LA

BORATORY MANUAL
in
PHARMACEUTICAL
BIOCHEMISTRY

Edited by:
Jeanie C. Majarucon, RPh,
MSPharm
TABLE OF CONTENTS

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Activity No. Title Page

GENERAL SAFETY RULES AND PROCEDURES IN THE 3

LABORATORY
1 Physical Processes 4
2 Colloids 10
3 pH and Buffers 13
4 Color Reactions of Proteins 17
5 Monosaccharides and Disaccharides 21
6 Detection of Carbohydrates in Food Samples 25
7 Reduction Test for Sugar 30
8 Isolation of Glycogen 33
9 Physical and Chemical Properties of Lipids 36
10 Saponification: The Preparation of Soap 42
11 The Chemistry of Milk 45
12 Nucleic Acids 49
13 Protein Digestion 53
14 Enzymes 56
15 Characterization of Salivary Amylase 61

General Safety Rules and Procedures in The Laboratory

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 Each student enrolled in Pharmaceutical Biochemistry laboratory at Tagum Doctors
College, Inc. must follow specific safety rules and procedures. Some of these rules and
procedures are listed in your previous laboratory manual. Others are listed below. Those rules
or procedures listed in your laboratory manual that appear to be in conflict with those given
below should be resolved by asking your course instructor for clarification.

General safety rules and procedures:

1. Ensure that you have the following items for use during the laboratory.
a. A laboratory gown, towel, gloves, and mask.
b. Germicidal soap and 70% alcohol
c. Rags, cotton, and tissue paper
d. Detergent and sponge
e. Pencil, colored pens, and paper
f. Masking tape and pentel pen
g. Plastic bags and newspaper for disposal of wastes
2. No food or drinks are permitted in the laboratory at any time.
3. Only closed-toe shoes are to be worn in the laboratory. Sandals are not permitted.
4. Keep hands and other objects away from your face, nose, eyes, ears, and mouth. The
application of cosmetics is prohibited in the laboratory.
5. Work areas/surfaces must be disinfected before and after use.
6. Laboratory coats must be worn and buttoned while in the laboratory. Laboratory coats
should not be worn outside the laboratory.
7. Protective eyewear must be worn when performing any exercise or procedure in the
laboratory.
8. Long hair should be secured behind your head.
9. Hands must be washed before leaving the laboratory.
10. All unnecessary books, purses, briefcases, etc., must be kept off the countertops.
11. Never pipette anything by mouth (including water). Always use pipetting devices.
12. Label all materials with your name, date, and any other applicable information.
13. Dispose wastes in their proper containers.
14. When handling chemicals, note the hazard code on the bottle and take the appropriate
precautions indicated.
15. Do not pour chemicals down the sink.
16. Return all chemicals, reagents, cultures, and glassware to their appropriate places.
17. Do not pour biohazardous fluids down the sink.
18. Glassware should be washed with soap and water, and then rinsed with distilled water.
19. Flame transfer loops, wires, or needles before and immediately after use to transfer
biological material.
20. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes containing
infectious material.
21. Be careful around Bunsen burners. Flames cannot always be seen.
22. Immediately, report any broken glass, especially those containing infectious materials.
23. If you are injured in the laboratory, immediately contact your course instructor. Any
chemical or biological fluid spills must be immediately reported to your course instructor.
Follow all instructions given by your course instructor for cleaning up any spills or broken glass.
24. Familiarize yourself with safety equipment in the laboratory and emergency escape routes.

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25. Always wipe and clean the lenses of your microscope before putting it away. Use the
appropriate tissue paper and cleaning solution for this purpose.
26. Use appropriate universal precautions with all biological fluids.
27. Do not remove any materials from the laboratory without the written permission of the
course instructor.

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No.1
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 PHYSICAL PROCESSES

OBJECTIVES:
At the end of this experiment on exploration of physical processes, the students should be able
to:
1. To enumerate the different physical processes in the human body.
2. _______________________________________________________________________
3. _______________________________________________________________________

ASSIGNMENT:
Define the following terms:
1. Dialysis –

2. Diffusion –

3. Absorption –

4. Surface Tension –

5. Surfactants –

REAGENT/MATERIALS:
Chorizo skin 1% freshly prepared starch AgNO3 solution
5% freshly prepared gelatin Methyl orange indicator Red litmus paper
Phenolphthalein indicator Ferric chloride solution String
Animal charcoal/Activated charcoal Freshly prepared diluted bile 95% ethyl alcohol
1% NaCl solution Lugol’s solution Copper sulfate sol.
Colloidal ferric hydroxide Sodium hydroxide sol’n
Potassium ferrocyanide sol’n 0.01% aqueous sol’n of methyl blue Filter paper
1% sol’n of brown sugar Sulfur powder

APPARATUS:
Beakers Glass rod Glass dropper test tube
Test tube rack Tripod Wire gauze Test tube holder
Bunsen burner Test tube brush Erlenmeyer flask Graduated cylinder
Spatula

PROCEDURE:
A. Dialysis
1. Wash a chorizo skin thoroughly and rinse with distilled water several times.
2. With one end closed, fill the chorizo skin 2/3 full with equal volume of 1% starch and
1% NaCl.

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 3. Tie a string around the top of the chorizo skin and suspend it on a glass rod laid
across a beaker containing distilled such that the levels of the fluids inside and
outside the chorizo skin are the same.
4. After 60 minutes, test 1 ml of the water in the beaker with 1ml of AgNO 3 solution
and another 1ml of water in the beaker with 1ml of Lugol’s solution. Note down the
results. Which of the substances inside the suspended chorizo skin diffuses out?
Explain.
B. Diffusion thru Gelatin
1. Fill 3 test tubes to about 1/3 with warm solution of 5% gelatin.
2. After the liquid has cooled and gelatinized, add one drop of the following colored
solution to each copper sulfate, methyl orange and colloidal ferric hydroxide.
3. After 2 hours, note the depths to which the colored substances have penetrated the
gelatin.
4. Observe until the next laboratory period.
5. Compute for the molecular weight of each of the colored substances.
Is there any relation between the extent of the penetration and the molecular weights of the
colored substances?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
___________________________________.
6. Fill a test tube 2/3 full with slightly alkaline solution of 5% gelatin.
7. Place a drop of phenolpthalein and a few drops of potassium ferrocyanide.
8. When the gelatin has set, add 3 drops of ferric chloride.
9. Observe until the next laboratory period. Explain the results.
C. Adsorption
1. Place 20ml of 0.01% aqueous solution in methylene blue in a flask.
2. Add 0.1grams of activated charcoal. Shake vigorously.
3. Filter and note the color of the filtrate.
4. Allow the filter paper and its content to dry on the funnel.
5. Pour 10ml of 95% alcohol over the animal charcoal and collect filtrate in a clean
flask.
Explain the behavior of the methylene blue towards the animal charcoal.
______________________________________________________________________________
______________________________________________________________________________
_____________.
6. Place 25ml of 1% solution of brown sugar in a beaker.
7. Add 1grams of powdered animal charcoal.
8. Boil for 5minutes with occasional stirring.
9. Filter while hot and note the color of the solution. Explain the results.

Is the phenomenon of adsorption of any commercial value? Explain.

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
D. Demonstration of Surface Tension

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 1. In a clean dry test tube, measure 5ml of diluted bile.
2. Sprinkle a small pinch of sulfur powder on the surface of the solution. Note the
descent of the sulfur powder.
3. Repeat the experiment using water instead of diluted bile. Note down the results.
4. Repeat the experiment using soap solution instead of diluted bile.
5. Repeat the experiment using NaCl solution instead of diluted bile.
Give the physiological significance of surface tension.
______________________________________________________________________________
______________________________________________________________________________
_________________________________________________________________________.
Which substance lowers the surface tension?
______________________________________________________________________________
_____________________________________________________.
Which substance increases the surface tension?
______________________________________________________________________________
_____________________________________________________.

DATA/RESULTS:
Physical Process Observations
A. Dialysis Test for NaCl: Water in beaker+AgNO3
Positive test for NaCl:
Positive test for Starch:
Test for Starch: Water in beaker+Lugol’s sol’n

CuSO4:
Methyl Orange:
Ferric hydroxide:
Alkaline gelatin with phenolphthalein, ferric
potassium ferrocyanide, ferric hydroxide.

Sol’n of methylene blue+Charcoal

*Color of 1st filtrate:

*Color of 2nd filtrate after adding alcohol:

Brown sugar+Charcoal

Color of filtrate:
Bile+Sulfur:
Water+Sulfur:
Soap so’ln+sulfur:
NaCl sol’n+sulfur:

CONCLUSION:

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POST LABORATORY QUESTIONS:
1. How does an artificial kidney machine function? Why must the solution in such a
machine be changed at regular intervals?

