BIOCHEMISTRY FOR

MEDICAL
LABORATORY
SCIENCE

NAME : _________________________________________
COURSE : _________________________________________
INSTRUCTOR : _________________________________________
SCHOOL YEAR: _________________________________________

(APRIL 2023)
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 Table of Contents

Preface………………………………………………………………………………………….…. 2
Laboratory Safety and Guidelines, Rules, and Regulations………………….…………….3
Learning Activity No. 1 Animal and Plant Cell ………………………………………………12
Learning Activity No. 2 Characteristics of Water……………………….…………………...14
Learning Activity No. 3 Analysis of Carbohydrates.…………………….………………….19
Learning Activity No. 4 Analysis of lipids…………………………….……………………......31
Learning Activity No. 5 Analysis of Proteins……………………………. ……………………38
Learning Activity No. 6 Isolation of RNA……………………...............................................50
Learning Activity No. 7 Analysis of vitamins .............……………….……………………….53
References………………………………………………………………………………………….61

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 PREFACE

This Laboratory Manual in Chemistry is meant for all students taking up a course
in biochemistry. This course deals with the structure, classifications, and functions of
the different biochemical substances in the body. This includes the chemistry of
carbohydrates, lipids, proteins, enzymes, nucleic acids, and its metabolism. This is a
five (5) unit course, within is two (2) unit laboratory. A total of six (6) contact hours per
week is dedicated to the laboratory.

The learning activities in this manual were carefully designed to provide the
student with a better understanding of methods and proper techniques in the
chemistry laboratory. The manual has 12 learning activities and covers practically the
basic principle and concepts of the chemistry laboratory.

The manual is made in a way to make a better understanding of the course

easily. Learning activities were explained and discussed properly for the students to
follow accurately and perform each procedure correctly. The availability of
instruments and chemicals was also considered in designing this manual.

This laboratory manual will serve the needs of the students and teachers and
enhance the ability of the students with the common techniques inside the laboratory
special dealing with chemicals. Thus, familiarization with the common names and uses
of the different instruments for their future laboratory subjects will be considered.

Instructor

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Introduction
Very rarely will an injury or accident occur in a well-supervised laboratory. When an
injury or accident does occur, it is generally brought about by complacency. In this
laboratory, you will hear a LOT about lab safety; you will be given safety instructions
at the start of each lab, and you will be told of the major hazards of each chemical
you will be using. Sometimes, such an emphasis makes a student nervous about what
may be a new learning environment for them. This is an unfortunate and unintentional
side effect, but it is important to give such emphasis on safety to reduce the odds of
injuries in the lab by being sure that students know what hazards exist, how to avoid
them and how to respond if something does go wrong. Knowledge is the best defense
against injury in any science laboratory.

The best way to prevent accidents is for you to know the possible hazards of the
laboratory. Any experiment, no matter how often it has been performed in the past,
has the potential to fail with hazardous results. By knowing the hazards, you will
develop a healthy respect for what is happening around you, and with this respect,
heightened levels of observation are sure to follow. This implies that potential
accidents can be spotted before they can occur. If there is ever anything that does
not seem right to you, it is not only your right, but also your obligation to point them
out to the instructor. The instructor will do his/her part to keep you safe, but he/she will
need your help.

The following sections present some general guidelines. These are not arbitrary rules
set down to make your life less enjoyable. Each one of them has a specific purpose,
which will hopefully be made clear to you. If not, ask!

Most of laboratory safety is COMMON SENSE. Remember that this is a general

guideline, and therefore may be incomplete. If you are ever unsure about safety,
please ask.

The organic chemistry laboratory is potentially one of the most dangerous of

undergraduate laboratories. That is why you must have a set of safety guidelines. It is
a very good idea to pay close attention to these rules, for one very good reason:

The penalties are only too real.

Disobeying safety rules is not at all like flouting many other rules. You can get seriously
hurt. No appeal. No bargaining for a flat 1 grade so you can get into medical school.
Perhaps as a patient, but certainly not as a student. So, go ahead. Ignore these
guidelines. But remember—

You have been warned!

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LABORATORY APPAREL
1. Safety goggles are ALWAYS required in the laboratory! Splash hazards are perhaps
the most significant danger present in the lab, and eyes are extremely sensitive.
2. Contact lenses are not permitted in the lab. Your goggles will protect your eyes
from spill hazards, but do nothing to protect you from fumes, which can dry your
contacts out and may result in the necessity of an operation for their removal.
Contact lenses can also absorb chemicals from the air (especially the new
“breathable” lenses), concentrate and hold them against the eye, and prevent
proper flushing of the eye should a chemical be splashed into the eyes.
3. Laboratory gowns/aprons must be always donned. In an event of a spill, these
gowns/aprons will protect your clothing and could save you from scar tissue.
4. Sandals, open-toed shoes, and high heels are not permitted in the lab. This is to
protect your feet from splashes and spills. The restriction on high heels is for
balance.
5. Shorts or skirts cut above the knee are not permitted in the lab. Again, should a spill
occur, it will be your clothing that will be your protection from direct exposure of
the skin to that chemical. The idea is to put as many layers of clothing as possible
between you and a chemical spill. The more clothing, the more diffuse the
chemical will be by the time it reaches the skin, and the greater the chance to
remove the chemical before it reaches your skin.
6. Careful consideration should be given before wearing any jewelry into the lab.
Some chemicals evaporate very quickly and therefore pose relatively little danger
should they go onto your skin. However, if they get beneath a ring, watch or some
other form of jewelry, they can be prevented from evaporating, held against the
skin longer and greatly increase the risk of injury. Should you decide to wear jewelry
to the lab, be particularly mindful of itching, burning or any other irritation under or
around your jewelry.
7. Never wear clothes that hang, such as loose sleeves. Be sure ties and scarves are
tucked well inside your laboratory gown/apron. These pose fire hazards (if you are
reaching or bending down near an open flame) as well as chemical hazards (if
they accidentally get dragged through a chemical, they can transport that
chemical directly to your skin).
8. Long hair is to be constrained. Like hanging clothes, long hair is subject to fire and
contact with chemicals. A rubber band will be used to constrain particularly long
hair if necessary.
9. No radios, tape players, CD players or any other devices of this type will be
permitted in the laboratory at any time. Loud music is distracting, and headphones
prevent you from hearing announcements or verbal warnings given in the lab.

SAFETY EQUIPMENT
1. Take the time to identify all the laboratory safety equipment and always keep their
location in your mind. You should be able to close your eyes any time during a lab
and point to such safety equipment as the fire extinguisher, the emergency
eyewash stations, the fire blankets, the safety shower, etc. If you were to splash a
chemical in your eyes, you’d better be able to find that eyewash station without
your eyes well before permanent damage can occur (which can be seconds
depending on the nature of the chemical).
2. Check all safety equipment. I’ll keep as close an eye on it as possible, but I need
your help as well. Is the fire extinguisher charged? Does it have the plastic “seal”?
Is there enough sodium bicarbonate in case there is a chemical spill. If anything
does not look right to you, report it to your lab instructor IMMEDIATELY!

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3. Material Safety Data Sheets (MSDS’s) are available in the stockroom. Basic safety
information will be given during safety lecture before each lab,

GENERAL BEHAVIOR
1. Absolutely no HORSEPLAY will be tolerated in the laboratory! Offenders of this one
will be unceremoniously cast out with a zero-resulting score for that day’s work. I
realize that at times it is awfully tempting to grab that water bottle and squirt your
friends, but many hazardous chemicals look like water. The humor will be lost if
something other than water is in that bottle.
2. Always read the upcoming experiments carefully and thoroughly, being sure to
understand all the directions before entering the lab. This will help you to be
prepared to handle any hazards of the experiment and will also help you to
perform the experiment more quickly resulting in less “fumbling around” and
reckless work as you rush to finish on time. To ensure that you have read the
upcoming experiment, you are required to complete the pre-lab assignment
before entering the lab. If you fail to complete the pre-lab assignment on time,
you will not be allowed to perform the experiment.
3. Be in the lab promptly and ready when the lab begins. The safety lecture (specific
to that day’s experiment) will be the first item of business each day. If you are not
present to get this important information, you will not be allowed to do the
experiment.
4. Absolutely no food or beverages will be permitted inside the lab. They can absorb
chemicals from the air (and concentrate them), or can pick them up from the
bench, causing ingestion of these chemicals. Everything possible will be done to
be sure that laboratory air is safe for working in without the use of special respiratory
equipment. Please don’t complicate the issue by eating these chemicals as well!
5. WASH YOUR HANDS! Wash your hands frequently during lab and wash your hands
twice at the end of the lab, once in the lab itself, and again outside of lab (as in a
public rest room), ESPECIALLY before eating. Once you get home, you should wash
your face as well. You don’t want to drag too many chemicals around with you
on your skin.
6. Do NOT apply makeup (including Chapstick and other lip balms) in the lab. In fact,
you may want to seriously consider not wearing makeup to the lab at all. Makeup
can also pick up and concentrate fumes from the air and hold them against the
skin causing irritation. Perfumes, colognes, or other fragrances may also interfere
with the olfactory senses when an experiment calls for “smelling” something.
7. Should an injury occur, regardless of how minor it is, report it IMMEDIATELY to the
lab instructor. The smallest puncture wound allows for chemicals to enter the blood
stream directly. By notifying your instructor, even if no action is taken, the incident
will be reported to the safety officer or to the appropriate person. If this wound
should become infected later, having this information on file may prove to be of
extreme importance for prompt treatment.
8. Never pick up broken glassware with your bare hands, regardless of the size of the
pieces. Typically, puncture wounds occur with the largest pieces in such a
situation, because they look to be the most harmless. A brush and dustpan are
available in the stockroom for broken glassware. Please place all broken glassware
in the appropriate broken glassware container, and never put caps, paper, or
other waste in this same container. Very small bits of broken glassware can be
picked up with a damp paper towel/tissue paper.
9. NEVER put broken glass in a regular garbage can/trash bin. A container is
provided that is especially designed for broken glassware.

