Chem Manual

General Education Department
Integrated Sciences Cluster
BIOCHEMISTRY
(WITH ORGANIC CHEMISTRY)
Laboratory Experiments
2013 EDITION
WARREN S. VIDAR
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Biochemistry (with Organic Chemistry) Laboratory Manual 2013
BIOCHEMISTRY
(with Organic Chemistry)
Laboratory Experiments
2013 Edition
WARREN S. VIDAR, RCh, MSc
Chemistry Unit
Integrated Sciences Cluster
General Education Department
De La Salle Health Sciences Institute
City of Dasmariñas, Cavite
Copyright 2013
Chemistry Unit, Integrated Sciences Cluster

General Education Department, De La Salle Health Sciences Institute, 2013
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Contents
Chemistry Laboratory Guidelines and Policies
Proper Practices and Safety in the Laboratory
Experiments
1 Qualitative Organic Analysis
2 Synthesis and Analysis of Aspirin
3 Isolation and Analysis of Lipids from Egg
4 Classification of Carbohydrates
5 Isolation of Casein from Milk and Analysis of Amino Acids
6 Isolation and Analysis of DNA from White Onion
7 Determination of Vitamin C Concentration by Titration

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Laboratory Guidelines and Policies
A. GENERAL REQUIREMENTS
1. Individual Requirements
a. laboratory manual
b. laboratory gown
c. face mask
d. gloves
e. laboratory ID with 1 x 1 picture
2. Group Requirements
a. large plastic container which will serve as the lab kit
b. dishwashing liquid
c. one roll of thick tissue paper or paper towel (preferred)
d. barbecue sticks
e. sponge and scrubbing pad
f. test tube brushes
g. cotton rags (preferably the round ones)
h. hand soap (preferably antibacterial soaps)
i. stapler
j. masking tape (preferably 10 mm width)
k. black permanent marking pen (fine tip)
l. scissors
m. matches or lighters
n. hand-shaped pot holders
o. spoon-type coffee stirrers
p. aluminium foil
B. GROUPINGS
1. There shall be a minimum of three (3) and a maximum of four (4)

members per group. In a class, a maximum of eight (8) groups is
allowed.
2. Each group shall have a laboratory kit that contains the items listed in
A.2.
3. A locker will be provided for each group for storage of laboratory
items. Items that are not related to the laboratory class cannot be
stored inside the locker. Only one key will be provided for the group.
The group shall be liable for any damage to the locker or loss of the
key. The key must be returned to the laboratory custodian before the
final examination week.

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C. BORROWING OF MATERIALS
1. Most of the items that will be used in every experiment are stored in the
Laboratory Stockroom.
2. To borrow items from the Laboratory Stockroom, each group must
accomplish the borrower’s form. This form will be issued by the
instructor in charge.
3. The laboratory custodian will dispense only the items that are written
on the approved borrower’s form. The list of items to be borrowed is
stated in the laboratory manual. In case the group decides to borrow
additional items, permission must be granted by the instructor in
charge.
4. It is the responsibility of the students to check the conditions of the
items being dispensed by the laboratory custodian. If the dispensed
items, in the presence of the laboratory custodian, were found to be in
poor condition (i.e. with cracks, huge scratches, damage, dirt, etc.),
the student/s may request for a replacement. Therefore, this must be
done by the students before bringing the dispensed items to their
working area.
D. RETURNING OF MATERIALS
1. After the laboratory period, the borrowed items must be returned to

the laboratory custodian in good condition (i.e. the same condition
when it was dispensed).
2. In case of breakage or loss of any laboratory property, the group must
report this immediately to the instructor in charge. A charge slip will
be issued by the laboratory custodian and the group must settle their
accountability at the cashier’s office as soon as possible. The
Integrated Sciences Cluster coordinator reserves the right to hold any
clearance of students who fail to settle their dues before the end of the
semester.

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E. ASSESSMENT
1. Requirements:
a. Pre-laboratory Quizzes
b. Experiment Report Sheets
c. Post-laboratory Assignments
d. Laboratory Performance
e. Problem Sets or Seatwork
f. Practical Exam
g. Major Exam
2. Grading System
Criteria Percentage Distribution
Major Exam 30%
Practical Exam 20%
Other Requirements 50%
Pre-Lab. Quizzes 10%
Experiment Reports 10%
Post-Lab. Assignments/
Problem Sets 15%
Performance 15%
Total 100%

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Proper Practices and Safety in the Laboratory

Adapted from the CHED Memorandum Order No. 18, Series of 2007, for the BS Chemistry Program
A. Management of Work Space and General Equipment Setup
1. Keep workspace uncluttered.

2. Set up clean, dry apparatus, firmly clamped and well back from the
edge of the lab bench. Have due regard to the proximity of reagent
bottles to burners, other workers, and their equipment. Choose sizes
that can properly accommodate the procedure or operations to be
performed, allowing at least 20% free space.
3. Be sure that your glassware and equipment are free from flaws such
as cracks, chips, and obvious defects. Chipped or cracked glassware
should be repaired or discarded.
4. Glass stopcocks in burets and separatory funnels should be freshly
lubricated prior to use. Separatory funnels should be properly
supported and oriented so that the stopcock will not be loosened by
gravity. A retainer ring should be used on the stopcock plug.
5. Condensers should be properly supported with securely positioned
clamps. The attached water hoses should be secured with wire or a
clamping device.
6. Apparatus attached to a ring stand should be placed such that the
center of gravity of the system is centered over the base with adequate
provision for removing burners or baths quickly. Stands bearing heavy
loads should be firmly attached to a bench top. Equipment racks
should be securely anchored at the top and bottom.
7. A pan properly placed under a reaction vessel or container can
confine spilled liquids in the event of glass breakage.
8. Fume hoods are recommended for all hazardous operations or
whenever hazardous gases, vapors, mists or fumes are likely to be
evolved. They are particularly necessary when working with
flammable vapors with a density greater than air. Heavy vapors can
settle on the benchtop or floor, where they may use diffuse to a distant
burner or ignition source.
9. Hoods should be operating properly and must be free from any
obstacles that prevent proper air movement.
10. Use a hood when conducting a reaction that can result in an explosion
or when using a vacuum system, which may implode. Close the hood
sash to provide a shield. If a hood sash is not available, use a standing
shield stabilized with weights or fasteners. Proper eye and face
protection, including goggles and a face shield must also be worn even
when using a standing shield to a hood with a closed sash.

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11. Chemicals should only be heated in a ventilated area or hood. Prior to

heating a liquid, place boiling stones in reaction vessels that cannot be
stirred. If a burner is to be used, distribute the heat with ceramic wire
gauze. Use a thermometer in a boiling liquid if there is a risk of an
exothermic decomposition. The setup should allow for quick removal
of heat.
12. When working with flammable gases or liquids, do not allow burners,
switches, unshielded motors or other ignition sources in the vicinity.
Use appropriate traps, condensers or scrubbers to minimize release
of material in the environment. If a hot plate is used, ensure that its
temperature is maintained below the auto ignition temperature of the
chemicals being handled or likely to be released.
13. Whenever possible, use electrical heaters instead of gas burners.
B. Personal Safety
1. Wear proper eye protection. Contact lenses should not be worn.

2. Always know the hazards and chemical properties of the chemicals
used, e.g., corrosiveness, flammability, reactivity, and/or toxicity.
Follow hazard precautions.
3. Wear appropriate protective clothing and footwear. Full lab gowns
made of cotton are preferred to aprons. Do not wear high-heeled
shoes, sandals or slippers. Lab gowns should be removed when
outside the laboratory.
4. Confine long hair and loose clothing. Jewelries and similar
accessories should be removed when working in the laboratory.
5. Never work alone in the laboratory. Make sure somebody is around
especially when handling hazardous substances.
6. Do not eat, drink, smoke, use medication, or apply cosmetics in the
chemical laboratory or storage areas.
7. Do not perform unauthorized work, preparations, and experiments.
Follow prescribed directions. If in doubt, ask your instructor.
8. Always wash hands, arms and face before leaving the work area. This
should be done even if gloves are used.
9. Never engage in pranks or other acts of mischief.
10. You should know the location of the fire extinguisher, fire escape,
safety shower and first aid kit.
11. Clean up all spills, broken glassware, etc., immediately.