2. What is the importance of surfactants in the body?

3. What is reverse osmosis? How can drinking salt water cause dehydration?

4. A solution that has a higher salt concentration than the blood is said to be
_________________?

5. Plasmolysis is defined as? Which solution cause plasmolysis? Isotonic, hypertonic,

hypotonic or normal solution?

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Name: ______________________________ Date Performed: ________________
Group number: ____________ Date Submitted: ________________

Experiment no. 2
COLLOIDS

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OBJECTIVES:
At the end of this experiment, students are expected to:
1. To describe the different properties of colloids.
2. __________________________________________________________________.
3. __________________________________________________________________.

ASSIGNMENT:
Differentiate between Emulsoid and Suspensoid in terms:
1. Affinity for solvent:

2. Foam formation

3. Precipitation with electrolysis

4. Reversibility

REAGENT/MATERIALS:
Gelatin 5% Trichloroacetic acid (TCA) Ethyl Alcohol
Gelatin Ammonium sulfate (saturated sol’n) solid MgSO4
0.45N NaCl sol’n 1N MgCl2 sol’n Ferric chloride (satrated sol’n)
10% MgSO4 sol’n 1N Na2SO4

APPARATUS:
Weighing balance Spatula Beaker 250ml Hot plate Wire gauze
Glass rod Test tubes Test tube holder test tube rack
Test tube brush Graduated cylinder 10ml Stopwatch Glass droppers

PROCEDURE:
A. Preparation of an Emulsoid
1. In a beaker, dissolve 10grams of gelatin in 200ml hot water by constantly stirring.
2. Pour 1ml of the prepared emulsoid into a test tube and cool under tap water. Note
what is produced.
3. Bring the test tube with the emulsoid into a water bath and heat again. Observe the
result. Record your observations in the Data Table.
B. Preparation of Suspensoid
1. Place 200ml of boiling water in a beaker.
2. Add 1ml of saturated ferric chloride solution. Note what is produced.
3. Repeat the last two procedures in Part A. Compare the results with those in Part A.
C. Foam Formation
1. Shake 10ml of the 5% gelatin sol’n (emulsoid) vigorously with air.
2. Stand and note the result after 15minutes. Does it form permanent foam?
3. Shake 10ml of the colloidal ferric chloride sol’n (suspensoid) vigorously with air.

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 4. Stand and note the result after 15minutes. Does it form permanent foam?
Is there a difference in the foam formation of the two sol’ns?
______________________________________________________________________________
_______________________________________________________________________.
D. Precipitation with electrolytes
1. Place 5ml of the 5% gelatin sol’n in a test tube.
2. Add saturated ammonium sulfate drop by drop counting the number of drops, until
a permanent precipitate is formed.
3. Repeat the procedure using colloidal ferric chloride instead of gelatin and note the
result. Observe and give the difference. Explain.
E. Reversibility
1. Place 5ml of the colloidal ferric chloride in a test tube and add 1ml of 10% MgSO 4
sol’n.
2. Allow to stand for ½ hour.
3. Treat 5ml of the 5% gelatin sol’n in the same way.
4. If no precipitate forms in the latter, add solid MgSO4 until saturated.
5. Decant the supernatant fluid from each of the two colloids.
6. Add an excess of water and note whether the two colloids are reversible. Note down
the results and explain.
F. Precipitation of Suspensoid Particle with Monovalent and Bivalent Ions which Carry
Charges of Opposite Signs.
1. Place 5ml of 1% colloidal ferric chloride sol’n in a test tube.
2. Add 0.45N NaCL sol’ a drop at a time counting the drops and shaking after each
addition until a permanent precipitate is formed. Note down the results.
3. Perform the same but using 1N sol’n of Na2SO4 and MgCl2. Compare the results with
those obtained with NaCL.

DATA/RESULTS:
Procedure Observations
A. Preparation of an emulsoid When Cooled:

When Heated:

B. Preparation of Suspensoid When Cooled:

When Heated:

C. Foam formation Gelatin(Emulsoid)

FeCl3(Suspensoid)
D. Precipitation with Electrolytes Number of drops of ammonium sulfate added to
form a permanent precipitate:
Gelatin(emulsoid):
FeCl3 (suspensoid):
Appearance of Precipitate
Gelatin(emulsoid):

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 FeCl3 (suspensoid):
E. Reversibility Number of drops of ammonium sulfate added to
form a permanent precipitate:
Gelatin(emulsoid):
FeCl3 (suspensoid):
Appearance of Precipitate
Gelatin(emulsoid):
FeCl3 (suspensoid):
F. Precipitation of suspensoid
particle with monovalent and
bivalent ions which carry
charges of opposite signs.

CONCLUSION:

POST LABORATORY QUESTIONS:

1. How can colloids be made to settle? What use is made of this process?

2. Why do colloids cannot pass through membranes but it do with filter paper?

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No.3
pH AND BUFFERS

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OBJECTIVES:
After this experiment, the students should be able to:
1. To describe how buffer works.
2. _______________________________________________________________.
3. _______________________________________________________________.

ASSIGNMENT:
1. What is a buffer?

2. What is Henderson-Hasselbach equation?

3. What makes eggwhite a good buffer?

REAGENT/MATERIALS:
Lemon juice Ice tea sol’n Distilled water Tomato juice
0.90% NaCL sol’n Eggwhite sol’n Acetic acid/acetate buffer 0.10M HCl
0.10M NaOH

APPARATUS:
Blender graduate cylinder 10ml & 50ml Pipette/glass droppers with calibration
Rubber bulb pH paper/pH meter Glass rod
Glass droppers Beaker 50ml

PROCEDURES:
A. Testing the pH of the solutions without buffer.
1. Prepare 5 clean beakers and add 15ml of lemon juice, ice tea sol’n, 0.90% NaCL sol’n,
tomato juice and distilled water to each beaker. Label.
2. Check the pH of each sol’n using pH meter/pH paper. Record your results.
3. Divide the solution in equal portion into two separate beaker then add 0.50ml,
1.00ml, 1.50ml, 2.00ml and 2.5ml of 0.10 M HCL to one beaker and add 0.50ml,
1.00ml, 1.50ml, 2.00ml and 2.5ml of 0.10 M NaOH to the other. Stir using a stirring
rod and read the pH after each addition.
4. Record your results for each addition.
B. pH of test sol’n with Acetic acid/acetate buffer.
1. Do the same procedure from 1-4 of Part A but this time, add 2.00ml of acetic
acid/acetate buffer to each beaker containing 15ml of different tests sol’n prior to
the addition of 0.10M HCl and 0.10M NaOH.
C. pH of test solution with Eggwhite sol’n.
1. Repeat the same procedure from 1-4 and this time used 2,00ml of eggwhite sol’n.

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DATE/RESULTS:
A. Test Solutions without buffer.
Test Solutions 0.10M HCl pH reading 0.10M NaOH pH reading
0.00ml 0.00ml
0.50ml 0.50ml
Lemon Juice 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
Ice Tea solution 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
0.90% NaCl 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
Distilled water 0.50ml 0.50ml
1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml

B. Test Solution added with Acetic acid/acetate buffer solution.

Test Solutions 0.10M HCl pH reading 0.10M NaOH pH reading
0.00ml 0.00ml
0.50ml 0.50ml
Lemon Juice 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
Ice Tea solution 1.00ml 1.00ml

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 1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
0.90% NaCl 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
Distilled water 0.50ml 0.50ml
1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml

C. Test Solution added with eggwhite solution

Test Solutions 0.10M HCl pH reading 0.10M NaOH pH reading
0.00ml 0.00ml
0.50ml 0.50ml
Lemon Juice 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
Ice Tea solution 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
0.90% NaCl 1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
0.50ml 0.50ml
1.00ml 1.00ml

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 1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml
0.00ml 0.00ml
Distilled water 0.50ml 0.50ml
1.00ml 1.00ml
1.50ml 1.50ml
2.00ml 2.00ml
2.50ml 2.50ml

CONCLUSION:

POST-LABORATORY QUESTIONS:
1. Why are buffer system important in the body?

2. How do antacids works?

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 4
COLOR REACTIONS OF PROTEIN

OBJECTIVES:

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After this experiment, the students should be able to:
1. To test the presence of proteins in the given sample.
2. _________________________________________________________.
3. _________________________________________________________.

ASSIGNMENT
1. Draw the general structure for amino acid. Encircle the amino group, Box the carboxyl
group, and Underline the other atom/group that are attach to the carbon atom bearing
all these groups.

