qualitative reagent, Seliwanoff’s reagent, Bials reagent and Phenyl hydrazine.

Theory: The identification of unknown carbohydrate sample includes several tests involving specific
reagents which undergo characteristic color/ppt forming reactions with carbohydrates. The following
general scheme may be followed for identifying the class and nature of the given carbohydrate sample.
GENERAL SCHEME

Procedure:

S.No. Test Observation Inference

1. Molisch’s Test
2-3 drops of beta-naphthol solution
(Molisch’s reagent) are added to 2ml of
the test solution. Very gently add 1ml
of Conc. H2SO4 along the side of the
test tube.
2. Iodine test
4-5 drops of iodine solution are added
to 1ml of the test solution and
A deep violet coloration is Presence of
produced at the junction of carbohydrates.
two layers.
a) Blue-black (intense) color is Presence of starch.
observed.
b) Brown-blue color is Presence of glycogen.

Department of Pharmaceutics, IIT (BHU) CY-103 Biochemistry Lab Manual

 contents are mixed gently. observed
3. Barfoed’s test A deep blue colour is formed Presence of reducing
To 2 ml of the solution to be tested with a red ppt. settling down sugars.
added 2 ml of freshly prepared at the bottom or sides of the
Barfoed's reagent. Place test tubes test tube.
into a boiling water bath and heat for 3 Appearance of a red ppt as a Presence of reducing
minutes. Allow to cool. thin film at the bottom of the mono-saccharide.
test tube within 3-5 min.
The red ppt formation takes
Presence of reducing
more time (~10 min)
disaccharide
4. Fehling's test A red precipitate is formed. Presence of reducing
About 2 ml of sugar solution is added sugar.
to about 2 ml of Fehling’s solution
taken in a test-tube. It is then boiled
for 10 min.
5. Benedict’s test Formation of a green, red, or Presence of reducing
To 5 ml of Benedict's solution, add 1ml yellow precipitate. sugars.
of the test solution and shake each
tube. Place the tube in a boiling water
bath and heat for 3 minutes. Remove
the tubes from the heat and allow
them to cool.
5. Modified Benedict’s Test Formation of red or green Non-reducing sugar,
Take 4 ml of test solution in a test tube precipitate. Sucrose is confirmed.
and add 4-5 drops of Conc. HCl. Heat
the solution in a water bath for 15 min,
cool and add 2-5 drops of Conc. NaOH.
Take 2 ml of this solution and 2 ml of
Benedicts solution, heat for 2 min and
cool.
6. Seliwanoff test a) A cherry red colored Presence of ketoses.
To 3ml of Seliwanoff’s reagent, add precipitate within 5 minutes
1ml of the test solution. Boil in water is obtained.
bath for 2 minutes.
b) A faint red Presence of aldoses.
colour produced.
7. Bial's test a) A blue-green product Presence of pentoses.
Add 3ml of Bial’s reagent to 0.2ml of
the test solution. Heat the solution in a Presence of hexoses.
boiling water bath for 2 minutes. b) A muddy brown to gray
product

8. Osazone Test (Fischer’s reaction) a) Formation of beautiful Glucose/fructose

To 2 ml of the test solution, add 3ml of yellow crystals of osazone.
phenyl hydrazine hydrochloride, 0.1 g b) Needle shaped crystals/ Presence of lactose/
sodium acetate and a drop of glacial Hedgehog crystals/ Presence of maltose
acetic acid and mix. Keep in a boiling Sunflower shaped crystals
water bath for 30mts. Cool rapidly and
observe the crystals under
microscope.
SCHEME II. GENERAL PROCEDURE FOR THE QUALITATIVE ANALYSIS OF PROTEINS/AMINO ACIDS

Department of Pharmaceutics, IIT (BHU) CY-103 Biochemistry Lab Manual

Objective: To identify the nature of proteins/amino acids by carrying out the systematic qualitative
analysis
Materials Required: Test tubes, test tube stand, test tube holder, water bath, spatula, and dropper.

