vegetable oils have

SPOTTING EFFECT With

VEGETABLE both saturated and
translucent
OVERVIEW OIL unsaturated fatty
spot
acids
Spotting effect may sometimes be called as
‘Greasy spot test’ to determine if the lipid-like the sample used
material contains lipids. The positive visual result is contains wax, a
the presence of a translucent spot because of the simple lipid which
lipid content that will penetrate into the filter paper. has long-chain of fatty
When the liquid is inside the paper, it diffracts light acids and long-chain
and then results to a translucent spot. With minimal of alcohol. The sample
PROCEDURES HAIR WAX translucent has only shown
spot minimal translucent
1. Draw 5 circles in a filter paper using a pencil spot however, as per
2. Label each circle with the available definition wax is a
samples simple lipid hence it
3. Put 1 drop of each sample within each of still indicates a
the labeled circular areas positive result.
4. Allow the spots to dry
5. Hold the filter paper against the light and
note the presence of translucent spots
6. Record observations
With
RESULTS mayonnaise contains
MAYONNAISE translucent
saturated fatty acids
spot

GUIDE QUESTIONS LEC

1. WHAT IS THE PURPOSE OF THIS TEST? lotion contains
- To detect presence of lipids in the 'lipid- ceramides (lipid) and
like' samples based from the result in With minimal essential fatty acids
the form of grease and translucent spot LOTION translucent (omega-3 and omega-
spot 6). It still has shown a
2. WHAT PROPERTY OF LIPIDS IS translucent spot
DETECTED BY THIS TEST? though only minimal.
- The test is also called Greasy Spot test.
The translucent and grease texture
indicate the presence of lipids and its lecithin or
property of being non-volatile. phosphatidyl choline
- Most grease fats have higher boiling is composed of
point making them non-volatile because phosphoric acid,
the With cholines, esters of
- grease cannot absorb enough heat to LECITHIN translucent glycerol, and two
be vaporized. When the liquid is inside spot fatty acids. It has
the paper it diffracts light resulting to a shown a translucent
translucent spot. spot because it is a
lipid or fat essential in
3. ARE THE RESULTS OF THIS TEST the body
CONCLUSIVE? EXPLAIN YOUR
ANSWER
- Yes, the test results were conclusive.
 SOLUBILITY
PROCEDURE
1. Prepare 4 clean dry test tubes 1. Solubility of vegetable oil in different solvents:
2. Label: water, methylene chloride, ether and
A. WATER
toluene
- Fats and oil are nonpolar, so they will
3. Put 1 ml of these solvents into each
remain separate from molecules of a
respective containers
polar solvent such as water. In general,
4. In each tube, add 3 DROPS OF
VEGETABLE OIL and mix thoroughly they do not follow the rule: “like dissolves
5. Compare the solubility of the vegetable oil in like”.
- Oil and water don’t mix because water
the different solvents
6. Repeat the procedure using A DROP OF molecules are more attracted to each
LECITHIN FROM A SOFT GEL CAPSULE other than to oil molecules.

OBSERVATION B. METHYLENE CHLORIDE

- Methylene chloride is polar, but not as
SAMPLES WATER METHYLENE ETHER TOLUENE
CHLORIDE much polar as water because of its
VEG. OIL NOT MIXED MIXED MIXED nonpolar C-H bond, thus it will still mix
MIXED with vegetable oil, but not completely.
LECITHIN SLIGHTLY MIXED MIXED MIXED
MIXED
GUIDE QUESTIONS:
1. With the aid of their structural formula,
explain the demonstrated
solubility/insolubility of vegetable oil and
lecithin to water, methylene chloride, ether C. ETHER
and toluene. - Since ether and vegetable oil are both
non-polar and insoluble in water, they will
mix together. The rule “like dissolves like”
applies.

D. TOLUENE
- Toluene is composed of C-H bonds
making it a non-polar, so vegetable oil
has the ability to dissolve in it. The rule
“like dissolves like” is being applied in the
mixture.