2. Define a peptide linkage and show its formation with two amino acids:

3. Complete the table below with structure of the following amino acids:
Amino Acid Structure Amino Acid Structure Amino Acid Structure

Glycine Alanine Valine

Phenylalanine Tryptophan Tyrosine

Cysteine Methionine Arginine

REAGENT/MATERIALS:
1% fresh egg albumin solution Millon’s reagent Concentrated nitric acid
Ammonium hydroxide sol’n Hopkin’s-Cole reagent Concentrated sulfuric
acid
10% sodium hydroxide solution 0.5% copper sulfate sol’n
0.1% aqueous sol’n of ninhydrin Dilute alcohol-napthol sol’n
Sodium hypobromide sol’n saturated sodium carbonate sol’n
2% sodium nitroprusside 1% sodium hydroxide sol’n

APPARATUS:

Page | 17
 Graduate cylinder 10ml, 50ml Test tube Test tube Rack Stirring rod
Test tube holder Hot plate Beaker 500ml, 250ml Glass droppers
Water bath

PROCEDURE:
A. Millon’s Test
1. Place 1ml of egg albumin solution in a test tube.
2. Add 1drop of Millon’s reagent.
3. Mix and heat in the water bath. Observe.

B. Xanthoproteic Reaction
1. Place 1ml of egg albumin sol’n in a test tube.
2. Add 0.5ml of concentrated nitric acid.
3. Heat.
4. Cool and add ammonium hydroxide solution in excess. Observe.

C. Glyoxylic Acid Reaction (Hopkins-Cole)

1. Mix 1ml of egg albumin solution and 1ml of Hopkins-Cole reagent.
2. Incline the tube and allow 1ml of pure concentrated sulfuric acid to flow on the side
of the tube to form a layer of acid beneath the protein mixture.
(Sulfuric acid must be Pure)
3. Observe the color produced at the point of contact of the two liquids. If no color is
produced, make a gentle shake to cause slight mixing.

D. Biuret Test
1. Mix 1ml of egg albumin sol’n and 1ml of 10% NaOH.
2. Add 0.5% CuSO4 drop wise thoroughly after each addition. Observe.

E. Ninhydrin Reaction
1. Place 1ml of egg albumin solution in a test tube.
2. Add 0.5ml of freshly prepare 0.1% aqueous solution of ninhydrine.
3. Heat to boiling.
4. Allow to cool and observe the color produce.

F. Sakaguchi Reaction for Arginine.

1. To 3ml of egg albumin sol’n, add 1ml of 10% NaOH.
2. Add 6 drops of dilute alcohol-napthol sol’n.
3. Mix well and add 10 drops of sodium hypobromide solution.
4. Note results.

G. Nitroprusside Reaction for Sulfhydryl Group

1. To 1ml of egg albumin sol’n, add 0.5ml of saturated sodium carbonate solution and 5
drops of 2% sodium nitroprusside.
2. If the test is negative, add 2 drops of 1% NaOH. Purple color indicates the presence
of disulfide compounds.

DATA/RESULTS

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 Color Reactions Observation + or -
A. Millon’s Test

B. Xanthoproteic
Reaction

C. Glyoxylic Acid Reaction

D. Biuret Test

E. Ninhydrin Reaction

F. Sakaguchi Reaction

G. Nitroprusside Reaction

CONCLUSION:

POST-LABORATORY QUESTIONS
1. All proteins contain which elements? Which additional elements may be present?

2. Amino acids exhibit which property? Allotropism, amphoterism, or amphipathism?

Page | 19
EXPECTED RESULTS:
Write the expected results of the following color reactions.
Tests Expected Color Reactions Due to Presence of
Millon’s Test Phenol group in tyrosine
Xanthoproteic reaction Aromatic Amino acid
Glyoxylic acid reaction Indole nucleus in
tryptophan
Biuret Test Peptide linkage
Ninhydrin reaction Alpha amino group
Sakaguchi reaction Guanidine group of Arginine
Nitroprusside reaction Sulfhydryl group of Cysteine

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 5
MONOSACCHARIDES AND DISACCHARIDES

OBJECTIVES:
After this experiment, the students should be able to:
1. To identify the different properties of carbohydrates.
Page | 20
 2. ______________________________________________________________.
3. ______________________________________________________________.

ASSIGNMENT
Complete the table below with the structures of the following carbohydrates and name the
products of their hydrolysis (if there is any). Also classify each as Monosaccharide (M) and
Disaccharide (D).

Carbohydrate Structure Product of Hydrolysis M or D

Fructose

Galactose

Glucose

Lactose

Maltose

Sucrose

REAGENT/MATERIALS
2% and 5% freshly prepared solutions of the following:
Fructose Glucose Galactose Sucrose Maltose Lactose
Phenylhydrazine mixture (mix 2 parts of phenylhydrazine HCl and 3parts of sodium acetate by
weight. It should be freshly prepared).
Seliwanoff’s reagent Filter paper Ethyl alcohol
Concentrated HCl Phloroglucinol solid Concentrated HNO3
Orcinol-HCl reagent 4% solution of benzidine in glacial acetic acid
Concentrated NaOH Molisch reagent Concentrated H2SO4

APPARATUS:
Graduated cylinders 10ml Stirring rod Hot plate Water bath
Wire gauze Small test tubes Test tube rack Test tube holder

Page | 21
 Beaker 500ml Microscope Weighing balance Droppers

PROCEDURE:
A. Moore’s Test (influence of concentrated alkali)
1. Mix 1ml of 5% glucose and 1ml of concentrated NaOH.
2. Boil and note the change of color and the odor produced.

B. Molisch Test (α-napthol reaction)

1. Place 1ml of 5% glucose sol’n in a test tube.
2. Add 1drop of Molisch reagent and mix thoroughly.
3. Incline the tube and allow 1ml of concentrated H2SO4 to flow in the side of the tube.
The H2SO4 forms a layer at the bottom of the tube.
4. Note the color produced at the juncture of the two liquids.

C. Phenylhydrazine Reaction
1. To 1ml of 5% glucose solution, add 0.5g of phenylhydrazine mixture.
2. Shake well and heat in a boiling water bath for ½ hour.
3. Allow to cool slowly and examine the crystals under the microscope.
Compare the results obtained with the following figure given by Mulliken:
Carbohydrate Results
Glucose 4 to 5 minutes
Fructose 2 minutes
Maltose Osazone soluble in hot water
Lactose Osazone soluble in hot water
Sucrose 30 to 35 minutes after being hydrolyzes.

D. Seliwanoff’s Test (Resorcinol HCl Reaction)

1. Place 1ml of Seliwanoff’s reagent in each of the 6 test tubes.
2. Add three drops of each of the following freshly prepared 2% sugar solutions:
Test tube 1st 2nd 3rd 4th 5th 6th
Sugar Sol’n Fructose Glucose Galactose Sucrose Maltose Lactose
3. Boil in water bath.
4. Note carefully the color produced and record the time for the development of the
pink color in each of the following test tubes.
5. Filter and dissolve the precipitate in ethyl alcohol. Does it impart a red color to the
solution?
E. Phloroglucinol-HCl Test (Tollen’s Test)
1. Prepare 6 test tubes and to each place 0.5ml of each of the following 5% solutions
of: Galactose, glucose, fructose, sucrose, maltose, and lactose.
2. Add to each 0.5ml of concentrated HCl and a little phloroglucinol.
3. Heat in a boiling water bath.
4. Observe. Which of the sugars give red color?

F. Mucic Acid Test

1. To 1ml of 5% galactose, add 1ml of concentrated HNO3.
2. Heat in a boiling water bath for 1hour.
3. Stand until the next laboratory period.

Page | 22
 4. Examine in the microscope and draw them.
5. Repeat the experiment using glucose, fructose, lactose, maltose and sucrose. State
your results in the data table.
Is there a possibility for lactose to give positive Mucic Acid Test? Explain.
______________________________________________________________________________
_____________________________________________________________________________.

G. Bial’s Test
1. Heat 3ml of orcinol-HCl reagent to boiling in each of 6 test tubes.
2. Add 5 drops of the sugar solution (glucose, fructose, galactose, sucrose, maltose,
lactose) to each of the test tubes.
3. Note the results.

H. Tauber’s Benzidine Test

1. Place 0.5ml of a 4% solution of benzidine in glacial acetic acid in each of the 6 test
tubes.
2. Add one drop of the sugar solution (glucose, fructose, galactose, sucrose, maltose,
lactose) to each of the test tubes.
3. Heat for 5 minutes in a boiling water bath.
4. Cool immediately under running water.

DATA/RESULTS:
Test Observation
A. Moore’s Test Color change: Odor:

B. Molisch Test Color produced at the

juncture of two liquids.

C. Phenylhydrazine Color of crystals: Appearance of the crystals:

Reaction

D. Seliwanoff’s test 1st Fructose: 4th Sucrose:

Note:
1. Color produced and 2nd Glucose: 5th Maltose:
2. Number of minutes for
the development of
pink color
3. Color after dissolving 3rd Galactose 6th Lactose:
in ethyl alcohol

E. Phloroglucinol-HCl 1st Galactose: 4th Sucrose:

Test.
Yes if Red color is 2nd Glucose: 5th Maltose:
observed, No if not.