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C. General Precautions for Handling Chemicals
All chemicals are potentially harmful. Avoid direct contact with any
chemical. Some substances now considered to be non-hazardous may, in the
future, be found to cause illness. It is especially important to keep chemicals
away from hands, face, and clothing, including shoes. Many substances are
readily absorbed through intact skin and inhalation. Chemicals can also
enter the body, through the mouth, and by contamination of the hands.
Chemicals can also be transferred to the eyes from the hands.
1. Do not use or handle any chemical until you have read and understood
the label and Material Safety Data Sheet (MSDS) for that chemical.
2. All containers of chemicals must be clearly labelled. Do not use any
substance from an unlabelled or doubtfully labelled container.
3. Keep your hands and face clean. Wash thoroughly with soap and
warm water whenever a chemical contacts your skin. Always wash
your hands and arms before leaving the work area.
4. Never taste a chemical. If it is necessary to smell the chemical,
cautiously waft a handful of vapor toward the nose. Smoking, drinking,
eating, taking medication, and applying cosmetics is forbidden in
chemical work or storage areas.
5. Some solvents, such as dimethyl sulfoxide (DMSO), serve as vehicles
for the rapid transport of other substances into the skin. Always wear
impervious gloves when handling such materials.
6. Many by-products of chemical reactions can be very hazardous.
Planning for the handling and control of these toxic by-products should
be part of the experimental procedure.
D. Safe Laboratory Techniques
1. Work neatly.
2. Study the experimental procedures beforehand. Familiarize yourself
with the chemicals, materials and apparatus to be used.
3. Always add a reagent slowly. Swirl and observe what happens when
the first small amount is added. Wait a few moments before adding
more; some reactions take time to start.
4. When adding liquids or powders, point the opening of a container away
from yourself and away from others. Use a funnel when pouring liquids
or powders into a small neck opening. Use a stirring rod to direct the
flow of the liquid being poured.
5. In case a stopper or lid is stuck, using extreme caution when opening
the container. Wear gloves and tap the neck of the container lightly to
loosen the stopper or lid.
6. Never look down the opening of a vessel.

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7. To avoid violent reactions and spattering while diluting chemicals,

always pour concentrated solutions slowly, while stirring, into water or
into less concentrated solutions to allow any heat evolved to dissipate.
Do not add water to concentrated acids as this may cause splattering;
instead, add the acid to the water. Wear a face shield or splash
goggles.
8. When using a separatory funnel with a ground glass stopcock, ensure
that the stopcock has been freshly lubricated and is closed. Keep a
breaker under the funnel in case the stopcock fails.
9. Large flasks must always be supported both at the neck and at the
base during use, such as when heating with a Bunsen burner.
10. Never use mouth suction to fill a pipette. Use an aspirator bulb or a
loose-fitting hose attached to an aspirator. Constantly watch the tip of
the pipette and do not allow it to draw air. Do not allow solutions to
contaminate the aspirator bulb.
11. When carrying large bottles of corrosive, toxic, or flammable liquids,
use impact resistant transport containers to protect them from
breakage and to contain spillage. Carry large bottles (e.g. 1 gallon)
with both hands; do not hold than by the neck alone.
12. Do not use diethyl ether in the presence of an open flame. Diethyl
ether boils at around 36°C, which is comparable to our normal room
temperature.
13. In general, electric heaters are preferred to gas burners.
14. Keep drawers and cabinets closed while working.
15. Do store materials, especially chemicals, on the floor – even
temporarily.
16. Work spaces and storage areas should be kept clear of broken
glassware, leftover chemicals, paper, dirt and debris.
17. Keep aisles free of obstructions such as pallets, carts, cylinders and
bottles of chemicals, boxes and waste receptacles.
18. Avoid slipping hazards by keeping the floor clear of ice, stoppers,
glass beads, glass rods and spilled liquids.
19. Use the required procedure for the proper disposal of chemical
wastes, biological wastes and solvents.

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Emergency Procedures
A. General Procedures for Spills
1. Immediately alert fellow workers and supervisor.

2. Avoid skin contact and minimize inhalation. All contaminated clothing
must be removed immediately to prevent skin penetration. The skin
must be washed with soap and water. Continue flushing the skin with
water for 15 minutes or more. Clothes should be laundered separately
from the other clothing before reuse.
3. If the material is not particularly volatile, has a low order of toxicity,
and there is no fire hazard, proceed clean-up operations as directed in
the MSDS. To facilitate cleaning up of liquids, and if applicable, use an
absorbent material that will neutralize the liquids (trisodium
phosphate, sand followed by sodium bicarbonate solution (powder for
acids), sodium thiosulfate for bromine, etc.).
4. A dustpan and brush should be used, and protective gloves should be
worn. Clean the contaminated area with soap and water, and mop it
dry. If the spill is on the floor, some absorbent should be sprinkled on
the spot to prevent slipping. Dispose of the residue properly.
5. If a volatile, flammable or toxic material is spilled, warn everyone
immediately to extinguish flames and turn off spark-producing
equipment. Shut down all equipment and vacate area until it is
decontaminated.
6. Many small liquid spills (<100 mL) can be absorbed with paper towels,
sand or an absorbent. However, paper towels are not suitable for
cleaning up flammable spills. Most solid spill can be brushed up and
disposed of in appropriate solid waste containers, but care must be
exercised to avoid reactive combinations. Do not leave paper towels
or other materials used to clean up a spill in open trashcans in the
work area. Dispose them properly.
7. Spills of Specific Type of Chemicals
a. Acids and other Acid Materials
i. Use calcined absorbent products. Avoid contact with
skin. Do not clean-up hydrogen fluoride (hydrofluoric
acid, HF) with silica-containing materials such as sand or
vermiculite.
b. Mercury
i. Because of the high toxicity of mercury vapor, spilled
mercury should be cleaned up immediately and
thoroughly using an aspirator or a vacuum device.
Domestic vacuum cleaners must not be used because

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they will only re-disperse mercury aerosols and spread

contamination. Mercury spilled into floor cracks can be
made non-volatile by amalgamation with zinc dust.
c. Alkali Metals
i. A spill of alkali metal should be smothered with
powdered graphite and removed to a safe location where
it can be disposed of by reaction with a long chain
primary alcohol such as n-butyl alcohol. Sodium-
potassium alloys present even greater hazards than
either sodium or potassium alone. Strict observation of
supplier’s recommendations must be followed. Particles
of alkali metal splattered on the skin should be removed
immediately. Flush the skin quickly with cool water. If
any metal on the skin becomes ignited, deluge it with
cold water immediately.
d. White (Yellow) Phosphorous

i. A spill of white phosphorous should be blanketed with
sand or wet absorbent. If any white phosphorous is
splattered on the skin, flush the skin with cool water and
remove adhering phosphorous.
B. Chemicals on the Skin
1. For spills covering a small amount of skin, flush immediately with water
for 15 minutes or more. Remove any clothing or jewelry adjacent to
the spilled area to facilitate removal of any residual materials. Check
the MSDS to determine whether any delayed effects should be
expected. If a delayed reaction is noted, seek medical attention
immediately.
2. For larger spills, quickly remove all contaminated clothing, shoes,

jewelry, etc. while using the safety shower. Do not attempt to wash off
chemicals from contaminated clothing. Instead, remove the clothing
quickly; no time should be wasted because of modesty. Be careful not
to spread the chemical on the skin, or especially into the eyes. Unless
the eyes are affected, do not remove safety goggles until all chemicals
are washed from the hair and face. Use caution when removing shirts
to prevent contamination of the eyes. It may be better to cut the
garments off. Immediately flood the affected body area with cool
water for at least 15 minutes. Resume if pain returns. Do not use
creams or lotions. Get medical attention as soon as possible.

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C. Chemicals in the Eyes
For chemical splashes, the following are recommended: flushing of the

eye for a minimum of 20 minutes, flushing the eye with copious amount of
water, and checking for and removing contact lenses at once. Eyeballs
should be rotated so that all surfaces are rinsed. Forcibly hold the eyelids
open, if necessary. Call for emergency medical assistance at the first
opportunity.
D. Releases of Acutely Toxic Vapors and Gases
Some vapors and gases can be permanently disabling or lethal when

inhaled even at low concentrations. These include the following:
Fluorine, F2
Chlorine, Cl2
Bromine, Br2
Iodine, I2
Arsine, AsH3
Stilbine, SbH3
Hydrogen fluoride, HF
Hydrogen sulfide, H2S
Hydrogen cyanide, HCN
Other Cyanides
Methyl isocyanate, CH3NCO
Phosgene, COCl2
Phosphorous trichloride, PCl3
Phosphorous oxychloride, POCl3
Phosphorous tribromide, PBr3
If you plan to work with any of these chemicals, consult the MSDS and
obtain additional guidance on emergency, first aid and handling procedures
before you begin working with the chemical.