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10. Always read the labels to reagents (chemicals used in an experiment) twice! Many
chemicals look identical and may differ only slightly in their spelling or
concentration. Sodium sulfate may look like sodium sulfite, but they are most
certainly different and confusing them in the lab may result in dire consequences.
Therefore, read the label as you grab the bottle, and holding it in your hand, look
carefully at the label a second time and verify that it is exactly what you want.
11. Never make unauthorized substitutions. If you are wondering what would happen
if you used this instead of that, ask your instructor. It is your instructor who can tell if
an undesirable reaction would happen.
12. Never use reagents from an unmarked bottle. All reagent bottles will have proper
labels, so if a reagent is unlabeled, it is an incorrect reagent.
13. In any emergency, the fastest way to get the lab in-charge or instructor’s attention
is to SCREAM! But do not PANIC.
14. If you are not feeling well, report it to your instructor immediately. If your instructor
should lose consciousness during a lab period, it may be due to chemical fumes.
Evacuate the lab immediately and seek another professor for help. Should
anybody else lose consciousness in the lab, your instructor or the lab in-charge will
determine whether evacuation of the lab is warranted.
15. Avoid bringing excess coats, books, backpacks, or other personal items to the
laboratory.
There is always the danger of spilling chemicals on them, and they create fire
hazard if left in the aisle, tabletop or anywhere.
16. Never smell a chemical straight out of a container. Some chemicals are extremely
caustic (fumes severely irritate delicate tissues) and the fumes should be avoided.
To safely smell a chemical, hold it two to three feet from your nose, and with your
other hand cupped, waft the fumes towards you. You may slowly move the
chemical closer to your nose if you cannot smell it all the while taking only small
sniffs.

FIRE
1. In the event of fire, DON’T PANIC! Follow the Fire Emergency Protocol which is
oriented to you by the school safety officer every year.
2. If a small portion of your clothes catches fire, the fire may be extinguished by
patting it out.
3. If a large portion of your clothes catch fire, there are three options for putting the
flames out. (1) Drop to the ground and roll. (2) Use safety shower. (3) Use the fire
blanket.
4. NEVER use a fire extinguisher on a person. Carbon dioxide fire extinguishers
(distinguishable by their flared-out nozzles) are extremely cold and may cause
shock to the person or frostbite of the eyes. Chemical fire extinguishers cause
excessive scarring by mixing of the chemicals in the extinguisher with the
damaged skin. All fire extinguishers have the potential of causing asphyxiation.
5. If a fire should occur in a beaker or some other container, cover it with a glass dish
or other flame-retardant item.
6. NEVER move ANY object that is burning. If you try to pick up a beaker that is on
fire, should you drop it, the burning chemical will spill making the situation even
worse.
7. Never use water to extinguish a chemical fire. Many flammable liquids float on
water, meaning that the water will have no effect but to spread the fire. Other
chemicals may even react explosively with water!
8. If a fire is large enough to warrant the use of a fire extinguisher, the proper use of
the extinguisher is as follows: (1) Be sure there is an exit behind you in case you

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 cannot get the fire under control; (2) pull out the restraining pin (which requires
breaking the plastic seal).
(3) point the extinguisher hose at the base of the fire; (4) holding the extinguisher
UPRIGHT, squeeze the handle to release the extinguishing media; (5) sweep the
spray back and forth at the front of the fire. There are two important things to
remember when using a fire extinguisher. (1) You may only have about a 30
second blast of extinguishing media, so extinguishers are only for use on relatively
small fires. (2) Some fires may be inappropriate for a fire extinguisher. Be sure you
have the right rating of the extinguisher, and never try to extinguish a fire on a
vertical surface!
CHEMICALS AND CHEMICAL SPILLS
1. Report all chemical spills IMMEDIATELY to your lab instructor. The chemical you will
be handling are NOT “scaled down” chemicals—they are the same chemicals any
professional chemist would order and use. Keep a healthy respect for them, or
they may bite you!
2. Should a chemical spill on you or anybody, immediately remove all affected
clothing (start from the back forward to avoid dragging the chemicals across your
face) and wash the affected body area with copious amount of water.
Unfortunately, chemicals have no respect for modesty and will cause permanent
damage if not treated immediately. If a large portion of your clothing is affected,
immediately get to the safety shower, and remove the contaminated clothing
while the water is running.
3. Small spills on the bench or floor must be cleaned up immediately. Sodium
bicarbonate and vinegar are included as part of the safety equipment for
neutralization of acids and alkaline (basic) solutions respectively. Neutralize all
acid and alkaline spills before cleaning. If you are not sure how to clean a spill, let
your lab instructor know immediately.
4. Be especially careful of spills around the balances. These electronic devices are
EXTREMELY sensitive to corrosion. A brush is kept near the balances so you can
brush the balances thoroughly after EACH use (even a single grain of a reagent
can cause irreversible damage). Clean up any spill near the balance IMMEDIATELY
and report it to your lab instructor.
5. Mercury, lead, and other heavy metals pose a particular health hazard in that the
human body cannot get rid of heavy metals. Any heavy metals you’ve ever been
exposed to are still with you today (including mercury if you ever played with it, or
lead if you’ve ever eaten lead paint, a favorite activity of children as it tends to
have a sweet taste). As a result, although most heavy metal poisons are not
particularly toxic, the effects of heavy metal poisoning are typically only seen long-
term, and can include uncontrolled trembling, insanity, and death. The only way
to combat these effects is through minimization of exposure to heavy metal
poisoning. Mercury poses a particular hazard as vapors from the liquid
accumulate in a room and quickly are at dangerous concentrations in the air. As
a result, report ANY spills of mercury, as for example, from a broken thermometer,
as quickly as possible so it can be cleaned up immediately.

LABORATORY EQUIPMENT

1. Never heat a piece of glassware (beakers, flasks, etc.) that is chipped or cracked
unless otherwise told to do so by your lab instructor. Heating defective glassware
can cause that glassware to break (or explode!), resulting in a spill.

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2. If you have chipped or cracked glassware, or glassware with sharp edges or
jagged edges, inform your lab instructor or lab in-charge before bringing the
materials into your designated lab room. The equipment will probably be
replaced, or you may simply be given special instructions on using that bit of
equipment.

GENERAL GUIDELINES
1. Epilepsy, pregnancy, dyslexia as well as other medical conditions can be
hazardous in the laboratory. If you have any medical condition which you think
may adversely affect your ability to safely perform in the laboratory, or that makes
you particularly at risk to be in the laboratory, please inform your instructor as soon
as possible. Many such conditions may be deemed personal, but the chemicals
themselves cannot tell the difference.
2. To turn on a Bunsen burner, first turn the nozzle on the bottom of the burner all the
way off, then turns it back on about 2 turns. With a LIT MATCH in one hand, slowly
turn on the gas at the spigot. Hold the match near the edge of the burner as you
do so the air being pushed out by the gas does not blow it out. Such a procedure
will avoid “explosions” when lighting the burner.
3. Before using a burner, be sure nobody else on the bench has any organic solvents.
Organic solvents are flammable, and heavier than air, meaning that as they
evaporate, they creep down the edge of their container to the bench top,
whereupon they spread out horizontally. Once these fumes reach an open flame,
they can ignite causing “flashback”, thereby causing the beaker of solvent to
catch fire from four feet or more away!
4. Before getting any organic solvent, be sure nobody on your entire lab bench has
an open flame.
5. Hot under the collar. Many times, you’ll be asked or told to heat something. Don’t
just automatically go for the Bunsen burner or alcohol lamp. That way lays fire.
Usually—No Flames!
Try a hot plate, try a heating mantle. But try to stay away from flames. Most of the
fires started when some bozo decided to heat some flammable solvent in an open
beaker. Sure, there are times when you’ll HAVE to use a flame but use it away from
all flammables and in a hood, and only with the permission of your instructor.
6. Never take more of a reagent than you need. This means that if you need about 5
mL of a solvent, use your 10 mL beaker to get it, NOT your 600 mL beaker.
7. NEVER return an unused portion of a reagent to its original container. See if
anybody else at your bench, or in the lab, needs it. If not, give it to your instructor,
who will look at you in a forlorn and sullen manner but will appreciate that you did
not put it back in the original container. Returning unused portions of reagent
greatly increase the odds of cross contamination, which is getting the reagent
contaminated with an unwanted chemical.
8. NEVER pour a waste chemical in the drain, or put it in the garbage container, unless
otherwise instructed to do so by your lab instructor. Waste bottles will be provided.
Always pour waste into the appropriate and labeled waste bottle (reading the
waste bottle label twice).
9. If you have glass stirring rods or glass tube with sharp or jagged edges, fire polish
them.
This means holding the sharp end in a Bunsen burner flame and rotating the rod or
tube until a bright orange flame begins to show on the end being heated.
Continue to heat while rotating another minute or so, effectively melting that end