Lecithin

2. Solubility of lecithin in different solvents:

A. WATER
- The structure of lecithin is very similar
with vegetable oil, but instead of the third
 fatty acid, it has a phosphate group, 2. Add 1.0mL of bromine water to both tubes
which is polar thus it is water soluble, and mix thoroughly.
which means it will slightly dissolve in 3. 3. Compare the change in color of the
water. Phospholipids, like lecithin, bromine water.
emulsify oil in water by forming little A. With the Vegetable oil, bromine water
protective envelopes around tiny droplets forms the upper layer.
of oil B. With Methylene Chloride, bromine water
forms the lower layer.
B. METHYLENE CHLORIDE
Recorded observations (ACTUAL RESULTS)
- Methylene Chloride is also a nonpolar
substance because of its C-H bond and SAMPLES Noted color change in bromine
most of the lecithin dissolved in it even water
though it has polar ends. Vegetable Oil 2 Layers:
(unsaturated) a) Colorless (Top Layer)
b) Colorless (Bottom Layer)

Lecithin 2 Layers:
(saturated) a) yellow (Top Layer)
b) orange-yellow (Bottom Layer)
C. ETHER
- Lecithin attracts both water and fatty
Guide Questions:
substances (and so are both hydrophilic
and lipophilic). 1. WHAT IS THE PRINCIPLE INVOLVED IN
- Ether is a nonpolar substance and most THE BROMINE WATER TEST? WHAT IS
of the lecithin dissolved in it even though ITS PURPOSE?
it has polar ends. - The principle of Bromine Water Test is
that it detects presence of unsaturated
D. TOLUENE lipids. Orange bromine water is
- Lecithin has a hydrocarbon which is a decolorized, when added to a compound
nonpolar and because the containing C=C double bonds
electronegativity difference of the two (unsaturated vegetable oils or alkenes).
elements are below 0.5, but it also However, it remains unchanged (orange)
consists of choline end which is a polar when added to a compound containing
bond, therefore, lecithin is somewhat a C-C single bonds (saturated vegetable
polar substance also. oils or alkanes).
- Toluene, also known as methylbenzene, 2. PROVIDE THE TYPE OF EQUATION
also consists of hydrocarbon chain USED IN THE BROMINE WATER TEST.
making it a nonpolar substance.
Addition Reaction
- Most of the lecithin dissolved in it even
though it has polar ends. Therefore, the
rule “like dissolves like" does apply
to them.