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Disposal of Chemicals
Government and local environmental regulations should govern the disposal

of laboratory wastes. In the absence of such guidelines, the following
procedures should be observed:
1. To minimize disposal problems and waste, always specify the smallest

practical quantities in laboratory experiments.
2. Chemicals should be purchased in the appropriate amounts in
practical container sizes. Storage of chemicals over long periods
should be avoided since these increases the hazards of chemical
accidents and is wasteful.
3. When disposing chemicals, keep different classes of waste chemicals
in a separate disposal containers. Incompatible chemicals spilled
together on a bench or placed into the same disposal container can
cause a violent chemical reaction.
4. Place non-hazardous waste in a wastepaper basket, separate from
chemical wastes. If the paper is contaminated, such as paper towels
used to clean up a spill, place the paper in a specially labelled,
combustible waste container with a self-closing lid. The combustible
waste should be treated as chemical waste, and should be emptied
daily.
5. Broken glass and glassware should be place in designated sharp
waste container. Place broken plastic apparatus in a marked waste
container different from the broken glass container. Broken mercury
thermometers and manometers may contain mercury in the fragments.
Dispose as waste mercury.
6. For discarded and unused chemicals, place each container of solid
and liquid chemicals in its own specially marked container, based on
hazard class compatibility. Close these containers after each use.
Chemicals may be put down the drain only if the laboratory supervisor
has approved this method of disposal, and the chemical is determined
to be non-hazardous and non-polluting. Solutions of acids or bases
should be neutralized before disposal. Do not pour water-insoluble
chemicals into the sink or down a drain. Chemicals poured into the
sink can damage plumbing and sewer systems, and can cause
environmental damage. If it happens accidentally, notify the
laboratory supervisor.

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EXPERIMENT NO. 1
Qualitative Analysis of Organic Compounds
I. BACKGROUND
Organic compounds are basically classified based on the type of

functional group that they possess. Different compounds with similar
functional groups can be classified together, having similar physical and
chemical characteristics. Organic reactions may undergo any of the
following types: substitution, addition, elimination, and rearrangement.
Hydrocarbons are compounds that contain exclusively of carbon and

hydrogen. They can be classified as aliphatic or aromatic. The former can be
further classified as cyclic or acyclic. Alkanes, alkenes, and alkynes are
aliphatic acyclic hydrocarbons; while cycloalkanes and cycloalkenes are
aliphatic cyclic hydrocarbons. Cycloalkynes may exist if the ring is big
enough to reduce the strain required to maintain an approximately 180
degree-geometry. Aromatic hydrocarbons are generally cyclic and must
contain alternating double and single bonds, though other rules must also be
followed. Alkanes and aromatic hydrocarbons typically undergo, though not
limited to, substitution reactions; while alkenes and alkynes usually undergo
addition, oxidation, and reduction reactions.
Alkyl halides are hydrocarbons with halogens (F, Cl, Br, I). They are
also known as haloalkanes. Alkyl halides are very reactive. They undergo
substitution and elimination reactions to form alcohols or alkenes.
Alcohols, having the hydroxyl functional group (-OH), mostly undergo

oxidation, reduction, and substitution reactions. They can be classified as
primary, secondary, or tertiary alcohols, based on the degree of substitution
around the carbon that contains the OH functionality.
Aldehydes and ketones belong to a group of compounds that contain

the carbonyl functional group. This functionality is made up of a carbon atom
that is doubly bonded to an oxygen atom. Small molecules of aldehydes and
ketones have a polar nature due to the increased electron density at the
oxygen atom, making them soluble in water. Carbonyl groups may undergo
various chemical reactions such as, oxidation, reduction, addition, and
substitution reactions.

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II. OBJECTIVES
At the end of this activity, the students should be able to:

 classify organic compounds as hydrocarbons, alkyl halides, alcohols,
aldehydes, or ketones, using qualitative chemical reactions; and
 understand the various nature of chemical reactions of these classes
of compounds.
III. MATERIALS
A. The students need to bring the following:

none
B. The instructors/technician/custodian need to prepare the following:

Reagents
hexane cyclohexene tert-butyl chloride
ethanol benzyl alcohol isopropanol
tert-butanol toluene phenol
formaldehyde benzaldehyde acetone
bromine water Tollens reagent

Jones reagent 1% ferric chloride
10% sodium hydroxide iodine solution
2,4-dinitrophenylhydrazine in HCl reagent Baeyer’s reagent
0.1 M ethanolic silver nitrate Lucas reagent
Instrument
water bath
C. The students need to borrow the following from the stockroom:

Item Qty. Item Qty.
test tube (13 x 100 mm) 24 stainless water bath 1
test tube holder/clamps 5 distilled water (in wash bottle) 1

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IV. PROCEDURE
A. Preliminary Preparations
Read and note these steps first before proceeding to each chemical test.
1. Label 13 test tubes with hexane, cyclohexene, tert-butyl chloride,
ethanol, benzyl alcohol, isopropanol, tert-butanol, toluene, phenol,
formaldehyde, benzeldehyde, acetone, and blank.
2. Place 10 drops of each sample into their respective and properly
labelled test tube. The blank contains distilled water only.
3. After each chemical test, make sure to wash all the test tubes with
liquid soap and water, before proceeding to the next test.
B. Bromine Test
1. Add five drops of bromine water into each test tube.
2. Shake well and note your observations.
C. Baeyer’s Test
1. Add 10 drops of Baeyer’s reagent into each test tube.
D. Jones Test
1. Add six drops of Jones reagent into each test tube.
E. Lucas Test
1. Add 20 drops of Lucas reagent into each test tube.
2. Shake well and note your observations. Note the time required for the
formation of a second layer or emulsion for alcohols (if any).
3. If there are no observable changes after 5 minutes, heat the sample
gently in a boiling water bath for 2 minutes and note any observable
changes.
F. Ethanolic Silver Nitrate Test

1. Add 20 drops of ethanolic silver nitrate into the walls of each test tube.
2. Note your observations. If no observable changes take place, heat the
solution in a boiling water bath for two 2 minutes and note any
observable changes.
G. Ferric Chloride Test

1. Add 10 drops of 1% ferric chloride into each test tube.

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H. Tollens Test
1. Add 20 drops of Tollens reagent into the walls of each test tube.
2. Note your observations. If a reaction does not occur, heat the solution
in a boiling water bath for 2 minutes and record any observable
changes.
I. Iodoform Test
1. Add 15 drops of 10% NaOH into each test tube.
2. Add 20 drops of iodine solution then shake thoroughly. Note the
production of a pale yellow-colored product.
3. Heat the test tubes in a warm water bath at around 60°C. If the yellow
color disappears, add more iodine solution.
4. Note if the yellow color persists after adding more iodine, followed by
heating.
J. Reaction with 2,4-dinitrophenylhydrazine (2,4-DNPH)

1. Add 25 drops of 95% or AR ethanol into each test tube.
2. Add 5 mL of 2,4,-DNPH reagent to each solution.
3. Shake well, allow to stand, and note your observations.
4. If a reaction does not occur, heat the solution in a boiling water bath
for 5 minutes and allow it to cool at room temperature. Note your
observations

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REPORT SHEET – EXPERIMENT NO. 1

Qualitative Analysis of Organic Compounds
CLASS NO.:
NAME:
SECTION: DATE PERFORMED:
GROUP NO.: DATE SUBMITTED:
INSTRUCTOR:
ASSIGNMENT
Research each test and write the expected positive and negative results.
Chemical Test Test for Positive Result Negative Result
A. Bromine Test
B. Baeyer’s Test
C. Jones Test
D. Lucas Test
E. Ethanolic AgNO3 Test
F. Ferric Chloride Test
G. Tollens Test
H. Iodoform Test
I. 2,4-DNP Test
ACTUAL OBSERVATIONS
Write (+) for positive results and (-) for negative results.
Sample A B C D E F G H I
Hexane
Cyclohexene
tert-Butyl chloride
Ethanol
Isopropanol
tert-Butanol
Benzyl alcohol
Toluene
Formaldehyde
Acetone
Blank

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POST-LABORATORY ASSIGNMENT – EXPERIMENT NO. 1

Reactions of Hydrocarbons, Alkyl Halides, Alcohols,
Aldehydes, and Ketones
CLASS NO.:
NAME:
INSTRUCTOR:
1. Can tertiary alcohols undergo oxidation? Explain.
2. Lucas test can only be used to alcohols with five (5) carbons or less.
Explain why.
3. Can ketones undergo mild oxidation? Explain.
4. What results would you expect if the following tests were carried out on 4-
hydroxy-3-methoxybenzaldehyde? Explain.
a. water solubility
b. Tollen’s test
c. Benedict’s test

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5. A screening test is common to test for phenylalanine or phenylpyruvate in

the urine. Show the structures of phenylalanine and phenylpyruvate and
indicate whether any of the tests you have performed in this experiment
would give positive results with this urinary metabolite. Is phenylpyruvate a
ketone? Is it a methyl ketone?