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 a little bit. Be SURE to let it cool COMPLETELY before attempting to fire polish the
other end.
10. Many items (glass, metal, etc.) when HOT look the same as they do cool. Be VERY
careful whenever working with all your equipment (beakers, flasks, evaporating
dish, iron ring, etc.) and make it sure to cool them before handling.
11. If you are inserting glass tubing into a rubber stopper, use the following technique
to avoid jamming a jagged piece of glass through your hand: (1) use glycerol or
water to lubricate either the end of the glass tubing being inserted, the hole in the
stopper where the tubing will be inserted into, or both, (2) protect your hands by
using a paper towel to hold both the glass tubing as well as the rubber stopper, (3)
hold the rubber stopper in such a way that the tubing cannot go through the hole
and into your palm (your fingers should actually curve, holding the edge of the
stopper, as if to make the letter “C”, (4) hold the glass tubing, also with your palm
away from the end, near the end being inserted into the stopper, (5) insert the
glass tubing with a twisting motion, and (6) clean up any excess glycerol.
12. Improper heating of a test tube can result in the chemicals within the test tube
shooting out, possibly resulting in injury to anybody in the path. When heating a
test tube, use the following procedure: (1) unless directed otherwise, always place
a few (five or six) boiling chips in the test tube, (2) use a test tube clamp to hold
the test tube, (3) hold the test tube at about 45° angle, (4) be sure the opening of
the test tube is pointing AWAY from anybody else (preferably towards a wall in a
low-traffic area of the lab), (5) NEVER heat the bottom of the test tube (unless
otherwise directed), instead heat the middle of the test tube just at the level of the
liquid in the test tube, (6) move the test tube horizontally back and forth across the
flame to prevent the liquid from heating too quickly, (7) should the liquid begin to
overheat (heat too rapidly), remove the test tube from the flame and allow the
contents to cool for a minute or so.
11. NEVER look down the opening of ANY container, including beakers, flasks, and test
tubes (as well as any other piece of equipment). Should something happen to
cause the chemicals to “blast out” of the container, they will go directly into your
face if you are looking down the opening at the time.
12. Do not use graduated cylinders for any purpose other than to measure a volume
of a liquid. Graduated cylinders should never be used to get reagent for an
experiment (use a beaker for this) or to run reactions (use a test tube for this).
13. Never put a dropper into a reagent bottle. Instead, put the reagent in a beaker so
you can bring it back to your desk and use a dropper.
14. In diluting acids, the acid should always be added to the water. This should be
taught as a safety habit rather than a “diluting technique”.

STUDENT RESPONSIBILITIES, DUTIES, AND CONDUCT IN THE LABORATORY

1. The presence of lab instructor is required in all laboratory sessions. Students will be
dismissed if an instructor fails to report to the laboratory after 30 minutes of waiting
time.
2. Cleanliness and orderliness in the laboratory must be maintained. Make it sure that
working tables/bench tops, sinks, and floor are clean. All electrical devices inside
the laboratory should be switched off before leaving the room and stools are
arranged accordingly.
3. Loitering in the laboratory corridors is not allowed.
4. The use of electronic devices such as cell phones and music players are
prohibited.
5. The laboratories and corridors shall not be used as practice area for student
activities like dramatics, dance contest, cheering contest or whatsoever.

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6. Work quietly. Avoid boisterous laughter or loud discussions inside the laboratory.
7. Vandalism on any surface is strictly prohibited. Defiance to this rule is subjected to
disciplinary action.
8. Care must be employed in using any school property. A student will be liable in
replacing a damaged property/item if done intentionally.
9. In case of breakages and losses of glassware and apparatuses, report immediately
to the laboratory in-charge for recording. The individual/group should replace the
broken or damaged item within two (2) weeks.

GENERAL RULES FOR REQUISITION

A. Borrowing of Laboratory Equipment and Apparatuses
1. Students who are officially enrolled in any science subject with lab are allowed
to borrow equipment/apparatuses from the science stockroom with the
approval of the instructor.
2. Students should fill-up the requisition form COMPLETELY and should be signed
by the instructor. The form should be forwarded to the stockroom 3-5 days prior
to the actual activity.
3. Students can only claim their requested materials during their activity period.
They are required to check the equipment/apparatus before bringing to the
laboratory if there is preexisting damage to the item. Complaints after bringing
the equipment/apparatus to the room are not entertained.
4. Additional or late requisition of materials will not be entertained.
5. All apparatuses should be CLEAN and DRY before returning to the stockroom.
B. Requesting of Chemicals
1. Instructors or any representative from the class authorized by the instructor must
submit a list of chemicals needed in the experiment indicating the exact
amount of each chemical that is good for the whole class 3-5 days prior to the
actual laboratory activity.
2. Instructors or any representative from the class authorized by the instructor must
verify the availability of the chemicals two (2) days before the activity.
3. Students can withdraw the requested chemicals only on the day of their
laboratory activity.
C. Use of Laboratory Rooms and Instrumentation Room
1. Instructors should reserve the use of laboratory room or instrumentation room
three (3) days before its scheduled class.
2. In the instrumentation room, students must write their names on the logbooks of
the corresponding instruments they will use.
3. Students should NEVER touch or operate instruments other than what they are
using.
4. Stools should be arranged accordingly before leaving the room.
5. All electrical devices should be properly turned off and plugged out before
leaving the rooms.

LABORATORY KIT
Students/groups must provide themselves with the following supplies before the start
of the laboratory activity:
1. tissue paper or paper towels
2. matches
3. medicine droppers
4. hand/body soap
5. detergent/dishwashing liquid soap
6. labeling tape

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 7. pencil/marker and ruler
8. pair of scissors
9. rugs and hand towels

Learning Activity 1
Animal and Plant Cell

Specific Learning Objectives

1. Observe animal and plant cell.
2. Identify the organelles seen under the microscope.
3. Compare and contrast plant and animal cell.

Time Allotment: 3 hours

Learning Resources
Microscope
Glass slides
Cover Slips
Toothpick
Onion
Methylene blue
Iodine solution
Forceps

Learning Content

All living things are made up of cells. Cells can be simple without a nucleus and other
membrane bound organelles. These cells are called prokaryotic cells. Cells can also
be more complex, containing a nucleus and other organelles that have specific jobs
and responsibilities. These larger, more complex cells are known as eukaryotic cells. All
organisms other than bacteria are made up of eukaryotic cells. Although there are
many other types of organisms, we will explore two basic types of eukaryotic cells:
those found in plants and those found in animals. In this lab, you will use a microscope
to observe a plant cell and an animal cell to identify similarities and differences
between the two types of cells.

Procedure

Plant Cell
1. Read all directions before beginning the investigation. If you do not
understand the procedure, ask your teacher before you complete the
step.
2. Obtain a microscope slide and a thin piece of onion tissue. The thinner
the tissue, the more likely you will be able to see the cells.
3. Using forceps, carefully place the onion tissue near the center of the
microscope slide. Check to make sure that the tissue is not folded, but

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 flat on the surface of the microscope slide.
4. Add a drop of iodine solution to the onion tissue.
5. Cover the tissue with a microscope slide coverslip and place the slide on
the stage of the microscope. Turn on the microscope.
6. On your microscope, move the low-power objective into place and
position the diaphragm so that the largest opening is used. This will allow
the maximum amount of light to pass through the specimen.
7. Use the coarse adjustment knob to get the objective lens as close to
the stage as possible. NOTE: After moving the coarse adjustment
knob, you should not have to move it again until the time to view your
next specimen.
8. Looking through the ocular lens, turn the fine adjustment knob to sharpen the
image. In other words, slowly
9. Turn this knob until the image becomes clear and visible to you.
10. Once the image is clear, move the objective lens to the next
magnification and use the fine adjustment knob again to sharpen the
image, just as you did in procedure 8. If the cells are visible at this point,
make observations.
11. Pay close attention to shape, size, position to other cells, barrier of the cell,
and structures inside of the cell. Magnify the cells even more by moving the
objective lens to the highest magnification if needed. Sketch and/or
describe what you see in the space provided in the results.
12. Once you are satisfied with your observations of the onion tissue, clean
your microscope slide and coverslip. Pat them both gently with a paper
towel or cloth to dry.

Animal Cell

1. Using a toothpick or a swab, select one member of the group to carefully

scrap the side of their cheek.
2. Swirl the end of the toothpick or swab, which now contains your cells,
near the center of the cleaned microscope slide. Immediately dispose of
the cheek swabbing tool.
3. Add a small drop of methylene blue near the center of the slide where
you swirled the swabbing tool.
4. Carefully cover the microscope slide with the coverslip and place it on
the stage of the microscope.
5. Find your cells using the same procedures as we did with the onion tissue.
Sketch the cheek cells in the space provided in your results. Remember to
pay close attention to shape, size, position to other cells, barrier of the
cell, and structures inside of the cell.
6. Once you are satisfied with your observations of the cheek cells, clean
your microscope slide and coverslip with soap, water, and a disinfectant
(if directed by your teacher). Pat them both gently with a paper towel or
cloth to dry.
7. Clean and tidy your workspace from the lab.

13 | P a g e
 Learning Activity 1
Animal and plant Cell

Name: ________________________________ Group No.: _______________

Course and year: _______________________ Date: ____________________

Results

Plant Cell Scanner Animal Cell Scanner

-The plant
- The
stretched o
circu 14 | P a g e
Plant Cell LPO Animal Cell LPO

-The plant
- The
Plant Cell HPO Animal Cell HPO

stretched
spreo
circu
Guide Questions

around th
1. Did the basic shape of the plant cell seem to differ from the animal cell?
If so, how? Explain your answer using complete sentences.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

2. One set of cells should have been arranged in more of a regular pattern.
Which cells? Explain your answer using complete sentences.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

3. Both plant cells and animal cells contain many organelles, which are
structures that are specialized to carry out jobs (much like the organs in our
bodies). These organelles may not have been visible. However, some may
have been seen. Were there any organelles or structures that were visible? If

15 | P a g e
 so, what do you suppose they were? Why do you suppose we couldn’t see
all the organelles? Explain your answer using complete sentences.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

Learning Activity 2
Characteristics of Water

Specific Learning Outcomes: At the end of the learning activity the students shall
1. Test the different Characteristics of water.
2. Determine the properties of water that make it a suitable medium for
sustaining life in biological system.
3. Explain dialysis.

Time Allotment: 6 Hours

Learning Resources:

Salt

Sugar

Gelatin

CuSO4

Lard

Ethanol

Chloroform

Citric Acid Powder

NaHCO3 Powder

1% NaCl in starch Solution

0.1 M AgNO3

10% sucrose solution

Deionize water.