Reagents required: Copper Sulphate, Ninhydrin solution, Ammounium sulphate, Mercuric nitrate,
Trichloroacetic acid, Litmus paper, Mercury, Glacial acetic acid, Glyoxalic acid, Lead acetate, Sodium
nitroprusside, Ammonia, α-Naphthol and Bromine Solution

Theory: Refer the textbook

Procedure:

S. No. Test Observation Inference

I. Proteins
1. Biuret Test: To 2 mL protein solution, Blue color Presence of casein,
add 5-6 drops of dilute CuSO4 albumin, gelatin
(Fehling’s solution A diluted 1/10 with
water) - Add 3 mL 40% NaOH solution.
(Use 3 mL acetone and 1.5 mL water,
1 drop of dilute NaOH if the protein is
insoluble in water.)
2. Ninhydrin Test: To 1 mL protein Appearance of blue color Presence of amino acid
solution, add 5 drops of 0.2% (Free amino group in the
ninhydrin solution in acetone. Boil protein)
over a water bath for 2 min. Allow to
cool.
3 Salting out test: Add solid ammonium Precipitate is formed Presence of proteins
sulfate to about 5 mL of protein (Protein aggregates)
solution in a test tube (the salt should
be added in quantities of
approximately 1 g at a time). Agitate
the solution gently after each addition
to dissolve the ammonium sulfate
4 Heavy metal precipitation test: Treat Formation of white Presence of proteins
3 mL of the protein solution provided precipitate
with a few drops of mercuric nitrate
5 Acid precipitation test: Treat 3 mL of Precipitate is formed Presence of proteins
protein solution provided with a few
drops of trichloroacetic acid solution.
II. Amino acids
1. Solubility test: a) Soluble in water a) Presence of amino acid
Place a small sample in a test tube, b)
add a few ml of solvent (water or c)
alcohol or dil HCl or dil NaOH) and
warming if necessary.
Also check the acidity or basicity of
solution by using a litmus paper.
2. Ninhydrin Test: To 1 mL amino acid Appearance of blue color Presence of amino acid
solution, add 5 drops of 0.2%
ninhydrin solution in acetone. Boil
over a water bath for 2 min. Allow to
cool.
3. Xanthoproteic Test: To 2 mL amino Formation of yellow color Presence of tyrosine,

Department of Pharmaceutics, IIT (BHU) CY-103 Biochemistry Lab Manual

 acid solution in a boiling test tube, add or ppt. tryptophan, phenylalanine
equal volume of concentrated HNO3 (a (Activated aromatic ring)
drop of H2SO4). Heat over a flame for 2
min and observe the color. Cool under
tap, alkalinize by carefully adding 40%
NaOH.
4. Millon’s Test: To 2 mL amino acid Appearance of brick red Presence of tyrosine
solution in a test tube, add 1-2 drops color (Phenolic OH)
of Millon’s reagent (Hg dissolved in
Con HNO3). - Warm the tube in a
boiling water bath for 10 min.
5. Hopkin’s Cole Test: To a few mL of Formation of purple color Presence of tryptophan
glacial acetic acid containing glyoxylic at the junction of two (Indole ring)
acid, add 1-2 drops of the amino acid layers.
solution. Pour 1-2 mL H2SO4 down the
side of the sloping test tube.
6. Lead-Sulfide Test: Add 5 ml dilute Formation of brown color Presence of cysteine and
NaOH to 2 ml dilute lead acetate. A or ppt. cystine (SH group)
white precipitate of lead hydroxide
forms. Boil until the precipitate
dissolves with the formation of
sodium plumbate. Boil 2 ml amino
acid solution with a few drops of 40%
NaOH for 2 min. Cool and add a few
drops of the sodium plumbate
solution.
7. Nitroprusside Test: Put 2 ml amino Red color!! Presence of cysteine
acid solution into the test tube. Add
0.5 ml nitroprusside solution and
shake thoroughly. Add 0.5 mL
ammonium hydroxide.
8. Sakaguchi Test: 1 ml NaOH and 3 ml Formation of red color Presence of arginine
amino acid (arginine) solution is mixed (guanidine group)
and 2 drops of α-naphthol is added.
Mix thoroughly and add 4-5 drops of
bromine solution.

SCHEME III. GENERAL PROCEDURE FOR THE QUALITATIVE ANALYSIS OF URINE SAMPLE

Objective: To identify the normal and abnormal constituents of urine by carrying out the systematic
qualitative analysis

Materials Required: Test tubes, test tube stand, test tube holder, water bath, spatula, and dropper.

Department of Pharmaceutics, IIT (BHU) CY-103 Biochemistry Lab Manual

Reagents required: Benedict’s qualitative reagent, Fehling’s reagent, Ammonia, Ammounium sulphate,
Sodium nitroprusside, Nitric acid, Glacial acetic acid, Sulphosalicylic acid, Sulphur powder, Potassium
Ferricyanide, Picric acid.