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EXPERIMENT NO. 2
Esterification: Synthesis of Aspirin
I. BACKGROUND
Salicylic acid was first discovered from the bark of a willow tree and it
was found to reduce pain and fever. Later, it was found that salicylic acid
damages the mucous membranes of the mouth and esophagus; as well as
bleeding of the stomach lining. These side-effects led to the synthesis of
aspirin.
Aspirin, also known as acetylsalicylic acid, is one of the most common

over-the-counter analgesics (pain-relievers). In addition, it also serves as an
antipyretic, anti-inflammatory agent, and anticlotting agent. This is formed by
acetylating the phenol group of salicylic acid, which is believed to be causing
the adverse side-effects. Aspirin works by inhibiting the formation of
compounds that controls hormone activity and stimulate immune responses
in the body, which are known as prostaglandins.
Esters are carboxylic acid derivatives with the general formula

RCOOR’. They are typically synthesized through the process known as
esterification, by reacting carboxylic acids or acid anhydrides with alcohols.
Aspirin is an ester which can be synthesized by esterification using salicylic
acid and an acid anhydride, catalyzed by a strong acid such as sulfuric acid.
The salicylic acid will be the source of the OH and the anhydride will be the
source of the acetyl group. It is important to note that salicylic acid is not
soluble in acetic anhydride. When the reactants are mixed together and
heated, the disappearance of solids (salicylic acid) indicates the formation of
aspirin; since aspirin is soluble under this condition. However, aspirin
becomes sparingly soluble in a cool solution of acetic anhydride and acetic
acid, thus it precipitates when the solution is cooled. The precipitate can be
collected by vacuum filtration.

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II. OBJECTIVES

 prepare/synthesize aspirin from salicylic acid through esterification;
and
 determine the purity of the synthesized aspirin using ferric chloride
test and using thin-layer chromatography.
III. MATERIALS

commercially available aspirin
B. The instructors/technician/custodian need/s to prepare the following:

Reagents
salicylic acid concentrated sulfuric acid (H2SO4)
acetic anhydride 1% ferric chloride (FeCl3)
dichloromethane ice-cold water
Instrument
water bath blower/dryer
vacuum filtration setup:

Item Qty. Item Qty.
Erlenmeyer flask (250-mL) 2 trough 1
graduated cylinder (100-mL) 1 stirring rod 1
test tube (13 x 100) 3 watch glass 1
graduated cylinder (10-mL) 1 beaker (50-mL) 2
mortar and pestle 1 dropper 2
beaker (250-mL) 1 stainless water bath 1
distilled water (in wash bottle) 1 filter papers 2

P a g e | 10
V. PROCEDURE
A. Synthesis of Aspirin
1. Weigh 2.0 g of salicylic acid to the nearest 0.01 g in a 250-mL
Erlenmeyer flask.
2. Add 5.0 mL of acetic anhydride into the flask. Do this in the fume hood.
3. Add five drops of conc. H2SO4 dropwise then carefully swirl the flask.
4. Heat the mixture in a boiling water bath with constant swirling until all
the solids dissolve.
5. After all the solids dissolve, remove the flask from the boiling water
bath then cool at room temperature.
6. Carefully add 1 mL of water to the mixture (after cooling) under the
hood.
7. Wait for about 5 minutes to allow completion of the reaction then add
50 mL of cold water.
8. Cool the mixture in an ice bath for 10 minutes.
9. In case no precipitate of aspirin appears, carefully scratch the walls of
the flask with a stirring rod.
10. Collect the precipitate (aspirin) on a pre-weighed filter paper using
suction filtration.
11. Rinse the flask with cold water to collect as much crystals.
12. Wash the aspirin crystals on the filter paper with cold water.
13. Spread the crystals out on the filter paper while the vacuum is still on
to facilitate drying.
14. A blower/dryer can be used to facilitate further drying.
15. Weigh the filter paper with the synthesized aspirin on a clean watch
glass. Record the mass of the synthesized aspirin to the nearest 0.01
g.
16. Calculate the theoretical yield of aspirin from salicylic acid by using
the following formula:
1 mole salicylic acid 1 mole aspirin 180 g aspirin

amt. of salicylic acid (g)     amt. aspirin (g)
138 g salicylic acid 1 mole salicylic acid 1 mole aspirin
17. Calculate the percent yield by using the following formula:

amount of aspirin synthesized (in g)
% yield   100
theoretical yield of aspirin (in g)

P a g e | 11
B. Checking the Purify of Aspirin using Ferric Chloride Test

1. Dissolve a small amount of the prepared aspirin and a small amount of
crushed commercially available aspirin using 5 mL of distilled water, in
separate and properly labelled 30-mL beakers.
2. Place 10 to 20 drops of the aspirin solution and the commercial aspirin
in separate, clean, properly labelled test tubes. Assign an additional
test tube for the blank.
3. Place 10 drops of 1% FeCl3 into each test rube and observe the color
formed.
4. The unreacted salicylic acid would react with the FeCl3 to give a purple
color. The darker (more intense) the purple color, the more salicylic
acid are left unreacted thus making the synthesized compound more
impure.
5. Note all your observations.
C. Checking the Purity of Aspirin using Thin-Layer Chromatography

1. Dissolve a small amount of the synthesized aspirin and commercially
available aspirin using 5 mL dichloromethane or chloroform into
separate, properly labelled 30-mL beakers.
2. Develop the TLC chromatogram of each sample using 9:1 chloroform-
MeOH or 9:1 DCM-MeOH.
3. Visualize the spots using available visualizing agents (i.e. iodine
crystals or 15% H2SO4 in ethanol).
4. Calculate the Rf of the samples and compare the two. The
commercially available aspirin will serve as the standard.

P a g e | 12

CLASS NO.:
NAME:
INSTRUCTOR:
A. Synthesis of Aspirin
A.1. Mass of salicylic acid g
A.2. Observation after adding acetic anhydride
A.3. Observation after adding conc. H2SO4
A.5. Observation after boiling
A.7. Observation after 5 minutes of waiting
A.8. Observation after cooling for 10 minutes
A.10. Color of precipitate
Mass of filter paper used to collect aspirin g
A.15. Mass of filter paper + synthesized aspirin g
Mass of synthesized aspirin g
A.16. Theoretical yield of aspirin to be synthesized g
Show solution below.
A.17. Percent yield of synthesized aspirin %

Show solution below.

P a g e | 13
B. Checking the Purify of Aspirin using Ferric Chloride Test

Samples Observations Inferences
Synthesized aspirin
Commercial aspirin
Standard aspirin
Blank
C. Checking the Purity of Aspirin using Thin-Layer Chromatography

Developing solution
Visualizing agent
Draw the TLC chromatograms.

P a g e | 14
POST-LABORATORY ASSIGNGMENT – EXPERIMENT NO. 5

CLASS NO.:
NAME:
INSTRUCTOR:
1. Why was it necessary to boil the reaction mixture containing salicylic acid
and acetic anhydride?
2. What was the purpose of adding concentrated sulfuric acid in the

experiment?
3. What was the purpose of adding cold water and cooling of the reaction
mixture?
4. What was the indication that the reaction between salicylic acid and acetic
anhydride was complete?

P a g e | 15
5. What was the purpose of washing the aspirin precipitate with cold water?
6. Explain the use of ferric chloride in the experiment? Write the chemical
equation showing the reaction of ferric chloride with salicylic acid.