16 | P a g e
 Dialysis bag

Beakers

Test tubes

Test tube rack

Iron stand and Rubber band

Learning Content:

With two thirds of the earth's surface covered by water and the human body
consisting of 75 percent of it, it is evidently clear that water is one of the prime
elements responsible for life on earth. Water circulates through the land just as it
does through the human body, transporting, dissolving, and replenishing nutrients
and organic matter, while carrying away waste material. Further in the body, it
regulates the activities of fluids, tissues, cells, lymph, blood, and glandular
secretions.

Procedure

Water as a Universal solvent

1. Put 0.5g of the following substances into six separate test tubes: Salt, Sugar,
Gelatin, CuSO4, Lard, and Ethanol. Add 1 ml of water and shake to dissolve
the substances. To substances that did not dissolve add another 1 ml of water
and shake again. To solid substances that did not dissolve add another 1 ml
of water and shake again.
2. Repeat the procedure using a chloroform.
3. Describe the solubility in both solvents as soluble, slightly soluble, and
insoluble.

Water a Good Medium for Biochemical Reactions

1. Mix 0.1 g of dry powder citric acid and sodium carbonate in a dry test tube.
Observe if chemical reaction occurs.
2. Add 10 ml of water to the mixture and note what happens.

Properties of Water Solutions

Obtain a dialysis bag about 20 to 25 cm long and soak in clean water for
about 10 minutes. Fill with 30 ml of 1% starch NaCl mixture, tie the bag and
rinse thoroughly with water. Put the bag in a beaker containing deionize
water. Adjust the set up such the level of fluids inside and outside the bags

17 | P a g e
 are the same. After 1-hour test 1 ml of dialysate with few drops of 0.1 M
AgNO3.

Learning Activity 2
Characteristics of Water

Name: ________________________________ Group No.: _______________

Course and year: _______________________ Date: ____________________

Learning Outcomes

A. Water as a Universal solvent

Substances Water Chloroform

Salt
Sugar
Gelatin
CuSO4
Lard
Ethanol

B. Observation in Water a Good Medium for Biochemical Reactions

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
C. Observation in Properties of Water Solutions

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

Guide Questions

18 | P a g e
 1. What are the different functions of water in living system?
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

2. Based on your observation, how would you define dialysis?

___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

Learning Activity 3
Analysis of Carbohydrates
Specific Learning Outcomes: At the end of the learning activity the students shall
1. differentiate different kinds of carbohydrates.
2. identify the chemical responses, reactions, and properties that characterize a
specific kind of carbohydrate; and
3. gain an insight of the importance of carbohydrates in the series of biochemical
reactions.

Time Allotment: 12 Hours

Learning Resources:
Equipment and glassware: Chemicals and reagents
Test tubes 5% of glucose
Test tube rack sucrose
Test tube brush Starch
Beaker (250 mL) galactose
Water bath ribose
Test tube holder lactose
Medicine dropper saccharin
Graduated cylinder (10 mL) cellulose
Spot plate Molisch reagent
Glass funnel conc. H2SO4
Filter paper 10% NaOH

19 | P a g e
 Watch glass Bial’s reagent
Tripod phosphomolybdic acid
1% iodine solution
Fehling’s reagent
conc. HCl
Barfoed’s reagent
Benedict’s reagent
Tollens’ reagent
Seliwanoff’s reagent

Learning Content
A carbohydrate is an organic compound with the general formula C n(H2O)n,
that is, consists only of carbon, hydrogen and oxygen, with the last two in the 2:1 atom
ratio. Carbohydrates make up the bulk of organic substances on earth and perform
numerous roles in living things. The carbohydrates (saccharides) are divided into four
chemical groups: monosaccharides, disaccharides, oligosaccharides, and
polysaccharides. Polysaccharides serve for the storage of energy (e.g., starch in
plants and glycogen in animals) and as structural components (e.g., cellulose in plants
and chitin in arthropods). Structural polysaccharides are frequently found in
combination with proteins (glycoproteins or mucoproteins) or lipids
(lipopolysaccharides). The 5-carbon monosaccharide ribose is an important
component of coenzymes (e.g., ATP, FAD and NAD) and the backbone of the
genetic molecule known as RNA. The related deoxyribose is a component of DNA.
Saccharides and their derivatives include many other important biomolecules that
play key roles in the immune system, fertilization, preventing pathogenesis, blood
clotting and development. This experiment aims to introduce you with the
identification of unknown carbohydrates. To gain maximum benefit, observations
should be related, as far as possible, to the structure of the substances examined.
Some important points:
1. Most of the tests and reactions described are not quantitative and volumes
are approximate, despite these facts some tests do not work if quantities
greatly more than those stated are used.
2. DO NOT place your pipettes in reagent bottles as this leads to
contamination.
3. In most tests, it is important to apply a control test using water instead of the
solution under examination.
4. When you need to boil your sample in a test tube, prepare a hot water in a
large beaker and put your test tube inside the beaker. DO NOT forget to put
boiling chips in the beaker.
5. Because test tubes are placed in a boiling-water bath, you will need to label
them by marking the etched glass portion on the tube using either a pencil

20 | P a g e
 or marking pen. Do not use paper labels, which can become loose in the
hot water.
TESTS ON CARBOHYDRATES:
Chemical Alert
Molisch reagent – toxic and corrosive
Iodine-potassium iodide – highly toxic, corrosive, and irritant
Barfoed’s reagent – corrosive and irritant
Tollens’ reagent – toxic and corrosive
Benedict’s reagent – toxic and irritant
Fehling’s reagent – toxic and irritant
Seliwanoff’s reagent – toxic and corrosive
Concentrated sulfuric acid – toxic and corrosive.

Preliminary Instructions
1. Label two sets of 6 clean, dry test tubes with the names of each of the six
carbohydrate solutions you are testing: glucose, sucrose, starch, galactose,
saccharin, and cellulose.
2. Obtain from your laboratory instructor 30 mL of the following 1% carbohydrate
solutions: glucose, sucrose, starch, galactose, saccharin, and cellulose. Label the
containers properly.
3. Prepare a boiling-water bath for future use. To do so, pour about 200 mL of tap
water into a 600-mL beaker. Add two boiling chips to the beaker.
1.) Molisch’s Test
Molisch’s Test is a sensitive chemical test for all carbohydrates, and some
compounds containing carbohydrates in a combined form, based on the
dehydration of the carbohydrate by sulfuric acid to produce an aldehyde (either
furfural or a derivative), which then condenses with the phenolic structure resulting in
a red or purple-colored compound.
Procedure:
1. Transfer 2 mL of each of the carbohydrate solutions into the first set of
labeled test tubes.
2. Add 2-3 drops of Molisch’s reagent (10% α-naphthol in ethanol) to each test
tube and mix well by gentle swirling of the tube.
3. Add 2 mL of concentrated H2SO4 to each of the test tubes in the second set.
4. One at a time and cautiously, hold the test tubes containing concentrated
sulfuric acid at an angle of about 30° from the vertical and slowly pour the
carbohydrate Molisch reagent solutions from the first set of test tubes into
them. Do not shake or mix the resulting solutions. Two separate layers will form.
5. Observe the color at the interface between two layers and record your results
on Data Sheet.

21 | P a g e
 6. A brown color due to charring must be ignored and the test should be
repeated with a more dilute sugar solution. a more dilute sugar solution.
7. Discard the test solutions containing Molisch reagent into the waste
receptacle.
8. Wash all test tubes with soap or detergent solution. Rinse three times with tap
water and once with distilled water. Allow the test tubes to drain to remove as
much of the water as possible.
2.) Carbohydrates as Reducing Sugars
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone
group. The enolization of sugars under alkaline conditions is an important
consideration in reduction tests. The ability of a sugar to reduce alkaline test reagents
depends on the availability of an aldehyde or keto group for reduction reactions.
Several sugars especially disaccharides or polysaccharides have glycosidic linkages
which involve bonding a carbohydrate (sugar) molecule to another one, and hence
there is no reducing group on the sugar; like in the case of sucrose, glycogen, starch
and dextrin. In the case of reducing sugars, the presence of alkali causes extensive
enolization especially at high pH and temperature. This leads to a higher susceptibility
to oxidation reactions than at neutral or acidic pH. These sugars, therefore, become
potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so forth. Most used
tests for detection of reducing sugars are Fehling’s Test, Be Fehling’s Test, Benedict’s
Test and Barfoed’s Test. nedict’s Test and Barfoed’s Test.
Fehling’s Test:
Fehling’s Solution (deep blue colored) is used to determine the presence of
reducing sugars and aldehydes.
Procedure:
1. Into the first set of labeled test tubes, prepare Fehling’s solution by adding 1
mL of Fehling’s solution A (aqueous solution of CuSO4) with 1 mL of Fehling’s
solution B (solution of potassium tartrate).
2. Transfer 2 mL of each of the carbohydrate solutions into the second set of
labeled test tubes. Pour the prepared Fehling’s solution from the first set of test
tubes into each of the carbohydrate solutions and mix well.
3. Using a test tube clamp, place the test tubes containing the reaction mixtures
in the boiling-water bath.
4. Use the clamp to remove each test tube from the bath as soon as you see
the red precipitate of cuprous oxide that forms at the end precipitate of
cuprous oxide of the reaction. Do not allow the test tubes to stand in the bath
for more than 5 min, even if no color change or precipitate forms.
5. Record your observations.
6. Discard the reaction mixtures into the waste container. Wash, rinse, and drain
all the glassware as before.
Barfoed’s Test
Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is
balanced so that is can only be reduced by monosaccharides but not less powerful
reducing sugars. Disaccharides may also react with this reagent, but the reaction is
much slower when compared to monosaccharides, thus this test can be used to
distinguish reducing monosaccharides from reducing dissacharides.