Page | 23
 3rd Fructose: 6th Lactose:
F. Mucic Acid Test 1st Galactose: 4th Sucrose:
Note:
1. The red color of
precipitate formed. 2nd Glucose: 5th Maltose:
2. Draw.

3rd Fructose: 6th Lactose:

G. Bial’s Test: 1st Galactose: 4th Sucrose:
Yes if green color is
observed; No if not 2nd Glucose: 5th Maltose:

3rd Fructose: 6th Lactose:

H. Tauber’s Benzidine 1st Galactose: 4th Sucrose:
Test:
Yes if Pink to Red is 2nd Glucose: 5th Maltose:
observed; No if not
3rd Fructose: 6th Lactose:

CONCLUSION:

POST-LABORATORY QUESTIONS:
1. How are carbohydrates formed in nature? Write the reaction for this process. Are
animals able to synthesize carbohydrates?

Page | 24
 2. What test should be performed to identify a disaccharide?

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 6
DETECTION OF CARBOHYDRATES IN FOOD SAMPLES

OBJECTIVES:
At the end of the experiment, students are expected to:
1. To detect the presence of carbohydrates in given food samples.
2. ______________________________________________________.
3. ______________________________________________________.

Page | 25
ASSIGNMENT:
1. List down foods that are commonly consumes by human, and classify them as to
Monosaccharide, Disaccharide, or Polysaccharide.
Monosaccharide Disaccharide Polysaccharide

2. What are the test that can be used to detect the presence of what carbohydrate in the
foods? Explain each.

REAGENTS/MATERIALS
1% glucose solution Low-fat fresh milk
1% sucrose solution Fruit juice
1% lactose solution Tea extract (soak the tea bag in hot water until the
Distilled water the water turns light brown)
Molisch reagent Saltine cracker
Iodine solution
Options:
Other food samples such as fruits, breads, noodles and other beverages could be used.
Depending on the size of the class (or how many students there are), the instructor may assign
different food samples to different groups.
APPARATUS:
18 test tubes 3 test tube racks 250ml beaker
Mortar and pestle Pipettes
Corks (to cover the test tubes for Molisch test) Vortex (optional)

PROCEDURES:
A. Preparation of Standards and Samples
1. Prepare and number nine regular-sized test tubes as directed below.
Note: two sets of these samples are needed for the two carbohydrate test.
1. 1ml of distilled water (control)
2. 1ml of 1% glucose solution
3. 1ml of 1% sucrose solution

Page | 26
 4. 1ml of 1% lactose solution
5. 1ml of 1% starch solution
6. 1ml of fruit juice (i.e., freshly squeezed orange juice or any commercially
available
fruit juice)
7. 1ml of tea extract (diluted to a light brown color)
8. 1ml Low-fat milk.
9. 1ml of saltine cracker mixture.
*To prepare a solid sample like saltine cracker, grind about half the standard piece of a
cracker using a mortar and pestle. Place about 100mg of sample in the test tube. Add 1ml of
distilled water. Shake to ensure adequate mixing.

B. Molisch Test
1. Add three drops of Molisch reagent to each sample.
2. Cover the test tube with a cork and shake well.
3. Incline the test tube at a 45oC angel and slowly add 20drops of concentrated sulfuric
acid (H2SO4). Notice the formation of two layers.
Note: Do not point the mouth of the test tube towards yourself or your classmates.
4. Observe the color produced where the two layers meet.
5. Record your observations. Remember that a purple color is a positive result for
monosaccharide.

C. Iodine Test
1. Add one drop of iodine solution to each test tube containing the standards and
samples.
2. Shake each test tube thoroughly.
3. Observe the control sample (the test tube with distilled or deionized water).
4. If none of the samples produced a different color compared with the control samples,
add 10 drops of distilled water to each test tube and another drop of iodine solution.
5. Shake the test tube well to ensure sufficient mixing of the sample and solvent.
6. Record your observations. Remember that polysaccharides can combine with iodine
to give a blue, red, violet, or purple complex.

DATA/RESULTS
MOLISCH TEST
Sample Observed Color Reaction to Molisch
reagent (+/-)
Distilled water (Control)
1% glucose solution
1% sucrose solution
1% lactose
1% starch solution
Fruit juice
Tea extract

Page | 27
 Low-fat fresh milk
Saltine cracker

IODINE TEST
Sample Observed Color Reaction to Molisch
reagent (+/-)
Distilled water (Control)
1% glucose solution
1% sucrose solution
1% lactose
1% starch solution
Fruit juice
Tea extract
Low-fat fresh milk
Saltine cracker

CONCLUSION

POST-LABORATORY QUESTIONS:
1. What are the products formed when each of the following carbohydrates is hydrolyzed?
a. Sucrose (table sugar)

b. Lactose

c. Maltose

d. Starch

2. What is the reaction of sucrose in Benedict’s test? Why is sucrose a non-reducing sugar?
Explain with a structural illustration.

3. Why can’t human digest cellulose?

Page | 28
4. One of the reasons why fructose is used in so many food products today is because it is much
sweeter than glucose. However some processed food manufacturers claim that their products
have “No high fructose syrup”, which gives the impression that fructose is not good for the
body. Explain the difference in the metabolism of fructose and glucose in the human body.

5. Among the carbohydrate tests performed in this experiment, which do you think is the
appropriate test to detect the presence of sugar in urine?

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 7
REDUCTION TEST FOR SUGAR

OBJECTIVES:
At the end of the experiment, students are expected to:
1. To enumerate the tests for reducing sugars.
2. ______________________________________________________.

Page | 29
 3. ______________________________________________________.

ASSIGNMENT
1. Differentiate between oxidation and reduction. In this experiment, which is oxidized?
Which is reduced? Is it the sugar of the reagent?

2. Complete the table below with indications for positive tests.

Reduction Test Positive Test
1. Fehling’s Test
2. Benedict’s Test
3. Nylander’s Test
4. Barfoed’s Test
5. Picric Acid Test

REAGENTS/MATERIALS:
Fehling’s solution A Nylander’s reagent
Fehling’s solution B Barfoed’s reagent
5% glucose solution Saturated Picric acid solution
Benedict’s solution 10% Na2CO3 solution

APPARATUS:
Graduated Cylinder 10ml Hot plate Dropppers Test tubes Test tube rack
Test tube holder Stop watch Beaker 500ml, 250ml Stirring rod
Water bath

PROCEDURES:
A. Fehling’s Test
1. Dilute 1ml of Fehling’s solution mixture (equal parts of Fehling’s solution A and B)
with 4ml of water.
2. Boil 1ml of the diluted mixture (if there is any change in color, discard and get a
freshly prepared solution).
3. Add 5% glucose drop by drop heating the mixture after each addition.
4. Note the changes.

B. Benedict’s Test
1. To 1ml of Benedict’s Solution, add 2 drops of 5% glucose solution.
2. Boil in a water bath for 2minutes and allow to cool. Observe.
C. Nylander’s Test
1. Mix 2 drops of 5% glucose solution with 1ml of nylander’s reagent
2. Heat for 5 minutes in a boiling water bath, and note the result.
D. Barfoed’s Test
1. Mix 1ml of Barfoed’s reagent with 2 drops of 5% glucose solution.
2. Heat for 30 seconds and allow to cool for 15 minutes. Observe.
E. Picric Acid Test

Page | 30
 1. To 1ml of 5% glucose solution, add 0.5ml of saturated picric acid solution and about
4 drops of 10% Na2CO3 solution.
2. Warm and note the result.

DATA/RESULTS: Describe in detail

Reduction Tests Observation
1. Fehling’s Test

2. Benedict’s Test

3. Nylander’s Test

4. Barfoed’s Test

5. Picric Acid Test

CONCLUSION: Give explanations for all your observation

POST-LABORATORY QUESTIONS:
1. What product is formed when the aldehyde end of glucose is oxidized?

2. How do hexoses affect alkaline Cu2+ complex ions, and what use is made of this reaction?

3. Do all three disaccharides act as reducing agents? Why or why not?

Page | 31
Name: ______________________________ Date Performed: ________________
Group number: ____________ Date Submitted: ________________

Experiment No. 8
ISOLATION OF GLYCOGEN

OBJECTIVES:
At the end of the experiment, students are expected to:
1. ___________________________________________________.
2. ___________________________________________________.
3. ___________________________________________________.

Page | 32
ASSIGNMENT:
1. What is glycogenesis? Glycogenolysis?