P a g e | 16
EXPERIMENT NO. 3
Isolation and Analysis of Lipids from Egg
I. BACKGROUND
Generally, lipids are macromolecules that are insoluble in water but

unlike carbohydrates, proteins, and nucleic acids, they are not polymeric in
nature. Most of the components of biological membranes are lipids. Other
lipids may be a good source of energy, may function as hormones,
antioxidants, pigments, growth factors, and vitamins (McKee & McKee 2003).
Since lipids exist in a variety of forms, they have many classes which can be
grouped either as saponifiable or non-saponifiable lipids.
Egg is a good source of nutrition for humans since it provides a

complete diet for a developing embryo and serves as the principal source of
food for the first few days of a chick’s life. An average egg may weigh
approximately 57 grams wherein 11% is attributed to the shell constituents,
58% for the egg white, and 31% for the egg yolk. One whole egg contains
approximately 74% water, 13% protein, 11% fat, and 1% ash. Meanwhile, the
egg white does not contain fats and they are normally found in the egg yolk
which is present in as much as 33%. The egg white is composed of
approximately 88% water and 11% protein. (Source: University of Illinois
Extension; Incubation and Embryology).
II. OBJECTIVES

 extract and isolate phosphorylated and non-phosphorylated lipids from
egg yolk using various separation techniques and
 characterize the isolated lipids using qualitative chemical tests.

P a g e | 17
III. MATERIALS

one whole egg

Reagents
dichloromethane 3 M nitric acid
methanol 2.5 % ammonium molybdate
1% sodium chloride solution acetic anhydride
anhydrous sodium sulfate conc. sulfuric acid
acetone potassium nitrate
hydroquinone sodium carbonate
ninhydrin in ethanol Kraut’s reagent
Molisch reagent ice
Instruments
water bath

Item Qty. Item Qty.
beakers (250-mL) 4 funnel 2
graduated cylinder (100-mL) 1 filter papers 2
stirring rod 1 iron ring 1
iron clamp 1 separatory funnel (250-mL) 1
Erlenmeyer flasks (250-mL) 4 evaporating dish 1
Bunsen burner/hot plate 1 trough 1
test tube (13 x 100 mm) 10 test tube holder/clamps 2

P a g e | 18
IV. PROCEDURE
A. Isolation of Lipids from Egg (Folch Method)

1. Crack to open one whole egg.
2. Separate the egg yolk and the egg white and discard the latter.
3. Place the egg yolk into a 250-mL beaker.
4. Add 100 mL of dichloromethane-methanol (2:1 v/v) solution.
5. Mix thoroughly by stirring for 15 minutes.
6. Allow to stand for 2 minutes.
7. Filter the homogenous mixture into a separate 250-mL beaker using a
filter paper.
8. Discard the residue.
9. Collect 15 mL of the filtrate.
10. Place the 15 mL filtrate into a separatory funnel.
11. Extract the lipids by adding 10 mL of 1% NaCl solution. Be sure to vent
frequently the air by opening the stopcock while the separatory funnel
is inverted and holding the cap tightly. If you neglect venting, pressure
might build up and blow the solution out of the funnel.
12. Collect the organic lower layer into a 250-mL beaker.
13. Discard the remaining aqueous upper layer from the separatory
funnel.
14. Return the collected organic lower layer back into the separatory
funnel.
15. Extract again by adding 10 mL of 1% NaCl solution. Be sure to vent
frequently the air.
16. Similarly, collect the organic lower layer and discard the aqueous
upper layer.
17. Add anhydrous Na2SO4 to remove the remaining traces of water.
18. Filter the mixture and collect the filtrate into a 250-mL Erlenmeyer
flask.
19. Add a pinch of hydroquinone to the filtrate.
20. Transfer the filtrate into an evaporating dish and heat it to dryness
under the fume hood.
21. Add 10 mL of acetone to the residue in the evaporating dish.
22. Cool the acetone-residue mixture in an ice bath for 15 minutes.
23. Decant the acetone-residue mixture through a filter paper into a 250-
mL Erlenmeyer flask. The decantate contains the non-phosphorylated
lipids.
24. Wash the remaining residue in the evaporating dish with 5 mL cold
acetone. This residue contains the phosphorylated lipids.
25. Decant the acetone-residue mixture through a filter paper into the 250-
mL Erlenmeyer flask used in A.23.
26. Transfer the residue in A.25 into a test tube and dissolve it in 3 mL
dichloromethane-methanol (2:1 v/v) solution.

P a g e | 19
27. Add a pinch of hydroquinone and label it as PL (phosphorylated lipids)

with your group number and section. Cover the test tube with foil and
keep it in the refrigerator until it is needed.
28. For the decantate in A.25, evaporate it to dryness under the fume
hood.
29. Add 3 mL of dichloromethane-methanol (2:1 v/v) solution and transfer it
into a test tube.
30. Add a pinch of hydroquinone and label it as NPL (non-phosphorylated
lipids) with your group number and section. Cover the test tube with
foil and keep it in the refrigerator until it is needed.
B. Analysis of Lipids
1. Libermann-Burchard Test
a. Place 20 drops of each lipid sample into two separate test tubes.
b. Place 20 drops of distilled water into another test tube (blank).
c. Add 10 drops of acetic anhydride into each test tube then swirl
gently.
d. Allow 10 drops of conc. H2SO4 to slide down the walls of each test
tube until it reaches the samples one at a time.
i) Note your observations. Check for the formation of a blue green-
colored product. This confirms the presence of cholesterol, which
is a non-phosphorylated and non-hydrolyzable lipid.
2. Test for Phosphate

a. Mix 20 drops of each lipid sample with 5 mL of KNO3:Na2CO3 (3:1)
solution into an evaporating dish.
b. Heat the mixture until it turns to a grayish or colorless liquid or
when a white or gray ash is obtained.
c. Allow it to cool for 5 minutes then add 6 mL of warm water to
dissolve the contents.
d. Transfer the solution into a test tube then add 3 M HNO3.
e. Heat the solution in a water bath at 65°C.
f. Add 6 mL of 2.5% ammonium molybdate then heat the test tube in a
boiling water bath.
g. Note your observations. Check for the formation of yellow
precipitate. It confirms the presence of phosphate group.
3. Ninhydrin Test
a. Place 15 drops of each lipid sample into two separate test tubes
and prepare a blank in a separate test tube.
b. Add 16 drops of ninhydrin in ethanol solution into each test tube.
c. Heat the test tubes in a boiling water bath.

P a g e | 20
d. Note your observations. Check for the formation of a blue or violet-

colored product. It confirms the presence of amino groups.
4. Molisch Test
b. Heat the samples until the liquid has evaporated.
c. Add 15 drops of distilled water into the test tubes with dried
samples.
d. Add 5 drops of Molisch reagent into each test tube.
e. Tilt the test tubes and allow 15 drops of conc. H2SO4 to flow down.
f. Return the test tubes in the rack and note your observations.
Check for the presence of a purple ring at the interface which
confirms the presence of carbohydrates.
5. Kraut’s Test
b. Heat the samples until the liquid has evaporated.
c. Add 15 drops of distilled water into the test tubes with dried
samples.
d. Add 20 drops of Kraut’s reagent and swirl gently.
e. Heat the test tube and note your observations. Check for the
formation of a dark orange to red precipitate. It confirms the
presence of choline.
6. Acrolein Test
a. Place a pinch of each lipid sample into two separate test tubes and
prepare a blank in a separate test tube.
b. Add a pinch of potassium hydrogen sulfate (KHSO4) into each test
tube.
c. Heat the test tubes in an open flame or in a boiling water bath.
d. Note your observations. Check for the formation of a burnt fat
odor. It confirms the presence of glycerol.

P a g e | 21

CLASS NO.:
NAME:
INSTRUCTOR:
A. Isolation of Lipids from Egg

A.5. Observation after mixing
A.6. Observation after 10 minutes
A.7. Color of the filtrate
A.11. Observation after adding 1% NaCl
Color of the organic upper layer
Color of the aqueous lower layer
A.20. Color of the residue
Describe the appearance of the phosphorylated lipids.
Describe the appearance of the non-phosphorylated lipids.
B. Analysis of Lipids
Test PL NPL Blank
Liebermann-
Burchard Test
Test for Phosphate
Ninhydrin Test
Molisch Test
Kraut’s Test
Acrolein Test

P a g e | 22

CLASS NO.:
NAME:
INSTRUCTOR:
1. What is the difference between hydrolyzable lipids and non-hydrolyzable

lipids?
2. Draw the structures of cholesterol, tripalmitin, lecithin, and choline.

Classify each as hydrolyzable or non-hydrolyzable. Identify which lipids are
phosphorylated.
3. What is the purpose of adding hydroquinone?
4. What is the purpose of washing the egg homogenate with 1% saline

solution?