22 | P a g e
Procedure:
1. Transfer 1 mL of each of your carbohydrate solutions into one set of the
washed, drained test tubes.
2. Add 3 mL of Barfoed’s reagent to each test tube. Using a test tube clamp,
place the test tubes containing the reaction mixtures in your boiling-water
bath.
3. Allow the test tubes to stand in the boiling-water bath for 5 min.
4. After 5 min, use the test tube clamp to cautiously remove the hot test tubes
and observe the contents.
5. You will observe a brick-red cuprous oxide precipitate if reduction has taken
place.
6. Replace in the boiling-water bath for 5 additional minutes any test tubes that
have no red precipitate.
7. Cool under running water any test tubes that contain a red precipitate. Place
the cool test tubes from the bath in a test tube rack.
8. Record your observations.
Benedict’s Test
Benedict’s test uses a mixture of copper (II) sulfate. Sodium citrate, and sodium
carbonate in a mildly basic solution. This reagent is used as a general test for detecting
reducing sugars. If the saccharide is a reducing sugar, it will reduce the copper (II)
ions to copper(I) oxide, a red precipitate.
Procedure:
1. Add 3 mL of Benedict’s reagent to each of the test tubes in one set. Then
transfer 10 drops of each of your carbohydrate solutions to the appropriately
labeled tubes. Using a test tube clamp, place the test tubes containing the
reaction mixtures in the boiling-water bath. After exactly 2 min, use the test
tube clamp to remove the test tubes, and place them in a test tube rack.
2. Record all observations and the color of any precipitate on Data Sheet.
3. Discard the test solutions containing Benedict’s reagent into the container
provided by your laboratory instructor.
4. Wash, rinse, and drain the test tubes as before.
Tollens’ Test
Tollens’ test uses a chemical reagent most used to determine whether a known
carbonyl-containing compound is an aldehyde or a ketone. It is usually ammoniacal
silver nitrate, but can also be other mixtures, if aqueous diamminesilver(I) complex is
present. A positive test with Tollens' reagent results in elemental silver precipitating out
of solution, occasionally onto the inner surface of the reaction vessel, producing a
characteristic and memorable "silver mirror" on the inner vessel surface.
Procedure:
1. Transfer 2 mL of each of your carbohydrate solutions into one set of the
washed, drained test tubes.
2. Add 3 mL of Tollens’ reagent to each test tube and mix well. Using a test tube
clamp, place the test tubes containing the reaction mixtures in your boiling-
water bath.
3. Allow the test tubes to stand in the boiling-water bath for 2-3 min.
4. Observe the change of color and record the results.

23 | P a g e
 5. Discard the test solutions containing Tollens’ reagent into the container
provided by your laboratory instructor.
6. Wash all test tubes with soap or detergent solution. Rinse three times with tap
water and once with distilled water. Allow the test tubes to drain to remove as
much of the water as possible.
3) Action of Alkali on Sugars
Procedure:
1. Heat 1 mL glucose solution with 1 mL 40% NaOH for 1 min.
2. Cool and apply test for reducing sugars (e.g., Fehling’s Test).
3. Apply a control test with glucose solution to observe the difference.
4) The Inversion of Sucrose
Sucrose is a disaccharide, which means that it is a molecule that is derived from
two simple sugars (monosaccharides). In the case of sucrose, these simple sugars are
glucose and fructose. Inverted sugar is a mixture of glucose and fructose. It is obtained
by splitting sucrose into these two components. The splitting of sucrose is a hydrolysis
reaction which can be induced simply by heating an aqueous solution of sucrose.
Acid also accelerates the conversion of sucrose to invert.

Procedure:
1. Add 5 mL of sucrose solution to two test tubes.
2. Add 5 drops of conc. HCl to one test tube one test tube.
3. Heat both tubes in boiling water bath for 10 min.
4. Cool and neutralize with diluted NaOH (use litmus paper).
5. Take 1 mL of the sucrose solution treated with conc. HCl (inverted sucrose)
and set it aside in one test tube for the Seliwanoff’s Test.
6. Test both solutions (first tube of sucrose solution with conc. HCl and sucrose
solution only) for the presence of reducing sugar with Fehling’s Test.

5) Seliwanoff’s Test
Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are
distinguished from aldoses via their ketone/aldehyde functionality. If the sugar
contains a ketone group, it is a ketose and if it contains an aldehyde group, it is an
aldose. This test is since, when heated, ketoses are more rapidly dehydrated than
aldoses.
Procedure:
1. Use the previously set aside inverted sucrose. To 1 mL of the inverted sucrose
solution add 3 mL Seliwanoff’s reagent (0.5 g resorcinol per liter 10% HCl).
2. To another two test tubes, prepare 1 mL of 1% glucose solution and sucrose
solution separately. Add 3 mL of Seliwanoff’s reagent to each test tube.
3. Using a test tube clamp, place the test tubes containing the reaction mixtures
in your boiling-water bath.

24 | P a g e
 4. Allow the test tubes to stand in the boiling-water bath for less than 30
seconds. A red color must appear for ketoses.
5. Upon prolonged heating, glucose will also give an appreciable color.
6. Discard the test solutions containing Seliwanoff’s reagent into the container
provided by your laboratory instructor.
7. Wash all test tubes with soap or detergent solution. Rinse three times with tap
water and once with distilled water. Allow the test tubes to drain to remove as
much of the water as possible.
6) Iodine Test:
Iodine test is an indicator for the presence of starch. Iodine solution (iodine
dissolved in an aqueous solution of potassium iodide) reacts with starch producing a
blue-black color. Apply this test to all the polysaccharides provided.
Procedure:
1. To 2 mL of the 6 carbohydrate solutions, add 1-2 drops of iodine solution.
2. Observe the different colors obtained for each of the polysaccharide
solutions
Learning Activity 3
Analysis of Carbohydrates

Name: ________________________________ Group No.: _______________

Course and year: _______________________ Date: ____________________

MolischTest

Observations Carbohydrates or not?

Glucose

Sucrose

Starch

Galactose

Saccharin

Cellulose

25 | P a g e
Fehling’s Test

Observations Reducing or non-

reducing
Glucose

Sucrose

Starch

Galactose

Saccharin

Cellulose

Barfoed’s Test

Observations Monosaccharide or
Disaccharide?
Glucose

Sucrose

Starch

Galactose

Saccharin

Cellulose

26 | P a g e
Benedict’s Test

Observations Reducing or non -

educing
Glucose

Sucrose

Starch

Galactose

Saccharin

Cellulose

Seliwanoff’ Test

Observations Aldose or Ketose

Glucose

Sucrose

Starch

Galactose

Saccharin

Cellulose

Tollens’Test

27 | P a g e
 Observations Reducing or
Nonreducing
Glucose

Sucrose

Starch

Galactose

Saccharin

Cellulose

Iodine Test

Observations With Starch or Non?

Glucose

Sucrose

Starch

Galactose

Saccharin

Cellulose

1. Explain why carbohydrates are important in cell membrane?

___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

28 | P a g e
 2. What is other importance of carbohydrates in the series of biochemical
reactions?
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

3. Using your textbook or a reference book, briefly explain the meanings of the
following terms as they relate to this experiment.

a. aldose
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

b. polysaccharides
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
c. reducing sugar
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
d. hemiacetal
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

4. Classify the following saccharides as either reducing or non-

reducing sugars.
Ribose __________________________
Sucrose __________________________
hydrolyzed starch __________________________

5. Complete the table below. For each test, indicate what you will observe if the
test is positive. Then state what a positive test will tell you about each of the
carbohydrates you are testing.

Test expected observation substance indicated

for a. positive test by a positive test
Potassium iodide/Iodine
(KI/I2) starch test

Benedict’s test

29 | P a g e
 Seliwanoff’s test

Bial’s test

6. When an aldose reacts with Barfoed’s reagent, what type of organic

compound forms? What type of chemical reaction is this?
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

7. Draw the Haworth structure for fructose. Label any special structural features,
such as hemiacetals, acetal linkages, and anomeric carbons.
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
8. Explain why fructose, an α-hydroxyketone, reacts with Benedict’s reagent.
What structural rearrangement is necessary for this reaction to occur?
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
9. Explain what happened when you mixed sucrose with Seliwanoff’s reagent.
Was this the result expected? Briefly comment.
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
10. The carbohydrate erythrose is classified as a four-carbon aldose. Suppose you
tested an erythrose solution using the procedures described in this experiment.
Describe what you would expect to see when you performed the following
tests.
a. KI/I2
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
b. Barfoed’s
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
c. Benedict’s
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
d. Seliwanoff’s
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

30 | P a g e
 e. Molisch
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
11. Test tablets using the chemistry involved in the Benedict’s test are used to
detect sugars in the urine of diabetics. When a tablet is added to the urine
sample, the solution will turn one of several colors, depending upon the amount
of reducing substances present. In contrast, urine strips contain an enzyme that
reacts specifically with glucose. Briefly explain the advantage of the test strips
over the tablets.
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

Learning Activity4
Analysis of Lipids
Specific Learning Outcomes: At the end of the learning activity the students shall
1. learn the technique of characterizing lipids.
2. identify the specific fats and oils; and
3. differentiate the different compounds and derived lipids.

Time Allotment: 6 Hours

Learning Resources:
Equipment and glassware: Chemicals and reagents
Beaker Olive oil
Crucible Coconut oil
Erlenmeyer flask Lard butter
flask with stopper ether
heating set-up phenolphthalein solution
test tubes solid KHSO4
Medicine dropper 20% NaOH
Graduated cylinder iodine test solution
Water Bath NaCl solution
conc. HCl
CCl4
CH3COOCOCH3
conc. H2SO4

31 | P a g e
 alcoholic KOH
anhydrous CHCl3
0.5 M HCl
Hanus iodine solution
15% Kl solution
0.1 M Na2S2O3
2% starch

Learning Content

Lipids are organic substances insoluble in water but soluble in organic solvents,
adopting the principle of “like dissolves like”. The physical and chemical properties of
lipids are due to the presence of carboxyl groups, number of double bonds, and
hydroxyl groups and the length of carbon atoms chain. In this experiment, the focus
is to characterize lipids.