Theory: Normal urine in humans is usually a transparent, yellow liquid. Normal urine contains 96 per cent
water and 4 per cent solids in solution. About half of the solids consist of urea, the main product broken down
in the metabolism of protein. The remainder includes nitrogen, chlorides, ketosteroids, phosphate, sulphur,
ammonia, creatinine, and uric acid.
Substances which are not present in easily detectable amounts in urine of normal healthy individuals but are
present in the urine under certain abnormal or diseased conditions are said to be abnormal constituents of
urine. The abnormal constituents include glucose, protein (albumin), ketone bodies (acetone, acetoacetic
acid, beta-hydroxy butyric acid), bilirubin etc. All the tests are characteristic to the nature of constituent
present in the urine sample and their presence is indicated by a characteristic observation.
Procedure:
S. No. Test Observation Inference
I. Physical Examination
9. Color a) Straw yellow to amber a) Normal urine
b) Red (urochrome/urobilin)
c) Yellow-brown b) Abnormal urine (RBC)
d) Orange or Bright yellow c) Bilirubin
d) Drugs or B-complex vitamins
2 Odour a) Urinoid odor a) Normal urine
b) Sweet or Fruity odor b) Diabetic due to acetone
c) Pungent c) Due to UTI infections
3 Turbidity a) Clear a) Normal Urine
b) Turbid b) May be due to urates,
calcium oxalate, cells
4 pH a) Acidic (freshly voided) a) Normal urine
b) Alkaline b) May be due to fermentation
by bacteria (UTI)
II. Chemical Tests
1 Glucose
a) Benedict’s test: in a test tube, a) Blue color a) Absence of sugar
add 3 ml Benedict’s reagent b) Change in color b) Presence of glucose
to 1 ml urine sample. Mix well Light green color 0.1 – 0.5 % of reducing sugar
and boil for 2-5 minutes. Cool Cloudy Green color 0.5 –1 % of reducing sugar
under tap water. Yellow to orange ppt 1 – 2 % of reducing sugar
b) Fehling’s test: In a test tube, Brick red ppt Above 2% of reducing sugar
add 1 ml Fehling A solution
and 1 ml Fehling B solution to Change in color Presence of glucose (Diabetes)
2 ml urine sample. Mix and
boil for 2 min.
2 Ketone bodies
Rothera’s test: Saturate 2-3 ml of Appearance of Presence of ketone bodies
urine in a test tube with permanganate color (ring) (ketosis, ketosuria)
ammonium sulfate. Add 2-3
drops of ammonia solution and
2-3 drops of freshly prepared 5%

Department of Pharmaceutics, IIT (BHU) CY-103 Biochemistry Lab Manual

 sodium nitroprusside and shake
well. Orange color which turns Presence of acetone (ketone
Legal’s Test: Add to the test tube cherry upon addition of conc bodies)
containing 3 ml of urine (study acetic acid
sample) 5 drops of 10% sodium
nitroprusside (Na2Fe(CN)5NO)
and 1 ml of 10% NaOH
3 Proteins
a) Heller’s test: in a test tube, A white coagulum is formed Presence of albumin
add 2 ml of urine slowly to 2 at the junction between the
ml of conc. nitric acid. 2 layers
b) Heat coagulation test: In a Formation of a coagulum is Presence of proteins
test tube, put 7-8 ml urine observed. It is stable after
sample. Incline the test tube the addition of glacial acetic
and heat the upper portion. acid & heating.
c) Sulphosalicylic acid test: Add
2-3 drops of (3%) Turbidity appears at upper Presence of albumin
Sulphosalicylic acid to 3-4 ml portion. (Proteinuria)
of urine
4 Bile salts/Bilirubin a) Sulfur powder will sink to a) Presence of bilirubin as it
Hay’s test: Sprinkle a little of the bottom of urine. lowers the surface tension
precipitated sulphur powder on of urine.
the surface of 2 ml urine. b) Sulfur remains on the b) Absence of bilirubin/bile.
surface of urine.
5 Thiamine (Vitamin B1)
To 1 ml of urine sample, add 5 ̶ 10 Yellow color Presence of thiamine
drops of 10% NaOH or KOH and
1-.2 drops of potassium
ferricyanide K3[FeCN)6], mix well
and heat.
6 Creatinine
Weill Test: Add 3- 4 ml of urine Pink to yellow color Presence of creatinine
and 3- 5 drops of 10% NaOH
solution in a test tube. Add a few
drops of sodium nitroprusside.
Jaffe reaction: Add 3- 4 ml of Orange color Presence of creatinine
urine, 3 ̶ 5 drops of 10% NaOH
and several drops of picric acid
into a test tube.

Department of Pharmaceutics, IIT (BHU) CY-103 Biochemistry Lab Manual