P a g e | 23
EXPERIMENT NO. 4
Characterization of Carbohydrates
Carbohydrates are the most abundant biomolecules in nature. They

have extensive roles in all forms of life. (Berg et al., 2002). They serve as
sources of energy (glucose) and metabolic intermediates. They are present
in the structural framework of RNA and DNA (ribose and deoxyribose).
Polysaccharides serve as structural elements in the cell walls of plants and
bacteria and other animals (i.e. cellulose for plants and chitin for insects).
Carbohydrates may also be linked to many proteins and lipids wherein they
play a key role in various cellular interactions (Berg et al, 2002; McKee &
McKee, 2003).
Carbohydrates are organic molecules that are basically composed of

carbon, hydrogen, and oxygen atoms, thus it is often abbreviated as CHO. It
is a polymeric molecule that is composed single units called monosaccharide.
There are two ways to classify a monosaccharide, either by the carbonyl
group head, or the number of its carbon chain. A monosaccharide can either
be an aldehyde (aldose) or a ketone (ketose). It can also be a triose (3
carbon), a tetrose (4 carbon), a pentose (5 carbon), a hexose (6 carbon) or
even a heptose (7 carbon). The two classifications can be combined wherein
a 3-carbon chain aldehyde monosaccharide is called an aldoriose and so on.
Other than the carbonyl groups, carbohydrates also contain hydroxyl groups
within its chain. When two monosaccharide are linked to each other by an
ether bond (glycosidic bond), it is known as a disaccharide.
Because of the presence of several functional groups in

carbohydrates, these can undergo several chemical reactions such as
oxidation, reduction, ring formation, hydrolysis, and many more which may
produce observable results. The products that will be formed may vary
based on the different class of carbohydrates.

P a g e | 24
II. OBJECTIVES

 classify carbohydrates based on the carbonyl functionality and based
on the number of carbons, using qualitative chemical tests.
III. MATERIALS

none

Reagents
glucose xylose lactose
amylose fructose sucrose
maltose galactose
Molisch reagent Barfoed’s reagent

conc. sulfuric acid Seliwanoff’s reagent
Iodine solution Bial’s Orcinol reagent
Benedict’s reagent
Instrument
water bath

Item Qty. Item Qty.
test ube (13 x 100 mm) 20 stainless water bath 1
test tube holder 5 distilled water (in wash bottles) 1

P a g e | 25
IV. PROCEDURE
A. Preliminary Preparations
1. Label 10 test tubes with glucose, amylose, maltose, xylose, fructose,
galactose, lactose, sucrose, unknown and blank (distilled water only).
2. Place 15 drops of each sugar sample into their respective, properly
labelled test tubes.
3. Prepare a boiling water bath setup.
4. Be sure to wash all the test tubes after each test before proceeding to
the next.
B. Molisch Test
1. Add 5 drops of Molisch reagent into each test tube.
2. Tilt the test tubes and allow 15 drops of concentrated H 2SO4 to flow
down.
3. Return the test tubes in the rack. Note your observations.
C. Iodine Test
1. Add 5 drops of iodine solution into each test tube.
2. Return the test tubes in the rack. Note your observations.
D. Benedict’s Test
1. Add 25 drops of Benedict’s reagent into each test tube.
2. Heat the test tubes in a boiling water bath for 2 minutes.
3. Remove the test tubes from the water bath and return them in the rack.
4. Note your observations.
E. Barfoed’s Test
1. Add 25 drops of Barfoed’s reagent into each test tube.
2. Heat the test tubes in a boiling water bath for 5 minutes.
3. Record the time (in minutes and seconds) when the orange or red
precipitate starts to appear.
i) Reducing monosaccharide will form orange or red precipitates
faster (in less than 2 to 3 minutes) than reducing disaccharides
(which may take up to 10 minutes).
F. Seliwanoff’s Test
1. Add 25 drops of Seliwanoff’s reagent into each test tube.
2. Heat the test tubes in a boiling water bath until an observable change
is observed. Note the time when the colored product is observed.
G. Bial’s Test
1. Add 25 drops of Bial’s Orcinol reagent into each test tube.
2. Heat the test tubes in a boiling water bath and note your observations.

P a g e | 26

CLASS NO.:
NAME:
INSTRUCTOR:
ASSIGNMENT:
Molisch Test
Iodine Test
Benedict’s Test
Barfoed’s Test
Seliwanoff’s Test
Bial’s Test
ACTUAL OBSERVATIONS
Observations
CHO
Molisch Iodine Benedict’s Barfoed’s Seliwanoff’s Bial’s
Sample
Test Test Test Test Test Test
Glucose
Amylose
Maltose
Xylose
Fructose
Galactose
Lactose
Sucrose
Unknown
Blank

P a g e | 27

CLASS NO.:
NAME:
INSTRUCTOR:
1. What are reducing sugars? Explain.
2. Explain why Benedict’s reagent is needed to be alkaline in nature?
3. Aside from Benedict’s test, what other test/s can be used to detect
reducing sugars? Explain the principle behind it/them.
4. How come Iodine Test only works for starch? Does it also produce a
positive result with glycogen? Explain and show illustrations.

P a g e | 28
5. How does Molisch Test produce its purple color when in reacted with
sugars? Show illustrations.
6. Seliwanoff’s Test is a test for the presence of ketoses. Does it also react
with aldoses? If yes, what particular aldoses and what color does it produce?
7. Bial’s Test is a test for pentoses. Does it also react with other sugars with
different carbon numbers? If yes, what particular carbon number and what
color does it produce?

P a g e | 29
EXPERIMENT NO. 5
Isolation of Casein from Milk and
Analysis of Amino Acids
I. BACKGROUND
Proteins are polymers composed of sub-units called amino acids that

are linked together by a peptide bond (amide bond). They serve as primary
structural components of muscles, connective tissues, hairs, nails, and other
parts of a living organism. They also serve as catalyst in several biochemical
reactions, material transport, and metabolism regulators in the body, as well
as in providing defenses for the body (McKee & McKee, 2003).
There are two major fractions of proteins in cow’s milk and these are
casein and whey protein. Caseins are phosphate-containing proteins that
occur as micelles in the native form, and precipitate at pH 4.6. Milk soluble
proteins remain in solution at pH 4.6 and constitute a heterogeneous group of
proteins (β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins,
lactoferrin, and other minor fractions). Caseins and milk soluble proteins
greatly differ regarding their amino acid (AA) composition. The latter
contains higher concentrations of total sulfur amino acids (methionine and
cysteine), lysine, threonine, and tryptophan (Lacroix et al., 2006).
II. OBJECTIVES

 isolate casein from milk by isoelectric precipitation; and
 characterize the amino acid present in casein using qualitative
chemical tests.

P a g e | 30
III. MATERIALS

non-fat milk
cheesecloth or old handkerchief

Reagents
glacial acetic acid ethanol
dichloromethane
L-glycine L-tryptophan
L-serine L-arginine
albumin L-tyrosine
3 M NaOH ninhydrin in ethanol

5% CuSO4 concentrated nitric acid
0.02% 1-naphthol bromine water
Hopkins-Cole reagent concentrated sulfuric acid
Instrument
pH meter
water bath
vacuum filtration setup

Item Qty. Item Qty.
beaker (250-mL) 4 graduated cylinder (50-mL) 1
stirring rod 1 thermometer 1
test tube (13 x 100 mm) 20 stainless water bath 1
distilled water (in wash bottles) 1 test tube holder 5

P a g e | 31
IV. PROCEDURE
A. Isolation of Casein from Milk

1. Place 100.0 mL of non-fat milk into a pre-weighed empty 250-mL
beaker. Determine and record the mass of the 100.0 mL milk.
2. Place it in a water bath.
3. Heat the water bath while constantly stirring the milk inside the
beaker.
4. Monitor the temperature of the milk sample using a thermometer.
5. Stop heating when the temperature reaches 40°C and remove the
beaker from the water bath.
6. Add 10 drops of glacial acetic acid while constantly stirring the warm
milk solution.
7. Note your observations. Check for the formation of any precipitate.
8. Measure the pH of the mixture after A.8 using the pH meter. The pH
should be 4.65 to 4.70. If the reading exceeds 4.70, add more glacial
acetic acid (approximately 5 drops).
9. Filter the heterogeneous mixture using a cheesecloth into another 250-
mL beaker.
10. Squeeze the cheesecloth gently to remove more traces of liquid on the
precipitate.
11. Discard the filtrate in the beaker.
12. Collect the precipitate from the cheesecloth and transfer it into a 250-
mL beaker.
13. Add 50 mL of 95% ethanol.
14. Stir the mixture for 5 minutes. Avoid hitting the walls of the beaker.
15. Allow the precipitate to settle to the bottom of the beaker.
16. Decant the mixture into a 250-mL beaker.
17. Discard the decantate.
18. Add 50 mL of dichloromethane-ethanol (1:1, v/v) to the residue.
19. Stir the mixture for 5 minutes. Avoid hitting the walls of the beaker.
20. Collect the solids using vacuum filtration.
21. Discard the filtrate.
22. Collect the casein precipitate on a dry pre-weighed filter paper.
23. Weigh the filter paper with the casein and determine the mass of the
casein.
24. Calculate for the percent casein present in 50.0 g of milk using the
following formula:
mass of casein precipitat e
% casein   100
mass of 100 - mL milk
25. Prepare a casein test solution by dissolving the precipitate in distilled
water.