2. What are the enzyme needed for the synthesis of glycogen? For the breaking of
glycogen?

3. Draw the structure of glycogen.

CHEMICALS/MATERIALS:
Chicken liver Bunsen burner Beaker
0.1% acetic acid Graduated cylinder Stirring rod
0.01M iodine solution Spatula Filter paper
Distilled water Funnel Two test tubes
Molisch’s reagent Test tube rack Dropper
Conc. Sulfuric acid Pipette
Water bath Balance

PROCEDURES:
A. Extraction of Glycogen from Animal Tissue (Chicken liver)
1. Mince a piece of clean, fresh chicken liver and place this in a beaker.
2. Weigh 3grams of minced chicken liver and place this in a beaker.
3. Pour 12m l of boiling water into the beaker containing the minced chicken liver. Stir
the mixture and boil it for two minutes.
4. Heat the mixture for 30 minutes in a boiling water bath. Add 3ml of distilled water to
the mixture.
5. Add 1ml of 0.1% acetic acid to the mixture. Filter the mixture.

B. Qualitative Tests for the Presence of Extracted Glycogen

Prepare two test tubes. Label test tube #1 as Molisch’s test and test tube #2 as iodine
test. Pour 1ml of glycogen solution obtained from Procedure A in each test tube.
1. Molisch’s Test
Add a few drops of Molisch’s reagent to test tube #1. Carefully pour down 2ml of
concentrated sulfuric acid on the side of the test tube. Observe the color at the junction of the
two liquids.

Page | 33
 2. Iodine Test
Add a few drops of 0.01M iodine to test tube #2. Observe.

DATA/RESULTS:
Extraction of Glycogen from Chicken Liver
Procedure Observation
1. 12ml of boiling water +3g of
mince chicken liver

2. Mixture boiled for 2 minutes

3. Mixture heated in a boiling

water bath for 30minutes

4. Addition of 0.1% acetic acid

to the mixture

Qualitative Tests for the Presence of Extracted Glycogen

Procedure Observation
1. Molisch’s Test
Glycogen solution + Molisch’s
reagent

2. Iodine Test
Glycogen solution + Iodine

CONCLUSION:

POST-LABORATORY QUESTIONS:
1. What is the difference between amylopectin and glycogen?

Page | 34
2. What is glycogenesis? Glycogenolysis?

3. Which between glycogen and amylopectin is more highly branched? Draw their structure.

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 9
PHYSICAL AND CHEMICAL PROPERTIES OF LIPIDS

OBJECTIVES:
At the end of the experiment, students are expected to:
1. ___________________________________________________.
2. ___________________________________________________.
3. ___________________________________________________.

ASSIGNMENT:

Page | 35
 1. Draw the general structure of fats and triglycerides and identify the components:
glycerol and fatty acids.
General Structure

Fat or Triglyceride

Glycerol

Fatty Acids

2. Differentiate between saturated and unsaturated fatty acid and give one example for
each. Draw the structure and give its name.
Fatty Acid Saturated Unsaturated

Name

Structure

3. Define the following terms:

Emulsion

Surface Tension

4. Write the equation for Acrolein Test.

REAGENT/MATERIALS:
Olive oil Cottonseed oil Lard Palmitic acid
Page | 36
 Oleic acid Ether Chloroform Ethyl Alcohol
Cooking oil Anise Oil Litmus paper Lugol’s sol.
0.5% Na2CO3 Albumin solution Acetone Potassium bisulfite
Filter paper Rancid oil Beef fat Distilled water

APPARATUS:
Test tube Test tube holder Glass slide Test tube rack Cork
Pipette 5-ml Graduated cylinder Wire gauze Stop watch Dropper
Crucible tong Weighing balance Evaporating dish
Hot plate Microscope

PROCEDURES:
A. Solubility
Test the solubility of the different fats and fatty acids in water, ether, chloroform, and
hot alcohol. Write down your observation as Soluble (Clear), Slightly soluble (Cloudy),
Insoluble (Two layers). Which are the best solvents for fats?

B. Formation of Translucent Spot

1. Place 1 drop of cooking oil on a piece of ordinary writing paper.
2. Note the formation of semi-transparent spot.
3. Allow to evaporate spontaneously. Does the translucent spot disappear?
4. Repeat the same experiment using Anise oil instead of cooking oil. Note the
difference in the results.

C. Reactions of Fats.
1. Test the reaction of cooking oil with Litmus (both blue and red) paper previously
moistened with water. What is the reaction?
2. Allow the oil to stand uncovered until the next laboratory period and test again with
litmus paper. Is there any change in the reaction? If no change is observed, allow it
to stand until another laboratory period or until a change in the reaction is observed.
How would your account for the change?

D. Acrolein Test
1. Place 0.5g of potassium bisulfite (KHSO4) in a clean dry test tube.
2. Add a drop of coconut oil and heat. Note the odor produced.

E. Emulsification of fats
1. Shake a drop of fresh cooking oil in 1ml water. Stand for sometime and observe the
result.
2. Place 1ml of water in a test tube and add one drop of 0.5% Na2CO3. Add a drop of
fresh cooking oil. Shake vigorously. Compare the results obtained with that of the
first.
3. Repeat the experiment in No. 2 using Rancid Oil. What kind of emulsion is produced?
Explain.
4. Shake a drop of fresh cooking oil with 1ml of freshly prepared dilute albumin
solution. How does it differ from those of the first three?

Page | 37
 F. Fat Crystals
1. Dissolve 1ml of melted lard in 3ml of ether.
2. Stopper loosely with filter paper.
3. Allow to evaporate until crystals begins to separate.
4. Examine crystal under microscope and draw them.
5. Repeat the experiment using beef fat instead of pork fat.

G. Surface Tension of Fats

1. Fill a clean 5ml pipette with water.
2. Hold the pipette perpendicular and allow each drop to fall slowly, counting the
number of drops which will fall from it per minute.
3. Dry the pipette with acetone
4. Repeat the procedure using cooking oil.
5. Drain the oil from the pipette thoroughly but do not wash.
6. Again fill the pipette with water and count the drops per minute. An increase in the
number of drops indicates that the surface tension has been lowered.

H. Iodine Absorption Test

1. Dissolve 5 to 10 drops of oleic acid in about 5ml of chloroform.
2. Add some Lugol’s iodine solution, a drop or two at a time, and shake between
additions. The solution will decolorize if unsaturated fatty acids are present. This is
due to the absorption of iodine.
3. Prepare a control tube with 5ml of chloroform. Add some Lugol’s iodine solution, a
drop or two at a time, and shake between additions.
4. Repeat the experiment using palmitic acid.

DATA/RESULTS:
Describe in detail
A. Solubility
Fats and Fatty Solubility
Acids Water Ether Chloroform Hot Alcohol
Olive oil
Cottonseed oil
Lard
Palmitic Acid
Oleic Acid

The Best solvent(s) is/are __________________________________________________.

B. Formation of Translucent Spot

Page | 38
 Fat Observation
Translucent Spot formation After Evaporation
Cookingg oil

Anise oil

C. Reaction of Fats
Time Observation
Blue Litmus Red Litmus
Immediately

Later

D. Acrolein Test
Odor: __________________________________________________________________.
Other Observation:
________________________________________________________________________
________________________________________________________________________

E. Emulsification of Fats
Mixture Observation
Fresh cooking oil in water

Fresh cooking oil in water with 0.5%

Na2CO3
Rancid cooking oil in water with 0.5%
Na2CO3.
Fresh cooking oil in dilute albumin
solution

F. Fat Crystals
DRWAING OF FAT CRYSTALS
LARD (pork fat) BEEF FAT

G. Surface Tension of Fats

Number of Drops per Minute
Water Oil Water with Oil

Page | 39
 H. Iodine Absorption Test
Test tube Number of Drops Other observations Saturated or
added to unsaturated
decolorize
Control Tube
Oleic Acid
Palmitic Acid

CONCLUSION: Give explanations for all your observations.

POST-LABORATORY QUESTIONS:
1. What is a fatty acids? What is the difference between unsaturated and saturated fatty
acids?

2. What causes Gaucher’s disease? Neiman-Pick disease? Respiratory distress syndrome?

Tay-Sachs disease?

Page | 40
Name: ______________________________ Date Performed: ________________
Group number: ____________ Date Submitted: ________________

Experiment No. 10
SAPONIFICATION: THE PREPARATION OF SOAP

OBJECTIVES:
At the end of the experiment, students are expected to:
1. _________________________________________________________.
2. _________________________________________________________.
3. _________________________________________________________.