BROMINE WATER TEST: A TEST FOR 3. SHOW THE SAMPLE EQUATION

UNSATURATION INVOLVED IN THE REACTION BETWEEN
BROMINE WATER AND YOUR SAMPLE/S
PROCEDURE: WHICH GAVE A POSITIVE RESULT.
1. Prepare 2 clean and dry test tubes. To 1 of
the tubes, place 1.0mL of vegetable oil and
in the other, the contents of the lecithin soft
gel capsule dissolved in 1.0mL methylene
chloride. Pierce the gel capsule
 Guide Questions:
1. WHAT IS THE PRINCIPLE INVOLVED IN
THE ACROLEIN TEST? WHAT IS ITS
PURPOSE?
- When lipids are subjected to dehydrating
agents such as Potassium bisulfate, an
unsaturated aldehyde (Acrolein) is
formed after the dehydration of the
Lecithin structure
glycerol portal of the molecule which then
4. PROVIDE THE POSSIBLE EXPLANATION leads to an unfavorable pungent and
FOR THE DIFFERENCE IN RESULTS irritating odor. Therefore, this test is
utilized in order to qualitatively detect the
BETWEEN THE VEGETABLE OIL AND
presence of glycerin in fats.
THE LECITHIN.
- Its principle is the oxidation and
- The vegetable oil changed to colorless dehydration reaction due to the exposure
meaning there was a formation of bonds of heat.
meanwhile, the lecithin turned out to be 2. SHOW THE SAMPLE EQUATION
saturated because the bromine remained INVOLVED IN THE REACTION BETWEEN:
its orange coloration which means there a. Glycerol + Potassium Bisulfate.
was no addition reaction of bonds. b. Cooking Oil + Potassium Bisulfate.
c. Lecithin A+Potassium Bisulfate.
ACROLEIN TEST
PROCEDURE
1. Prepare three clean and dry test tubes.
a. To the first tube, add a drop of
glycerol.
b. To the second tube, add a drop of
cooking oil.
c. To the third tube, add a drop of
lecithin from the soft gel capsule.
Pierce the gel capsule with a pointed
object, e.g., a needle or copper wire.
3. WHAT IS THE COMPOUND
2. 2. Mix a pinch-sized amount of powdered RESPONSIBLE FOR YOUR
potassium bisulfate to all the tubes. OBSERVATION?
3. Heat the mixture gently over a small flame - Acrolein- unsaturated aldehyde which
then note the odor of the vapor and record gives pungent odor
your observations.
RESULTS:
SAMPLES OBSERVATIONS
Glycerol No pungent or irritating
(Undergoes dehydration) odor was observed.
-
Cooking Oil (undergoes Burnt odor 4. WHAT IS THE PURPOSE OF THE
oxidation and POTASSIUM BISULFATE USED?
dehydration) - It acts as a dehydrating agent.
Lecithin (undergoes Burnt odor Whenever fat is heated in the presence
oxidation and of a dehydrating agent, the fat molecule
dehydration) shed its glycerol in the form of the
unsaturated aldehyde-acrolein. It was
used in the first world war as a chemical
weapon
5. WHAT GROUP OF LIPIDS WAS SCHEMATIC DIAGRAM
IDENTIFIED BY THIS TEST?
- Triglycerides and Fatty Acids.
Triglycerides are the major dietary fat and
are compose of three molecules of fatty
acids combined with a molecule of
glycerol. Fatty acids contain long chains
of monocarboxylic acids found in fats and
oils