P a g e | 32
B. Analysis of Amino Acids and Proteins

1. Preliminary Preparations for the Analysis of Proteins
a. Label seven (7) test tubes with glycine, serine, arginine,
tryptophan, tyrosine, albumin, and blank.
b. Place 15 drops of each sample into their respective, properly
labelled test tubes.
c. Be sure to wash all the test tubes after each test before proceeding
to the next.
2. Biuret Test
a. Add five drops of 3 M NaOH solution into each sample.
b. Add two drops of 5% CuSO4 solution then swirl gently.
c. Note your observations.
3. Ninhydrin Test
a. Add 16 drops of ninhydrin in ethanol solution into each sample.
b. Heat the test tube in a boiling water bath.
4. Xanthoproteic Test
a. Add 10 drops of concentrated nitric acid into each sample. Shake
the test tubes after each drop.
b. Heat the test tubes in a water bath.
5. Sakaguchi Test
a. Add three drops of 3 M NaOH into each sample.
b. Add three drops of 0.02 % α-naphthol solution then swirl gently.
c. Allow the test tubes to stand for 3 minutes.
d. Add three drops of freshly-prepared bromine water. Check if the
bromine water is still color orange. Ask your instructor for
replacement if the reagent is colorless.
e. Note your observations.
6. Hopkins-Cole Test
a. Add 2.0 mL of Hopkins-Cole reagent into each sample then swirl
gently.
b. Tilt the test tubes and allow 40 drops of concentrated H 2SO4 to
slide slowly down the walls until it reaches the sample. Do not mix
the solution.

P a g e | 33

Isolation of Casein from Milk and Analysis of Amino Acids
CLASS NO.:
NAME:
INSTRUCTOR:
A. Isolation of Casein from Milk
A.1. Mass of empty 250-mL beaker

Mass of 250-mL beaker + 100-mL milk
Mass of 100-mL milk
A.7. Describe the sample after the addition of glacial acetic acid.
A.8. pH of the sample

A.22. Mass of the filter paper
Mass of the filter paper + casein
Mass of casein
A.24. Percent casein present in milk
mass of casein precipitate __________

% casein   100   100  __________
mass of 100 - mL milk __________

P a g e | 34
ASSIGNMENT
Biuret Test
Ninhydrin Test
Xanthoproteic Test
Sakaguchi Test
Hopkins-Cole Test
ACTUAL OBSERVATIONS
Observations
Sample Biuret Ninhydrin Xanthoproteic Sakaguchi Hopkins-
Test Test Test Test Cole Test
Glycine
Serine
Arginine
Tryptophan
Tyrosine
Albumin
Casein
Blank

P a g e | 35

Isolation of Casein from Milk and Analysis of Amino Acids
CLASS NO.:
NAME:
INSTRUCTOR:
1. Why was it necessary to use a non-fat milk in the isolation of casein?
2. In section A.5, why were you asked to stop heating at 40°C?
3. In A.6, what was the purpose of adding glacial acetic acid?
4. In A.8, why was it important to have a pH of 4.6 to 4.7?
5. How does Biuret reagent react with proteins? What product is formed that
causes the purple color?

P a g e | 36
6. How does Xanthoproteic test detect aromatic amino acids? What chemical
reaction is involved and what products are formed?

P a g e | 37
EXPERIMENT NO. 6
Isolation and Analysis of DNA from White Onion
I. BACKGROUND
Nucleic acids are polymers of nucleotides linked by 3’ to 5’

phosphodiester bond. Nucleotides are the building blocks of nucleic acids
and each one is composed of a nitrogeneous base, a pentose sugar
backbone, and a phosphate group. The nitrogeneous bases can be grouped
into two as either a purine or a pyrimidine. Adenine and guanine are purine
bases while cytosine, thymine, and uracil are pyrimidine bases. Thymine is
only found in DNA and it is replaced by uracil in RNA.
There are two types of nucleic acids, the DNA and the RNA. DNA
serves as repository of genes which carry the biological instruction for the
synthesis of proteins while RNA is directly involved in protein synthesis.
II. OBJECTIVES

 isolate DNA from white onion; and
 analyze the isolated DNA using Dsche reaction.

P a g e | 38
III. MATERIALS

white onion
B. The instructors/technician/custodian need/s prepare the following:

Reagents
homogenizing solution
ice-cold ethanol (95% or absolute)
1 M sulfuric acid
Dische reagent
Instrument
water bath
laboratory blender
C. The students should borrow the following from the stockroom:

Item Qty. Item Qty.
beaker (250-mL) 4 stirring rod 1
graduated cylinder (100-mL) 1 trough 1
distilled water (in wash bottles) 1 test tube (13 x 100 mm) 3
dropper 1 ice
thermometer 1 watch glass 1

P a g e | 39
IV. PROCEDURE
A. Isolation of DNA from Onion

1. Chop an onion into small pieces. Weigh 20.0 g (to the nearest 0.01 g)
directly into a 250-mL beaker.
2. Place approximately 40.0 mL of homogenizing solution (or simply for
every 1 gram of sample, place 2 mL of homogenizing solution) into a
separate 250-mL beaker. Heat the solution in a warm water bath (set
at 60ºC) until the temperature reaches 60°C.
3. When the temperature reaches 60°C, immediately add the chopped
onion.
4. Stir and incubate the mixture for 15 minutes. Maintain the 60°C
temperature.
5. After the incubation period, place the beaker containing the mixture in
an ice bath and incubate for 5 minutes with mild stirring.
6. Homogenize the mixture using a blender for 45 seconds at low speed.
7. Pour the homogenate (the product of homogenization) into a 250-mL
beaker and incubate it in an ice bath for 15 to 20 minutes.
8. Filter the cooled homogenate through 2 layers of cheesecloth over a
250-mL beaker placed in an ice bath. Try to estimate the volume of the
filtrate based on the reading on the beaker.
9. Tilt the beaker at a certain angle and slowly add ice-cold 95% or
absolute ethanol down the side of the beaker. The amount (volume) of
the ethanol must be twice the amount of the filtrate.
10. Allow the mixture to stand for 3 minutes. Collect the floating DNA in
one direction only, using a spooler. The spooler can be a clean wire
that has a hook shape at one end.
11. Place the isolated DNA on a pre-weighed watch glass and allow it to
dry.
12. Calculate the percent yield.
mass of recovered DNA
% yield DNA   100
20.0 g white onion
B. Hydrolysis and Analysis of DNA using Dische Reaction

1. Place the isolated DNA into a test tube and add 20 drops of 1.0 M
H2SO4.
2. Heat the mixture in a boiling water bath for one (1) hour while shaking.
Cover the test tube with a foil.
3. Cool the mixture at room temperature and add 2.0 mL of water.
4. Place 20 drops of the hydrolyzed DNA sample into a clean test tube.
5. Add 2.0 mL of Dische reagent into the test tubes (including the blank).
6. Heat the solutions for 10 minutes in a boiling water bath.
7. Cool the solutions and note your observations.