ASSIGNMENT
1. Define the following terms:

Page | 41
 Saponification

Soap

2. Write the general chemical equation for saponification of fat.

REAGENTS/MATERIALS:
Ethyl alcohol Concentrated NaCl solution Filter Paper
Lard/Vegetable oil Dilute NaCl solution pH paper
Concentrated NaOH sol’n Cheese Cloth Distilled water

APPARATUS:
Graduated cylinder 10ml Tripod Balance Wire gauze
Evaporating dish Stopwatch Dropper Crucible tong
Stirring rod Beakers Hot plate

PROCEDURES:
A. Making of Soap
1. Add 5ml of alcohol to about 5grams of Lard or vegetable oil in an evaporating
dish.
2. Add 10 to 15drops of concentrated sodium hydroxide solution.
CAUTION!!!
Conc. NaOH solution causes severe skin and eye burns. If spillage occurs, clean up
immediately. Wash immediately with plenty of water if the solution comes in contact with
skin.

3. Stir the mixture and warm gently until all the alcohol evaporates. It may be
difficult to determine this precisely, but 10 minutes of heating should be enough.
CAUTION!!!
Heating should be done with hot plates of available. If Bunsen burner are used, keep
the flames low since alcohol will burn. Should the alcohol catch fire, cover the dish with your
asbestos board until the fire is extinguished.

4. After the alcohol has evaporated, allow the mixture to cool with stirring.
Formation of a solid indicates the production of soap.
Separate the solid from the rest of solution.
CAUTION!!!
Do not handle as the soap still contains large amount of sodium hydroxide.

5. To remove NaOH and glycerol from the soap, wash it several times with 10ml
portions of concentrated sodium chloride solution.

Page | 42
 Break up any large lumps of soaps to maximize the removal of glycerol and
sodium hydroxide. After washing, dry your soap with paper towel or filter paper.
Describe it.

B. pH of Soap
1. Dissolve a small iece of your soap in distilled water and check the pH with pH
paper.
2. Do the same thing with a commercial soap such as Ivory. Is soap acidic, basic or
neutral?

C. Lathering Ability
1. Take a small piece of your soap and wash your hand with it. Does it lather?
2. Repeat using a dilute salt solution instead of tap water. Does it still lather?
3. Try this test with commercial soap. Give the brand name.

DATA/RESULTS:
Describe in detail
A. Making of Soap
Describe:

Make a packaging of your soap with brand name and group name. Submit to your
instructor.

B. pH of Soap
Soap - pH - Acidic, Basic, Neutral
Your Soap - -
Commercial Soap - -
C. Lathering Ability
SOAP LATHERING ABILITY
In Tap Water In Dilute Salt Solution
Your Soap
Commercial Soap
CONCLUSION:

POST-LABORATORY QUESTIONS:
1. What is the difference between soap and detergents?

Page | 43
 2. Explain in details the cleansing action of soap.

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 11
THE CHEMISTRY OF MILK

OBJECTIVES:
At the end of the experiment, students are expected to learn how to:
1. _______________________________________________________.
2. _______________________________________________________.
3. _______________________________________________________.

ASSIGNMENT:

Page | 44
 1. Put a check (/) it its component of milk or (x) if milk is deficient in it.
______1. Fats ______4. Sugars ______7. Proteins ______10.
Vitamin A
______2. Calcium ______5. Phosphorous ______8. Vitamin B ______11.
Vitamin C
______3. Vitamin D ______6. Copper ______9. Iron ______12.
Water

2. Fill in the blank to complete the statement. Choose from the given options below. Write
the letter of your answer on the blank. Alphabetize the letter if there are more than two
answers in a blank.
A. Casein D. Glucose G. Lactose
B. Calcium caseinate E. LactalbuminH. Lactoglobulin
C. Galactose F. Lactic Acid I. Sucrose

1. The proteins in milk are _________. They are sufficient to meet all the protein
requirements of animals.
2. The salt, _________, is partially responsible for milk’s white color.
3. The carbohydrate in milk is ________, it is found exclusively in milk.
4. Lactose is made up of ________.
5. Upon standing, certain bacteria convert lactose to ________. The milk is said to
become sour. The formation of this causes the casein to precipitate from milk in the
form of curds.

REAGENT/MATERIALS:
Evaporated milk Filter paper Ether Potassium bisulfite crystals
Dilute acetic acid Benedict’s reagent Ammonium oxalate solution
Ethanol Ammonium molybdate sol’n 15% sodium hydroxide sol’n
Copper solution Nitric acid

APPARATUS:
Weighing balance Graduated cylinder 10ml, 50ml Beaker 100ml, 250ml
Stirring rod Funnel Erlenmeyer flask
Wash bottle Evaporating dish Test tubes
Test tube holder Test tube rack Hot plate
Wire gauze Droppers Water bath

PROCEDURES:
A. Properties
1. Test milk with a piece of pH paper. Record the pH.
2. Determine the density of milk.

B. Isolation of and Test for Casein

1. Prepare a solution by diluting 3ml of milk with 20ml of water. While stirring slowly,
add 20ml of dilute acetic acid. A precipitate of casein and milk fat should form.
2. Filter the precipitate and save the filtrate (this Liquid is called whey) for part D.
3. Wash the precipitate several times with water.

Page | 45
 4. Transfer the solid to a small beaker, cover with ethanol, and stir for a couple of
minutes (this removes traces of water)
5. Pour off the alcohol, and cover the solid casein with about 10ml of ether in order to
extract the butterfat.
CAUTION!!!
No flames should be present since ether vapors are VERY FLAMMABLE.
6. Pour the ether extraction into an evaporating dish and add 10ml more of ether to
the casein. Combine the second ether extract with the first. The extraction separates
the butterfat from the casein.
7. Place the evaporating dish containing ether aside. Away from the flame, in the hood
if possible, and allow the ether to evaporate. Save for PART C.
8. The solid casein remaining in the beaker should be pressed dry between pieces of
filter paper. Test the casein for protein by the Biuret Test.
9. Biuret Test:
Place the solid casein in the test tube. Add 5ml of 15% sodium hydroxide solution
and mix by tapping the tube against the palm of your hand. Add 5 drops of dilute
copper (II) sulfate solution to the tube, observe and record the color. A purple
color indicates the presence of Protein.

C. Acrolein Test for Butterfat

After all the ether has evaporated, test the residue for fat by the Acrolein Test.
1. Transfer a little amount of the residue into a test tube and add a few crystals of
potassium bisulfite.
2. Carefully heat the test tube until a color change begins.
3. Cautiously smell the odors that arise from the test tube. Record your observations.

D. Isolation of Lactalbumin and Lactoglobulin

1. Boil the filtrate obtained from the initial filtration of milk until lactalbumin and
lactoglobulin coagulate.
2. Filter and save the filtrate for Parts E, F and G. test the solid for Protein by Biuret
Test.

E. Test for Lactose

Add 5 drops of the filtrate from part D to 3ml of Benedict’s reagent and heat in a boiling
water bath. A brown color indicates the presence of sugar.

F. Test for Calcium Ion

Mix together 1ml of ammonium oxalate and 1ml of filtrate from Part D. a precipitate
indicates the presence of Calcium.

G. Test for Phosphate Ion

Mix together 4 drops of nitric acid, 2ml of ammonium molybdate solution, and 3ml of
the filtrate from Part D and heat in water bath. Formation of precipitate indicates the
presence of phosphate.

DATA/RESULTS:

Page | 46
 Test Observation
Properties

pH

Density

Biuret Test for Casein

Acrolein Test for Butterfat

Biuret Test for Lactalbumin and

Lactoglobulin
Test for Lactose

Test for Calcium Ion

Test for Phosphate Ion

CONCLUSION:

POST-LABORATORY QUESTIONS:
1. Define and state the function of the following:
A. Estrogen:

B. Progesterone:

C. Alveoli:

D. Oxytocin:

E. Prolactin:
Page | 47
 2. What is lactose intolerance? What causes lactose intolerance?

3. What importance do breastfeeding give to the infants compared with that of infant
formula milk?

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 12
NUCLEIC ACIDS

OBJECTIVES:
At the end of this experiment, students are expected to:
1. _________________________________________________________.
2. _________________________________________________________.
3. _________________________________________________________.