The following are the tests to be performed, with their corresponding underlying
principles:

1. Test for unsaturation. Palmitic acid is a saturated fatty acid. Oleic acid is a
monoenoic fatty acid. When unsaturated fatty acids are treated with halogens
like iodine, they add across the double bond. Color of the halogen disappears.

2. Acrolein formation. When lipids are heated in the presence of KHSO4, a

charactersitic odor of acrolein is produced.

3. Saponification. Coconut oil is a triacyl glycerol. On treatment with alkali, it is

hydrolyzed to glycerol and salts of fatty acid. When NaCl is added, sodium salts of
fatty acids preciptate. On acidification, few fatty acids separate and float.

32 | P a g e
4. Liebermann-Burchardt reaction. A chloroform (carbon tertachloride) solution of
sterol. When treated with acetic anhydride and concentrated sulfuric acid, it gives
a characteristic color.

5. Salkowski test. Sterols undergo dehydration process in the presence of sulfuric acid
to give 3,5-cholestadiene, which dimerises to bis-cholestadiene.

Procedure

Prepare the following: 1) liquid olive oil, 2) coconut oil, 3) lard and 4) butter.

1. Qualitative tests

a. Test for unsaturation

1. Shake 1 mL of oil sample with 5mL of ether and add 3 drops of iodine test
solution.
2. Shake again and note the changes in iodine test solution.

b. Acrolein test

1. Place a small amount of oil samples into the crucible and add 0.5g of solid
KHSO4.
2. Heat and note the odor.

c. Saponification

1. To a few drops of oil sample in a test tube, add 6mL of 20% NaOH and mix.
2. Keep in a boiling water bath for 20 minutes. Mix occasionally and cool.
3. Divide the clear solution into 3 test tubes.
4. To the first one, add 10mL of water, shake vigorously and add 2mL
phenolphthalein. Observe any change color.
5. To the second test tube, add 2mL of NaCl solution. Note the precipitate formed.
6. To the third test tube, add concentrated HCl drop by drop with mixing. Note
the scum forms at the top.

d. Liebermann-Burchardt test

33 | P a g e
 1. Prepare 3mL of CCl4 solution and 5mL of concentrated sulfuric acid and 0.5mL
CH3COOCOCH3.
2. Shake gently and allow to stand for a few minutes.
3. Note the colors produced.

e. Salkowski test

1. Dissolve few milligrams of butter to be tested in 3mL anhydrous CHCl3.

2. Add an equal volume of concentrated H2SO4 and shake the tube gently.
3. Let the liquid layers separate and observe any color change at interface.

B. Quantitative tests

a. Saponification number

1. Weigh a 5-gram fat sample into a 250-mL Erlenmeyer flask.

2. Dissolve the fat sample in 50 mL of 0.1ml alcoholic KOH and boil for 30 minutes.
3. Cool and titrate with 0.5 M HCl using phenolphthalein as indicator.
4. Perform a blank determination. Subtract the amount of 0.5M HCl obtained
during the determination of the sample from the amount obtained in the blank
to obtain the number of mL of 0.5M HCl equivalent to the KOH used in the
saponification of the sample.
5. Calculate as the saponificant number the milligrams of KOH required to
saponify 1 g of fat.

b. Iodine number

1. Weigh 0.25 g of solid fat samples in a 500 mL of glass-stoppered flask.

2. Dissolve the sample in 10 mL CCl4.
3. Add 30 mL Hanus iodine solution and allow to stand for 30 minutes with
occasional shaking.
4. Add 10 mL 15% KI solution and shake thoroughly.
5. Add 100 mL of freakly-boiled and cooled water washing down any free iodine
that may be found in the stopper.
6. Titrate iodine with 0.1 M Na2S2O3 until the yellow color of the solution has
almost disappeared.
7. Add 2 mL of 2% starch and continue the titration until blue color disappear.
8. Run 2 blank determinations on an equal portion of the Hanus iodine solution.
The number of mL of 0.1 M Na2S2O3 required by the blank less the quantity

34 | P a g e
 used in the determination gives the thiosulfate equivalent of the iodine
absorbed by the fat.
9. Calculate the grams of iodide by 100 g of the fat. This is the Hanus iodine
number.

Learning Activity 4
Analysis of Lipids

Name: ________________________________ Group No.: _______________

Course and year: _______________________ Date: ____________________

A. Observation

Test Coconut oil Olive OIl Lard Butter

Unsaturation

Acrolein

Saponification

Liebermann-
Burchard

Salkowski

35 | P a g e
B. Saponification number

1. Weight of sample ________________________

2. Volume of 0.5 M HCl used in blank determination ________________________

3. Volume of 0.5 M HCl used in actual determination ________________________

4. Saponification value ________________________

5. Computation

B. iodine

1. Lipid sample used ________________________

2. Weight of sample ________________________

3. Volume of 0.1 M Na2S2O3 used in blank ________________________

4. Volume of 0.1 M Na2S2O3 in actual determination ________________________

5. Computation

Questions

1. State the relevance of saponification with hardness of water.

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

2. Clinical significance of:

a. Fats and oils

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 ________________________________________________________________
________________________________________________________________
________________________________________________________________

b. Cholesterol
________________________________________________________________
________________________________________________________________
________________________________________________________________

3. Fats may be identified by their respective iodine and saponification numbers. Name
other quantitative methods.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

4. What is the biological importance of fatty acids?

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

5. What are essential fatty acids and name them?

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

6. What are sterol compounds? Give examples.

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

7. Differentiate fats and oils.

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

37 | P a g e
 Learning Activity 5
Analysis of Proteins
Specific Learning Outcomes: At the end of the learning activity the students shall
1. compare the different classes of proteins is based on their properties.
2. apply the knowledge gained in analyzing other food nutrients.

Time Allotment: 6 Hours

Learning Resources:
Equipment and glassware: Chemicals and reagents
Beaker 5% egg albumin
Graduated cylinder 5% casein
heating setup 5% gelatin
litmus paper 5% peptone
medicine dropper 5% flour
test tubes ethanol
water bath NaOH solution
filter paper CCl4
0.5% CuSO4 solution
soda lime

38 | P a g e
 conc. HNO3
10% AgNO3 solution
mercuric chloride solution
copper sulfate solution
lead acetate solution
barium chloride solution
picric acid
tannic acid
phosphomolybdic
potassium mercuric iodide
bromocresol green indicator
acetic acid
ammonium molybdate
ammonium sulfate
40% NaOH, Molisch reagent
Ninhydrin
carbon tetrachloride
conc. H2SO4 solution
Learning Content

The name protein is derived from Greek word proteios, which means “first”. The
name is so given because proteins are the first among natural polymers essential for
growth and maintenance of life. Classes of proteins can be differentiated by their
physical properties and specific reactions with a variety of reagents to form colored
products.

Below is the list of the tests, accompanied by a description of the underlying

principles.

4. Biuret test. This method is since cupric ion in an alkaline medium forms a colored
complex (due to peptide bond formation with the nitrogen in protein).
5. Isoelectric precipitation. Proteins have minimum solubility at their isoelectric point.
Since the net charge at the isoelectric point is zero for proteins, repulsion between
individual molecules is minimal.
6. Heat and acetic acid (HA) test. Proteins in solution are denatured when subjected
to heat treatment.

39 | P a g e
7. Half and full saturation tests. When an organic salt like ammonium sulfate or
copper sulfate is added to a solution containing proteins, the effective
concentration of water available for the solubility of the protein is decreased with
an increase in salt protein. Protein can be precipitated at full or half saturation.
8. Test for organic phosphate. The organic phosphate group in phosphoprotein is
converted to phosphate upon treating with strong NaOH solution. Inorganic
phosphate around neutral pH reacts with ammonium molybdate to yield colored
ammonium phosphomolybdate.
9. Molisch test. The presence of oligosaccharide units in the sample gives a positive
test.

10. Aldehyde test for indole nucleus. Sulfuric acid, in the presence of mercuric sulfate,
oxidizes the indole nucleus of trytophan. The product reacts with aldehydes to give
a chromogen.
11. Sakaguchi test. Arginine residue in protein reacts with α-napthol and sodium
hypobromite found to give a chromogen.

12. Sulfur test. When proteins are boiled with strong alkali, organic sulfur is converted
to sulfide.
13. Ninhydrin test. Ninhydrin reacts with alpha-amino groups of proteins and amino
acids to form a color complex.

Procedures

The instructor must provide five (5) unkown protein samples containing any of these
substances: egg albumin, casein, gelatin, peptone and flour, prepared either dry or
in 5% solution.

1. Physical properties
Note the odor, color, and solubility of the unknown protein samples in alcohol,
carbon tetrachloride, and water.

2. Chemical properties

a. Biuret test
1. To 3 mL protein sample in a test tube, add 2mL NaOH and 2 drops of CuSO4.
2. Note the color changes.

b. Test for nitrogen in protein sample

40 | P a g e
1. Prepare a mixture of 1g soda lime and 0.3g of dried protein sample.
2. Heat gently.
3. Note the odor evolved and test these vapors with moistened red and blue litmus
paper.

c. Coagulation tests

By heat

1. Place 3mL of the protein sample solution in a test tube, heat the closed end and
note the appearance of the substance formed.
2. Test its solubility in water.

By alcohol

1. To 3 mL of the protein sample solution in a test tube, add 5 mL of ethyl alcohol.

2. Note the appearance of the product and test its solubility in H2O.

d. Precipitation of protein

By strong mineral acids (Heller test)

1. To 2 mL of the protein solution in a test tube, carefully add (by slowly pouring along
the inside walls of the inclined tube) 2 mL of concentrated HNO3.
2. Bring the test tube in an upright position. Do not shake.
3. Note the color formed at the junction of two liquids.