P a g e | 40

CLASS NO.:
NAME:
INSTRUCTOR:
A. Isolation of DNA from Onion

A.1. Mass of white onion g
A.4. Observation after 15 minutes of incubation
A.6. Observation after homogenization
A.8. Estimated volume of the filtrate mL

A.9. Estimated volume of ethanol mL
A.10. Observation after 3 minutes
A.11. Percent DNA yield:
mass of collected DNA __________

% yield DNA   100   100  __________
20.0 g onion __________
B. Hydrolysis and Analysis of DNA using Dische Reaction

Observation
Test
DNA Sample Blank
Hydrolysis
Dische Reaction

P a g e | 41

CLASS NO.:
NAME:
INSTRUCTOR:
1. Identify the composition of the homogenizing solution used in the

extraction of DNA from white onion. Describe the role of each reagent found
in this mixture. You may use the back page for extra space.
2. Why was the incubation temperature set to 60°C?
3. What was the purpose of incubating the homogenate in an ice bath?
4. What was the purpose of adding ice-cold ethanol?
5. Explain the principle behind the Dische reaction.

P a g e | 42
EXPERIMENT NO. 7
Determination of Vitamin C Concentration
by Titration
I. BACKGROUND
Vitamin C, also known as ascorbic acid, is a water-soluble vitamin that

plays a role in oxidation reactions in the body. It is used to fight infection and
repair damaged tissues. Good sources of vitamin C are citrus fruits, grapes,
broccoli, lettuce, and green peppers.
Detection of vitamin C in fresh produce and packaged beverages can

be done by reaction with iodine. It will act as a reducing agent by being
oxidized and converting iodine to iodide. Starch solution can be used as an
indicator in this reaction. Iodine forms a deep-blue to black color when
reacting with starch. While iodine is being added to ascorbic acid, it is being
converted to iodide thus, a colored product cannot be observed. Once all of
the ascorbic acid has been consumed, the excess iodine will then form the
deep-blue to black end point.
Figure 8.1 Reaction between ascorbic acid and iodine
Figure 8.2 Formation of the dark-blue to black complex of starch with iodine
Retrieved from http://www.biosci.ohiou.edu/introbioslab/Bios170/170_2/iodine.htm

P a g e | 43
II. OBJECTIVES

 quantitatively determine the amount of vitamin C in fresh fruit juices
and packaged fruit juices using redox titration
III. MATERIALS

packaged fruit juice
ascorbic acid tablets or capsules

Reagents
0.005 M Iodine solution
1% Starch indicator
Instrument
none

Item Qty. Item Qty.
buret (50-mL) 2 beaker (250-mL) 1
graduated cylinder (25-mL) 1 beaker (100-mL) 2
mortar and pestle 1 buret clamp 2
Erlenmeyer flask (250 mL) 4 iron stand 2
stirring rod 1

P a g e | 44
IV. PROCEDURE
A. Titration of Standard Vitamin C

1. Crush the ascorbic acid tablets using mortar and pestle and dissolve
100 mg (0.100 g) of ascorbic acid in 100 mL water.
2. Using the 25-mL graduated cylinder, measure 20 mL of the sample
solution. Transfer this solution in a 250-mL Erlenmeyer flask.
3. Add 20 drops of starch indicator in the flask that contains the sample.
4. Fill the 50-mL buret with 0.005 M iodine solution and use this to titrate
the sample. The endpoint of the titration is identified as the first
permanent trace of a dark-blue to black color.
5. Perform this in two (2) trials and note the results in your experimental
report sheet.
B. Titration of Packaged Fruit Juice

1. Repeat A.2. to A.5. using the packaged fruit juice.
C. Treatment of Results
1. Calculate the average volume of iodine solution used in Parts A and B.
2. Given the concentration of iodine and the volume used, calculate the
moles of iodine reacting.
3. Using the equation of the titration (below), determine the number of
moles of ascorbic acid reacting.
ascorbic acid + I2 → 2I− + dehydroascorbic acid
4. Calculate the molar concentration of ascorbic acid in the solution

obtained from fruit/vegetable/juice.

P a g e | 45

Determination of Vitamin C Concentration by Titration
CLASS NO.:
NAME:
INSTRUCTOR:
A. Titration of Standard Vitamin C

Trial 1 Trial 2
Concentration of Iodine (M)
Volume of sample (mL)
Final reading of titrant (mL)
Initial reading of titrant (mL)
Volume of titrant used (mL)
No. of moles of iodine (mol)
No. of moles of ascorbic acid (mol)

Concentration of ascorbic acid in
the sample solution (M)
B.2. Titration of Packaged Fruit Juice

Trial 1 Trial 2
Concentration of Iodine (M)
Volume of sample (mL)
Final reading of titrant (mL)
Initial reading of titrant (mL)
Volume of titrant used (mL)
Volume of iodine used (mL)
No. of moles of iodine (mol)
No. of moles of ascorbic acid (mol)

Concentration of ascorbic acid in
the sample solution (M)

P a g e | 46
C. Calculations
 Write all your calculations below and box your final answers.

P a g e | 47

Determination of Vitamin C Concentration by Titration
CLASS NO.:
NAME:
INSTRUCTOR:
1. A solution of iodine was standardized with ascorbic acid. Using a

0.1000-g sample of pure ascorbic acid, 22.46 mL of iodine were required
to reach the starch endpoint. What is the molarity of the iodine solution?
2. A sample of fresh dalandan juice was filtered and titrated with the iodine
solution given in problem 1. A 100-mL sample of the juice took 10.75 mL of
the iodine solution to reach the start endpoint.
a. What is the concentration of vitamin C in the juice in mg vitamin C

per 100 mL of juice?
b. What quantity of juice will provide the RDA amount of vitamin C?


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General Education Department

Integrated Sciences Cluster

WARREN S. VIDAR, RCh, MSc

Chemistry Unit, Integrated Sciences Cluster

Chemistry Laboratory Guidelines and Policies

Proper Practices and Safety in the Laboratory

1 Qualitative Organic Analysis

2 Synthesis and Analysis of Aspirin

3 Isolation and Analysis of Lipids from Egg

5 Isolation of Casein from Milk and Analysis of Amino Acids

6 Isolation and Analysis of DNA from White Onion

7 Determination of Vitamin C Concentration by Titration

Chemistry Unit, Integrated Sciences Cluster

Laboratory Guidelines and Policies

1. There shall be a minimum of three (3) and a maximum of four (4)

Chemistry Unit, Integrated Sciences Cluster

1. After the laboratory period, the borrowed items must be returned to

Chemistry Unit, Integrated Sciences Cluster

Chemistry Unit, Integrated Sciences Cluster

Proper Practices and Safety in the Laboratory

A. Management of Work Space and General Equipment Setup

1. Keep workspace uncluttered.

Chemistry Unit, Integrated Sciences Cluster

11. Chemicals should only be heated in a ventilated area or hood. Prior to

1. Wear proper eye protection. Contact lenses should not be worn.

Chemistry Unit, Integrated Sciences Cluster

C. General Precautions for Handling Chemicals

D. Safe Laboratory Techniques

Chemistry Unit, Integrated Sciences Cluster

7. To avoid violent reactions and spattering while diluting chemicals,

Chemistry Unit, Integrated Sciences Cluster

A. General Procedures for Spills

1. Immediately alert fellow workers and supervisor.

Chemistry Unit, Integrated Sciences Cluster

they will only re-disperse mercury aerosols and spread

d. White (Yellow) Phosphorous

B. Chemicals on the Skin

2. For larger spills, quickly remove all contaminated clothing, shoes,

Chemistry Unit, Integrated Sciences Cluster

C. Chemicals in the Eyes

For chemical splashes, the following are recommended: flushing of the

D. Releases of Acutely Toxic Vapors and Gases

Some vapors and gases can be permanently disabling or lethal when

Chemistry Unit, Integrated Sciences Cluster

Government and local environmental regulations should govern the disposal

1. To minimize disposal problems and waste, always specify the smallest

Chemistry Unit, Integrated Sciences Cluster

Organic compounds are basically classified based on the type of

Hydrocarbons are compounds that contain exclusively of carbon and

Alcohols, having the hydroxyl functional group (-OH), mostly undergo

Aldehydes and ketones belong to a group of compounds that contain

Chemistry Unit, Integrated Sciences Cluster

At the end of this activity, the students should be able to:

A. The students need to bring the following:

B. The instructors/technician/custodian need to prepare the following:

bromine water Tollens reagent

C. The students need to borrow the following from the stockroom:

Chemistry Unit, Integrated Sciences Cluster

F. Ethanolic Silver Nitrate Test

G. Ferric Chloride Test

Chemistry Unit, Integrated Sciences Cluster

J. Reaction with 2,4-dinitrophenylhydrazine (2,4-DNPH)

Chemistry Unit, Integrated Sciences Cluster

REPORT SHEET – EXPERIMENT NO. 1