ASSIGNMENT:

Page | 48
 A. Complete the table below with expected results for positive test of the following
substances.
Substances Test/Reagents Indication of Positive Result
Purine

Pentose

Deoxyribose

Phosphorous

B. Draw the structure of DNA and RNA and Label the components as sugar, base and
phosphate.
DNA RNA

REAGENT/MATERIALS
Liver 5% Trichloroacetic Acid (TCA) Ethyl Alcohol Ether
2N HCl 2N NaOH Acetate buffer 10% CuSO4
Saturated sodium bisulfite Orcinol reagent Diphenylamine reagent
1N H2SO4 Concentrated HNO3 Molybdate-napthol sulfonic acid

APPARATUS:
Blender Graduated cylinder 10ml Centrifuge Centrifuge tubes
Glass rod Beakers 100ml, 250ml Test tubes Test tube rack
Test tube holder Test tube brush Hot plate Wire gauze
Water bath Crucible tong Thermometer Evaporating dish
Droppers

PROCEDURE:
A. Extraction of Nucleic Acid from Liver
Liver is suspended in 5% TCA to extract the acid soluble substances. The residue is
extracted with alcohol-ether mixture to remove the lipids. From the residue DNA
and RNA extracted with hot 5% TCA.
Page | 49
 1. Homogenize the liver with 1 volume of water.
2. Place 1ml of the homogeneous mixture in centrifuge tube.
3. Add about 5ml of 5% TCA, stir and centrifuge for 5 minutes,
4. Decant and discard the supernatant fluid. Repeat the washing using alcohol-
ether mixture (3:1)
5. Suspend the residue in 7ml of 5% TCA and heat at 90oC for 15minutes with
occasional stirring.
6. Cool, centrifuge and decant the supernatant to a clean test tube and add water
to make up to 10ml. Use this solution to test for purine and determination of
RNA and DNA.

B. Test for Purine

1. Place 1ml of the solution prepared in Part A in a test tube and add 1ml of 2N HCl.
2. Prepare a similar tube containing 1ml of water and 1ml of 2N HCl (Blank)
3. Place both tubes in a boiling water bath for 20 minutes. This hydrolyzes the
nucleic acid to form free purines.
4. Add 1ml of 2N NaOH and 2ml of acetate buffer to the tubes.
5. Again place in boiling water bath and heat.
6. Add 0.5ml of 10% copper sulfate. A bluish-brown precipitate form in both tubes.
7. Add 0.5ml of saturated sodium bisulfite and mix.
8. Heat in a water bath for few minutes. Observe.

C. Determination of RNA –Orcinol Test for Pentoses

1. Dilute 0.5ml of the solution prepared in Part A with 2.5ml of water.
2. Add 3ml of the Orcinol reagent and heat in water bath.
3. Cool the tube under tap water and note the result.

D. Test for Phosphorous

Precaution: To avoid irritating vapors, do this procedure under the hood or near the
window.
1. Evaporate 0.5ml of the prepared solution in Part A mixed with 1ml of 1N H 2SO4
until the mixture chars or blacken.
2. Cool and add 2 drops of concentrated HNO3.
3. Heat until brown fumes disappear and the solution clears.
4. If the solution does not clear, allow to cool and add 2 drops of concentrated
HNO3 and heat.
5. To the residue, add water to make 2ml volume.
6. Add 1ml of the molybdate-napthol sulfonic acid reagent.
7. Note the result.

DATA/RESULTS:
Test Observation
A. Test for Purine With Solution - Blank
Note color of precipitate

B. Orcinol Test for Pentose in RNA

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 C. Diphenyl Test for Deoxyribose in DNA

D. Test for Phosphorous

CONCLUSION:

POST-LABORATORY QUESTIONS:
1. What is sickle cell disease? What causes it? What is the difference in the shapes of the
red blood cells? What effect does this have on their ability to carry oxygen?

2. The insertion of wrong nucleotide into a DNA code group could result in? Mutation,
Recombination, Splicing or Cloning? Explain.

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Name: ______________________________ Date Performed: ________________
Group number: ____________ Date Submitted: ________________

Experiment No. 13
PROTEIN DIGESTION

OBJECTIVES:
At the end of the experiment, students are expected to:
1. ______________________________________________________.
2. ______________________________________________________.
3. ______________________________________________________.

ASSIGNMENT
1. What organ is responsible for the protein digestion?

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2. What are the products after the digestion of protein?

MATERIALS
4 test tubes
1 hard-boiled egg white divided into 4 pieces (discard the yolk)
Test tube rack
One 25ml graduated cylinder
10ml of 5% pepsin solution
10ml of 0.2% HCl solution
10ml of distilled water
10ml of the 5% pepsin solution and 0.2% HCl solution (5ml pepsin +5ml HCl)

PROCEDURES:
1. Prepare four test tubes as indicated below:
Test tube #1: 10ml of distilled water.
Test tube #2: 10ml of the 5% pepsin solution
Test tube #3: 10ml of the 0.2% HCl solution
Test tube #4: 10ml of the 5% pepsin solution and 0.2% HCl solution (5ml pepsin + 5ml
HCl)
2. Put one piece of egg into each tube.
3. Cover the test tube.
4. Let the tubes stand at room temperature for 48hours. Observe which solution is most
effective in breaking down the egg white.

DATA/RESULTS:
Observation of Protein Digestion
Mixture Observations with egg white
Test tube #1
(10ml of distilled water)

Test tube #2
(10ml of the 5% pepsin solution)

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 Test tube #3
(10ml of the 0.2% HCl solution)

Test tube #4
[10ml of the 5% pepsin solution and 0.2% HCl
solution (5ml pepsin + 5ml HCl)]

CONCLUSION:

POST-LAB GUIDE QUESTIONS:

1. What are the major sources of protein?

2. What is the average daily protein requirement of humans?

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3. If proteases such as pepsin and trypsin digest protein, why do they not digest the stomach
and small intestine, which organs are made from protein?

4. The stomach has a very low pH. Does this indicate that pepsin works effectively in an acidic or
basic environment?

5. Why is it important to know the level of the protein creatinine in the blood?

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 14
ENZYMES

OBJECTIVES:
At the end of the experiment, students are expected to:
4. ______________________________________________________.
5. ______________________________________________________.
6. ______________________________________________________.

ASSIGNMENT:
A. Give the function of the following enzymes and name two examples for each.
Enzyme Function Examples

Page | 55
 Oxidase
Catalase
Peroxidase

B. State the effect of the following factors on enzyme activity:

Factor Effect
Concentration of enzyme

Concentration of Substrate

Temperature

pH

REAGENT/MATERIALS:
Potato 1N AgNO3 1% Phenol Pyrogallol solution
Ground Chicken Liver Molisch Reagent 1N BaCl2 solution 1% Catechol
Alpha-napthol sol’n 3% Hydrogen Peroxide Benedict’s sol’n 1N CaCl2 sol.
Guaic solution Nadi reagent Paraphenylenediamine hydrochloride sol’n

APPARATUS:
Peeler Knife Blender Beaker 250ml, 500ml Test tube
Test tube rack Test tube holder Test tube brush Glass dropper
Hot plate Wire gauze Water bath Gradated cylinder 10ml Glass rod
Centrifuge Wash bottle

PROCEDURES:
DEMONSTRATION OF POTATO OXIDASES
It is convenient to combine the study of oxidase with that of the potato. This shows light
on the value of the potato as a food. It also gives information as to the composition of a typical
vegetable cell.
A. Preparation of Potato Extracts
1. Wash and peel a medium-sized potato.
2. Grate and transfer the gratings at once to a piece of cheesecloth which is suspended
in a beaker containing about 200ml of distilled water. Work gently with the hand to
get out as much of the starch a possible. Keep this extract in a beaker and label it as
Water Extract No. 1
3. Make a second extraction using 200ml of distilled water and label it as Water Extract
No. 2 (if the extract is to be kept overnight, it must be preserved with toluene)
4. Make a third extraction, and if it does not contain an appreciable amount of starch
anymore which is indicated by its slight cloudiness, discard the extract. Work the
pulp until it is practically starch free or when the collected water extract turns clear.

B. Test on Pulp
Work a portion of the pulp very thoroughly with water until is practically free from
starch and perform Molisch Test for carbohydrates.
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 Molisch Test (α-napthol reaction)
1. Place 1ml of the clear water extract in a test tube.
2. Add 1 drop of Molisch reagent and mix thoroughly.
3. Incline the tube and allow 1ml of concentrated H2SO4 to flow in the side of the tube.
The H2SO4 forms a layer at the bottom of the tube.
 Note the color produced at the juncture of the two liquids

C. Test on Water Extract No. 1

1. Into another beaker, pour off the supernatant liquid from water extract no.1 when
the starch has settled out; or use a centrifuge to facilitate settling or separation.
2. Test the supernatant liquid for reducing sugars by Benedict’s Test.
Benedict’s Test
- To 1ml of Benedict’s solution, add 2drops of the supernatant liquid.
- Boil for 2minutes and allow to cool. Observe.
- Formation of brick red precipitate indicates presence of reducing sugar.
3. Test the supernatant liquid for protein by Biuret Test.
Biuret Test
- Mix 1ml of supernatant liquid and 1ml of 10% NaOH.
- Add 0.5% CuSO4 dropwise mixing thoroughly after each addition. Observe.
- A violet color is obtained with long chain proteins while a pink color is
obtained with shorter chain.
4. Test the supernatant liquid (1ml) for chloride by adding dropwise 1N of AgNO 3
solution. Formation of white precipitate indicates the presence of chloride.
5. Test the supernatant liquid (1ml) for sulfate by adding dropwise of 1N BaCl 2 solution.
Formation of precipitate indicates the presence of sulfate.
6. Test the supernatant liquid (1ml) for phosphate by adding dropwise 1N of CaCl 2
solution. Formation of precipitate indicates the presence of phosphate.