By salt of heavy metals

1. Place 2 mL of the u known protein sample in the test tube and add the following
reagents:
TT 1: 1 mL of 10% AgNO3

TT 2: 1 mL of HgCl2

TT 3: 1 mL of CuSO4

TT 4: 1 mL of lead acetate

TT 5: 1 mL of BaCl2
2. Shake all the test tubes.
3. Note the color end test the solubility of the precipitate in H2O.

41 | P a g e
By alkaloidal reagents

1. In a separate fine test tube, place 2 mL of the protein sample and add the
following reagents:

TT 1: 1 mL of any picric acid (2,4,6-trinitrophenol)

TT 2: 1 mL of tannic acid

TT 3: 1 mL phosphomolybdic acid (H3PMo12O40)

TT 4: 1 mL phosphotungstic acid (H3PW12O40)

TT 5: 1 mL of potassium mercuric iodide with 1 mL of 6N HCl.

2. Shake all these test tubes gently. Note the color of precipitate formed.
3. Add the excess of their respective reagents and observed if the precipitates are
dissolved.
By isoelectric precipitation test

1. To 3 mL of the protein sample, add 3 drops of bromcresol green indicator solution.

2. Mix and add 1% acetic acid drop by drop until a green color is obtained.
3. Note the precipitate formed:

In the test tube with a curdy white precipitate, proceed to the test for organic
phosphate.

In the test tube with no precipitate, proceed to the heat and acetic acid tests

e. Heat and acetic acid tests

1. Take 10 mL of protein solution in a test tube.

2. Heat directly over a flame in a slanting position and allow the upper part to reach
boiling point. The lower part serves as the control.
3. If the upper part becomes cloudy, add a few drops 1% acetic acid without mixing.
4. Note the presence of coagulation:

If there is no coagulation, proceed to the full saturation test.

If there is coagulation, proceed to the saturation test before doing the full saturation
test.

f. Test for organic phosphate

1. To 5 mL of protein sample, add 0.5mL of 40% NaOH.

2. Heat strongly for 2-3 minutes and cool.
3. Add 0.5 mL HNO3 and filter.

42 | P a g e
4. To the filtrate, add a pinch of ammonium molybdate.
5. Heat gently over the flame.
6. Observe the change in color of the solution.

g. Full saturation test

1. To 3 mL of protein solution, add solid ammonium sulfate in small quantities at a

time, mixing until the solution is saturated. Allow to stand for 5 minutes and filter.

2. Perform the Biuret test using the filtrate and note the change in color.

h. Half saturation test

1. To 3 mL of protein solution, add an equal volume of saturated ammonium sulfate

solution to make the system half-saturated with respect to the salt.
2. Mix the solution and allow to stand for 5 minutes and filter.
3. To the filtrate, do the Biuret test using equal volume of 40% NaOH and 2 drops of
1% copper sulfate. Note the color of the solution and the precipitate.

i. Molisch test
1. To 1mL of protein solution, add 2-3 drops of 1% α-naphthol solution in ethanol and
mix.
2. Pour carefully along the sides of the test tube 2mL of concentrated H2SO4 in an
inclined position.
3. Observe the color of ring formed at the junction of two liquids.

j. Ninhydrin test

1. Place 2 mL of protein sample in separate test tubes.

2. Add 0.5 mL of ninhydrin solution.
3. Heat in a water bath for 5 minutes. Observe the color changes.

43 | P a g e
 Learning Activity 5
Analysis of Proteins

Name: ________________________________ Group No.: _______________

Course and year: _______________________ Date: ____________________

Results

A. Physical Properties

Sample Color Odor

Egg albumin

Casein

Gelatin

Peptone

44 | P a g e
 Flour

Solubility
Sample Ethanol Carbon Water
Tetrachloride
Egg albumin

Casein

Gelatin

Peptone

Flour

B. Chemical

Properties
Sample Observation (Biuret Test)
Egg albumin

Casein

Gelatin

Peptone

Flour

1. The minimum requirement for a positive reaction is:

___________________________________________________________________________

___________________________________________________________________________

2. The violet color of the solution is due to:

___________________________________________________________________________

___________________________________________________________________________

45 | P a g e
 b. Test for nitrogen in protein

Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

c. Coagulation of protein

Observation
Sample By heat By Alcohol
Egg albumin

Casein

Gelatin

Peptone

Flour

d. Precipitation of protein

Observation
Sample Salts of heavy
Heller’s ring test metals Alkaloid reagents
Egg albumin

Casein

Gelatin

Peptone

46 | P a g e
 Flour

The formation of the ring indicates: ______________________________________________

e. Isoelectric precipitation test

Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

f. Heat and acetic acid test

Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

g. Test for organic phosphate

Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

47 | P a g e
h. Full saturation precipitation test
Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

i. Half saturation precipitation test

Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

j. Molisch test

Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

48 | P a g e
 k. Ninhydrin test

Sample Observation
Egg albumin

Casein

Gelatin

Peptone

Flour

Guide Questions
1. What are zwitterions? Do they interfere with the precipitation test and color
reactions?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

2. Do all proteins react positively with Biuret test?

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

3. What are peptides? State the physiological significance of breaking down

proteins into peptides?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

4. What is the salting in and salting out processes? Are they involved in the formation
of the precipitate?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

49 | P a g e
5. What is isoelectric point? Is it related to the full and half saturation points of protein?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

6. What is the purpose of adding acetic acid drop by drop in isoelectric

precipitation test?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

7. What indicates the disappearance of cloudiness upon adding acetic acid in heat
and acetic acid test?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

Learning Activity6
Isolation of RNA
Specific Learning Outcomes: At the end of the learning activity the students shall
1. learn the technique of characterizing lipids.
2. identify the specific fats and oils; and
3. differentiate the different compounds and derived lipids.

Time Allotment: 6 Hours

Learning Resources:
Apparatus Chemicals
Beaker 10% NaOH
Mortar and Pestle Copper Sulfate
Filter paper Ammonia
Funnel 10% Nitric acid
Stove Ammonium Molybdate
Dropper Glucose
Graduated cylinder Benedicts Reagent
Watch glass. 3M NH4OH

50 | P a g e
Thermometer Yeast

Procedure:

A. Isolation of RNA
1. Weight 2 grams of yeast and 2 grams of sand and grind together in a
mortar and pestle.
2. Then transfer it in a beaker and add 15 ml of 10% NaOH.
3. And dilute it distilled water until 50 ml.
4. Then heat the solution for 30 minutes for about 90 oC.
5. After heating allow it to cool and filter.
6. Filtrate is the RNA.

B. Qualitative test for Nucleic Acids

A. Test for Nucleoproteins
1. Add 1 ml of filtrate and 1 ml of 10% NaOH in a test tube.
2. Then add 5-10 drops of 10% CuSO4.
3. Observe.

B. Mild Acid Hydrolysis

1. Add a 20 ml of 10% H2SO4 to the remaining filtrate.
2. Boil it for 3 minutes.
3. Observe.

C. Test for Inorganic Phosphate

1. Add 1 ml of filtrate (from mild acid hydrolysis) and 1 ml of ammonia
into a test tube.
2. Acidify the solution with a 10% HNO3.
3. Then add 2 ml of ammonium molybdate.
4. Then placed it in a water bath for 5 minutes.

D. Test for the presence of pentose.

1. Add 1 ml of filtrate (from mild acid hydrolysis) and 1 ml of glucose in
a test tube.
2. Add 3 ml of Benedict’s reagent into the solution.
3. Then heat it in a water bath.
4. Observe.

E. Test for presence of Purines.

1. Add 3ml of filtrate (from mild acid hydrolysis) and 3 ml of 3M NH 4OH
in a test tube.
2. Then add 2-3 drops of 0.1M of AgNO3.
3. Observe.

51 | P a g e
 Learning Activity 6
Isolation of RNA

Name: ________________________________ Group No.: _______________

Course and year: _______________________ Date: ____________________

Results

A. Isolation of RNA:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
B. Qualitative test for Nucleic Acids:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
C. Mild Acid Hydrolysis
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
52 | P a g e
 D. Test for Inorganic Phosphate:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
E. Test for the presence of pentose:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
F. Test for presence of Purines:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________

Learning Activity 7
Analysis of Vitamins
Specific Learning Outcomes: At the end of the learning activity the students shall
4. Identify the vitamin in a variety of foodstuffs,
5. Identify vitamins A, D, E, B1, B2, B6, and C qualitatively by observe the color of
reaction.
6. Describe the basic chemical reactions for the identification of vitamins in
food, and
7. Gain an understanding of how vitamins from natural organic extracts compare
with commercially prepared vitamins.

Time Allotment: 6 Hours

Learning Resources:
Equipment and glassware: Chemicals and reagents
Scissor Fish Oil Capsules
Drop Pipette Nature-E capsules.
Test Tube Carr-Price Reagent

53 | P a g e
 Clamp H2O2
Water bath Ethanol 95%
pH Paper Conc. Nitric Acid
Vitamin B1 Solution
1 N NaOH
Potassium ferricyanide
Isobutanol
Vitamin B Complex
2 M AgNO3
FeCl3 solution
Vitamin C Liquid
Fehling A
Fehling B
cupric sulfate solution
phosphomolybdic acid soln

Learning Content

Vitamins are organic compounds needed by the human body to run the
function. Human body needs really small amount of vitamin, around some
microgram until milligram. However, vitamin must be exist followed with food
because body can’t produce vitamin, except vitamin K. Deficiency of vitamin in
the human body caused failure process of vital organ in the body. This is because
vitamins are used as coenzyme from the enzyme that run the chemical reaction
inside the human body.
Vitamins have unique chemical structures that affect their solubilities in different
parts of the human body. Vitamins B and C are water soluble, whereas vitamins A,
D, E, and K are soluble in nonpolar solvents and in the fatty tissue of the body
(which is nonpolar). Because of their water solubility, vitamins B and C are not
stored to any appreciable extent in the body, and so foods containing these
vitamins should be included in the daily diet. In contrast, the fat-soluble vitamins
are stored in sufficient quantities to keep vitamin-deficiency diseases form

54 | P a g e
appearing even after a person has subsisted for a long period on a vitamin-
deficient diet.