Indicate in the Data table what information you have obtained as to the food value of the
potato.

D. Separation of Starch
1. Combine the starch from water extract no. 1 and water extract no. 2 (Collect the
supernatant liquid from water extract no. 2 n a clean beaker for Part E)
2. Wash by decantation with distilled water.
3. Drai off the water and turn the beaker upside down so that the starch will drain,
otherwise molds will develop.
4. Dry the starch then describe it and submit to your teacher.

E. Experiment on Potato Oxidase Using Water Extract No. 2

1. Into each of a series of 5 clean test tubes, introduce 5ml of the supernatant liquid
from water extract no. 2.
2. Add other reagent according to the following series.

Test Tube # Add

Supernatant Other reagent
Liquid from

Page | 57
 water extract no.
2
1st 5ml 10 drops of 1% phenol
2nd 5ml 10 drops of 1% catechol
3rd 5ml 10 drops of guaiac solution
4th 5ml 10 drops of pyrogallol solution
5th 5ml 5 drops of alpha-napthol solution + 5drops of
paraphylenediamine hydrochloride (AKA Nadi reagent)
3. Mix the contents of the test tubes by shaking. Watch for any color change.
4. If necessary, let stand until next laboratory period (add toluene as preservative) and
examine again.

DEMONSTRATION OF CATALASE
1. Mix about 1gram of ground liver with 3ml of water and 3ml of 3% hydrogen
peroxide.
2. Test the gas evolved for oxygen using the glowing match. Observe.

DEMONSTRATION OF POTATO PEROXIDASE

1. Prepare a series of a 5 test tubes containing 5ml portion of potato extract and 10 drops
of oxidase reagent as in Experiment on Potato oxidase.
2. Prepare another series but use potato extract previously boiled for 5 minutes.
3. To each of the 10 test tubes, add 10 drops of 3% hydrogen peroxide solution.
Note whether oxidation takes place more rapidly than in experiment above where no
hydrogen peroxide is added. Does boiling destroy the peroxide?

DATA/RESULTS:

DEMONSTRATION OF POTATO OXIDASE

Procedure Observation
Test on Pulp – Molisch
Test
* Color at the juncture
of the two liquids
Test on Water Extract Benedict’s Biuret Test: Test for Test for Test for
No.1 Test: Chloride; Sulfate: Phosphate:

* Note the precipitate

formation and color
change

*  If positive; x if
negative
Separation of Starch
Experiment on Potato + Phenol + Catechol + Guaiac + Pyrogallol + Nadi
oxidase using Water
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Extract No. 2 reagent
* Note the precipitate
formation and color
change

DEMONSTRATION OF CATALASE
Observation:

DEMONSTRATION OF POTATO PEROXIDASE

Procedure Observation
Potato Extract + Phenol + catechol + Guaiac + Pyrogallol + Nadi
* Note the precipitate + H2O2 + H2O2 + H2O2 + H2O2 Reagent +
formation and color H 2 O2
change
* Compare the rate of
formation of the
products with those
without hydrogen
peroxide.
* Does oxidation take
place faster?

Potato Extract (Boiled) + Phenol + catechol + Guaiac + Pyrogallol + Nadi

* Note the precipitate + H2O2 + H2O2 + H2O2 + H2O2 Reagent +
formation and color H 2 O2
change
* Compare the rate of
the products with those
without boiling the
extract.
* Yes or No. Does boiling
as indicated by slower or
no reaction destroy the
potato peroxide?

CONCLUSION:

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POST-LABORATORY QUESTIONS:
1. What are carbohydrates? Esterases? Proteases? Name two of each, indicating where
they are found in the body and the substrate they act on.

2. What is coenzyme A? Coenzyme Q? What are their functions?

3. What is an apoenzyme? Coenzyme? Optimum temperature? Optimum pH?

Name: ______________________________ Date Performed: ________________

Group number: ____________ Date Submitted: ________________

Experiment No. 15
CHARACTERIZATION OF SALIVARY AMYLASE

OBJECTIVES:
At the end of the experiment, students are expected to:
1. _______________________________________________________.
2. _______________________________________________________.
3. _______________________________________________________.

ASSIGNMENT:
1. What is ptyalin? State its digestive function.

2. Arrange the following carbohydrates from the most complex to the least: dextrin,
glucose, maltose and starch.

Page | 60
 3. Complete the table below.
Qualitative Test Test for the presence of Indication
Iodine Test

Benedict’s Test

Biuret’s Test

MATERIALS:
Saliva Paraffin wax Filter paper/Cheese cloth
Methylene blue sol’n Red/blue litmus paper Congo red indicator
Phenolphthalein indicator pH indicator paper strips Dilute acetic acid
10% NaOH 0.5% CuSO4 Starch
Benedict’s solution 0.1M sodium chloride 0.01N iodine solution
Graduated cylinder 10ml, 50ml Glass dropper Microscope
Glass plate Funnel Erlenmeyer flask
Test tubes Test tube rack Test tube brush
Test tube holder Beakers Glass rod
Weighing scale Hot plate Water bath
Thermometer Stop watch Wire gauze

PROCEDURES:
A. Collection of Sample
Rinse the mouth with water, and chew a small piece of paraffin wax to stimulate the
flow of saliva. Collect about 30ml of saliva and filter for use in the following experiment

B. Microscopy
1. Obtain a drop of saliva from your filter paper before the completion of your filtration,
and put it on a slide with cover glass.
2. Run some methylene blue solution under the cover glass and examine
microscopically.

C. Acidity
1. Test the color reaction of saliva to red and blue litmus papers, congo red indicator and
phenolphthalein indicator. State if saliva is acidic, basic or neutral.
2. Determine its approximate pH with pH indicator paper strip.

D. Test for Mucin

1. Place 5ml of saliva in a test tube and add dilute acetic acid. A white stringy precipitate
is formed which is insoluble in excess of the acid. This is mucin, it is any of a class of

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glycoprotein found in saliva, gastric juice etc., that form viscous solution and act as lubricants or
protectants on external and internal surfaces of the body.
2. Remove some of the mucin by a glass rod and test with the Biuret reaction.
Biuret Test (General test for proteins):
- Mix mucin and 2 dorps of 10% NaOH
- Add 0.5% CuSO4 drop by drop, mixing thoroughly after each addition.

As positive reaction is obtained with all native proteins and most of the derived proteins,
a violet color is obtained with long chain proteins while a pink color is obtained with
shorter chain.

E. Digestion of Raw Starch

1. Shake about ½ gram of raw starch with about 2ml of water.
2. Divide the mixture into two test tubes. Test one of the test tubes with Benedict’s
solution.
Benedict’s Test (Test for glucose)
- To 1ml of Benedict’s solution, add two drops of the mixture.
- Boil for 2minutes and allow to cool.
Yellow or red precipitate of cuprous oxide indicates the presence of reducing sugar.
3. Take the other test tube and mix it with equal volume of saliva. After one-half hour,
test the mixture with Benedict’s reagent. Record your observations in the Data Table.

F. Inorganic Phosphate
Mix 1ml of saliva and 1ml of ammonium molybdate solution. Heat and observe.

H. Estimation of Amylase in Saliva

1. To 10ml of 1% starch in a test tube, add 2ml of 0.1M sodium chloride, and place the
tube in a water batch maintained at 38oC.
2. Add 1ml of diluted saliva (1:10) to the tube containing the starch solution and mix the
contents. This called the digestion mixture.
3. Prepare 8 test tubes so that each contains 3ml of water and 3drops of 0.01N iodine.
4. In an interval of 3 seconds, add two drops of digestion mixture to one of the tubes
until the iodine ceases to produce any change in color.
5. If a time taken to reach this point (called the “achromatic point”) is less than four
minutes, further dilute the saliva with an equal volume of water and repeat the experiment.
6. Compute for the units of amylase.
Assuming that 1unit of amylase is the quantity required to digest 10ml of 1% starch
solution to the achromatic point in 4 minutes, calculate the number of units of amylase in 1ml
of your saliva.

Unit of amylase equals:

4minutes X dilution of saliva

Time in minute until iodine ceases to produce color change

I. Influence of NaCl on Salivary Amylase

Repeat the preceding experiment but replace the NaCl solution by water.

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 A longer time should be required showing that NaCl accelerates the action of salivary
amylase. If saliva is dialyzed from chlorides, it becomes inactive.

DATA/RESULTS:

CONCLUSION:

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