The different solubility patterns of the water-soluble vitamins and the fat-soluble
oned can be rationalized in terms of the structures of the molecules. The chemical
structures of vitamin A (retinol) and of vitamin C (ascorbic acid) are shown below.

Retinol Ascorbic acid

Notice that the vitamin A molecule is an alcohol with a very long carbon chain.
Because the OH group is such a small part of the molecule, the molecule
resembles the long-chain alcohols. This vitamin is nearly nonpolar. In contrast, the
vitamin C molecule is smaller and has more OH groups that can form hydrogen
bonds with water.

Some examples of vitamin:

1. Vitamin A
Vitamin A is an alcohol compound which has large molecule mass. Vitamin A
is the name of a group of fat-soluble retinoids, including retinol, retinal, and
retinyl esters. Vitamin A is involved in immune function, vision, reproduction, and
cellular communication. Vitamin A is critical for vision as an essential
component of rhodopsin, also supports cell growth and differentiation. Two
forms of vitamin A are available in the human diet: preformed vitamin A (retinol
and its esterified form, retinyl ester) and pro-vitamin A carotenoid. Vitamin A is
unstable compound, so Vitamin A should be preventing from contact with
oxygen in the air. Existence of vitamin A can be shown by the Carr-Price
reagent. Carr-Price reagent is a mixing solution between SbCl3 and chloroform
which will give blue color solution.
2. Vitamin D

55 | P a g e
 Vitamin D refers to a group of fat-soluble secosteroids responsible for
enhancing intestinal absorption of calcium, iron, magnesium, phosphate and
zinc. In humans, the most important compounds in this group are vitamin D 3
(also known as cholecalciferol) and vitamin D2 (ergocalciferol). A diet deficient
in vitamin D in conjunction with inadequate sun exposure causes osteomalacia
(or rickets when it occurs in children), which is a softening of the bones. Carr-
Price reagent also can be used to identify the occurrence of Vitamin D in a
sample. But, before adding the reagent, must be added by H2O2 5% and
heating to damage the vitamin A in the sample.
3. Vitamin E
Vitamin E is the collective name for a group of fat-soluble compounds with
distinctive antioxidant activities. Naturally occurring vitamin E exists in eight
chemical forms (alpha-, beta-, gamma-, and delta-tocopherol and alpha-,
beta-, gamma-, and delta-tocotrienol) that have varying levels of biological
activity. Alpha- (or α-) tocopherol is the only form that is recognized to meet
human requirements. In addition to its activities as an antioxidant, vitamin E is
involved in immune function and as shown primarily by in vitro studies of cells,
cell signaling, regulation of gene expression, and other metabolic processes.
Diseases and disorders because of deficient in Vitamin E are heart disease,
cancer, eye disorders, and cognitive decline.
4. Vitamin B1
Thiamine is a vitamin, also called vitamin B1. Vitamin B1 is found in many foods
including yeast, cereal grains, beans, nuts, and meat. It is often used in
combination with other B vitamins, and found in many vitamin B complex
products. Thiamine is also used for digestive problems including poor appetite,
ulcerative colitis, and ongoing diarrhea. Thiamine is also used for AIDS and
boosting the immune system, diabetic pain, heart disease, alcoholism, aging,
a type of brain damage called cerebellar syndrome, cancer sores, vision
problems such as cataracts and glaucoma, motion sickness, and improving
athletic performance.
5. Vitamin B2
Vitamin B2, also known as riboflavin, is arguably the only vitamin that gives you
a visual clue as to its passage through your body. When there is a lot of vitamins
B2 in the diet (or in a supplement), your urine turns bright yellow to show you it

56 | P a g e
 is there. In fact, the —flavin in riboflavin comes from flavus, the Latin word for
yellow. Vitamin B2, like the other B vitamins, is involved in energy metabolism. It
has also recently been found to affect the metabolism of iron in important
ways. Vitamin B2 is one of many nutrients required to recycle glutathione, which
is one of the most important antioxidants in the human body. Examples of good
vitamin B2 sources include spinach, beet greens, and broccoli.
6. Vitamin B6
Vitamin B6 is also called pyridoxine. It is involved in the process of making
serotonin and norepinephrine, which are chemicals that transmit signals in the
brain. Vitamin B6 is also involved in the formation of myelin, a protein layer that
forms around nerve cells. Vitamin B6 deficiency in adults may cause health
problems affecting the nerves, skin, mucous membranes, and circulatory
system. In children, the central nervous system is also affected. Deficiency can
occur in people with kidney failure complications, alcoholism, liver scarring,
overactive thyroid, problems with absorbing nutrients, and heart failure, as well
as those taking certain medications. Major sources of vitamin B6 include cereal
grains, legumes, vegetables (carrots, spinach, peas, and potatoes), milk,
cheese, eggs, fish, liver, meat, and flour. Vitamin B6 is often used with other B
vitamins in vitamin B complex formulas.

7. Vitamin C
Vitamin C, or known as ascorbic acid is a water-soluble vitamin, which is
needed by the body to form collagen in bones, cartilage, muscle, and blood
vessels. Dietary sources of vitamin C include fruits and vegetables, particularly
citrus fruits such as oranges. These days, vitamin C is used most often for
preventing and treating the common cold. Some people use it for other
infections including gum disease, acne and other skin infections, bronchitis,
human immunodeficiency virus (HIV) disease, stomach ulcers caused by
bacteria called Helicobacter pylori, tuberculosis, dysentery (an infection of the
lower intestine), and skin infections that produce boils (furunculosis). It is also
used for infections of the bladder and prostate. Some people use vitamin C for
depression, thinking problems, dementia, Alzheimer's disease, physical and
mental stress, fatigue, and attention deficit-hyperactivity disorder (ADHD).

57 | P a g e
 Ascorbic acid decreases gradually during storage especially at temperature
above 0oC.

Procedure:

Vitamin A Test
1. Cut off two fish oil capsule and drop the fish oil into test tube.
2. Add 1 mL (±20 drops) of Carr-Price reagent into the test tube (consist of CHCl 3
and SbCl3)
3. Observe the color changes.

Vitamin D Test
1. Cut off two fish oil capsule ad drop the fish oil into test tube.
2. Add 5 drops of H2O2 solution, then heat the solution in the water heater but
don’t boil it
3. Chilled with flowing tap water.
4. Add 1 mL of Carr-Price reagent.
5. Observe the color changes.

Vitamin E Test
1. Cut off the Nature-E capsule and drop the vitamin into test tube.
2. Add 0,5 mL (±10 drops) of ethanol 95% then shake the test tube.
3. Add 1 mL of conc. nitric acid.
4. Observe the color changes.

Vitamin B1 Test
1. Add 1 mL of Vitamin B1 liquid into test tube.
2. Add around 1-2 mL of NaOH solution until alkaline (pH = 10), for the precise
pH, check with universal indicator paper.
3. Mix thoroughly, then add potassium ferricyanide [K(Fe(CN)6)3] solution and
shake again.

58 | P a g e
 4. Observe the color changes, then add isobutanol solution, then check the
color that occur.

Vitamin B2 Test
1. Add 2 mL of vitamin B complex liquid into test tube.
2. Add ethanol 95% until the volume fill a half of the tube (Positive result turn
yellow)
3. Add 2 mL of vitamin B complex liquid into another test tube.
4. Add 1 mL of 2 M AgNO3 solution (Positive result turn red cherry)
5. Shake well all the solution and observe the color changes and fluorescence.

Vitamin B6 Test
1. Add 2 mL of vitamin B complex liquid into test tube.
2. Add 5 drops of FeCl3 solution.
3. Observe the color changes, check its fluorescence.
Vitamin C Test
1. Add 1 mL of vitamin C liquid into test tube.
2. In another test tube, mix 1.5 mL of Fehling A reagent with 1.5 mL of Fehling B
reagent.
3. Then mix the first tube into the second tube.
4. Shake the mixture then heat in the water bath.
5. Observe the changes happened.
Learning Activity 7
Analysis of Vitamins

Name: ________________________________ Group No.: _______________

Course and year: _______________________ Date: ____________________

Results

No. Identification Treatment Observation (+/-)

1. Vitamin A

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2. Vitamin D

3. Vitamin E

4. Vitamin B1

5. Vitamin B2

6. Vitamin B6

7. Vitamin C

60 | P a g e
 References

Introduction to General, Organic, Biochemistry. 12th edition Frederick A. Bettelheim,

William H. Brown, Mary K. Campbell, Shawn O. Farrell, and Omar Torres Chemistry

Stoker, H, Stephen (2016). General, Organic, and Biological Chemistry. 3rd Edition.

Curry J. (2012) Organic Chemistry Biological Approach

Kirby H. (1950). Materials and Method in Study of Protozoans. University of California

Press: Berkeley and Los Angeles pp.53

Nucum, Zenaida T. (2010). Laboratory Manual for Simplified Biochemistry

Orlans B. F. (1977). Animal care from protozoa to small mammals. Addison-Wesley

Publishing Inc.: USA

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http://www.starsandseas.com/SAS%20Cells/SAS%20cellphysiol/Osmosis.htm

https://www.cabrillo.edu/~ncrane/bio1c/botPDFs/Bacteria-cells.pdf

www.tricountyschools.org/Scheiding/science/.../CELL%20LAB.doc

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