Extraction of Brain Lipids

Procedure
1.In an Erlenmeyer or Florence flask, place
the homogenized brain and add enough
ether to completely immerse the brain
sample.
2. Cover tightly and set aside until the next Type of lipids that was extracted by:
laboratory period.
1. Ether
3. Decant the incubated brain sample. - Lipids, in general, are insoluble in water
hence they are usually labelled as
To the DECANTATE: hydrophobic. Solvents such as ether,
a. Add acetone gradually until the chloroform and benzene which are non-
precipitation is complete. The precipitation is polar solutions can extract lipids that are
complete when no turbidity can be observed hydrophobic. (note: not all lipids are
after adding more acetate to the clear liquid hydrophobic though, lipids found in the
above the precipitate. cell membranes are usually amphiphatic)
2. Acetone
b. Filter the mixture. - This chemical is partially polar and non-
polar organic solvent hence it has the ability
c. The residue, labeled as RESIDUE B, is to
to extract amphiphatic lipids(with hydrophilic
be divided into 3 equal portions.
and hydrophobic parts) like phospholipids
d. Each portion of RESIDUE B will be used in and sterols in the form of cholesterol which
the NINHYDRIN TEST, SODA LIME TEST, are found in the plasma membrane.
and the AMMONIUM MOLYBDATE TEST.
3. Hot 95% alcohol
e. Evaporate the filtrate until it dries using a
steam bath (use an electric water bath). - Ethanol disrupts the physical structure of
cell membranes , extracting even the
f. Label the residue obtained as RESIDUE C
glycolipids that are attached to the outer
and use this in the LEIBERMANN
leaflet of the membrane
BURCHARD TEST.
To the RESIDUE (After Decantation):
1. Get a small portion of the incubated brain
sample and add 10 ml of hot 95% ethanol
(use an electric water bath in heating the
alcohol).
2. Mix thoroughly and decant.
3. Discard the Residue and use the
decantate, labeled as DECANTATE A in the
MOLISCH TEST.
Molisch Test - Dehydrate sugar into hydroxy
methylfurfural w/c condense with alpha
Procedure
naphthol
1. To 2.0 ml of DECANTATE A, add 5 DROPS of 5. Equation
MOLISCH'S REAGENT. MIX THOROUGHLY.
2. Tilt the tube and carefully add 1.0 ml of
CONCENTRATED SULFURIC ACID, drop by drop,
by allowing the acid to flow by the side of the tube.
DO NOT MIX!
3. Return the tube to its original position and note the
color of the ring formed at the junction of the liquids.
4. Record your observations.
QUIDE QUESTIONS
1. What is the principle involved in the
Molisch test?
- Carbohydrates when reacted with conc.
H2SO4 get dehydrated to form furfural
and its derivatives.
- When monosaccharide is treated with
conc H2SO4 or conc HCl, -OH group of 6. Compound responsible for the
sugar are removed in the form of water observation
and furfural is formed from pentose sugar
and hydroxymethyl furfural is formed
from hexose sugar. These products react
with sulphonated α- naphthol to give a
purple (violet red) colored complex.
2. What is its purpose?
- The purpose of the Molisch test is to
detect the presence of Carbohydrates in
Lipids
3. What is the purpose of the reagent used?
- Molisch reagent is a solution of alpha-
naphthol in 95% ethanol. The alpha- 7. Group of lipids identified
naphthol is condensation reagent that - Glycolipids
reacts with the cyclic aldehydes to form - are carbohydrate-attached lipids, which
purple colored condensation products. are associated with cell membranes.
Although this test will detect compounds
other than carbohydrates (i.e.
glycoproteins, glycolipids), a negative Ninhydrin Test
result indicates the ABSENCE of PROCEDURES
carbohydrates
4. What is the purpose of the reagent used? 1. Dissolve a portion of residue B in 2.0 mL
- Concentrated H2SO4 is a water
dehydrating agent. Normally, without
2. Add 1.0 mL of ninhydrin reagent. Mix and
concentrated sulfuric acid, molisch test,
heat in boiling water bath for 2 minutes.
Pentoses and above don't yield Furfural
derivatives 3. Note the color of the resulting solution.
Expected Result
 - The color of the solution should have the blood clotting process as they are
turned into deep blue-purple found in blood platelets. Cephalins are
(Ruhemann’s Purple). phosphoglycerides that contain
ethanolamine or the amino acid serine
GUIDE QUESTIONS attached to the phosphate group through
1. What is the principle involved in the phosphate ester bonds. A variety of fatty
Ninhydrin test? What is its purpose acids make up the rest of the molecule.
- The principle in which this test lies is the This test is only positive for cephalin
reaction between the amino group of free because it is the only lipid with free serine
amino acids and ninhydrin reacts with an group. Free amino groups will react with
–NH2 group, a purple-blue complex is the ninhydrin reagent to yield a purple
formed. solution.
2. What is the purpose of the reagent used? 6. Account for the precaution with the
- Ninhydrin Reagent is used in the ninhydrin reagent
detection and assay of peptides, amino - Account for the precaution with Ninhydrin
acids, amines, and amino sugars yielding reagent: The Ninhydrin reagent will stain
highly fluorescent ternary compounds the skin blue because of amino acid on
with aldehydes and primary amines. the skin and cannot be immediately
Reaction with sarcosine or proline gives washed off completely if it comes in
azomethine ylides. It is also used for the contact with the skin. Ninhydrin is most
detection of free amino groups in amino commonly used to detect fingerprints.
acids, peptides and proteins.
3. Show the equation involved in the
Soda Lime Test
reaction between the Ninhydrin reagent
and your sample/s which gave a positive PROCEDURE
result
1. Grind a pinch of soda lime and a portion of
residue B.
2. Transfer the mixture in a dry test tube and
heat gently over a small flame. After a
minute, test the vapor using small pieces of
red and blue litmus paper.
Note the change in color of the pieces of
litmus paper. Record your observation
Expected Result:
- The red litmus paper should have
turned into blue.
4. What is the compound responsible for GUIDE QUESTIONS:
your observation?
1. What is the principle involved in the soda
- Diketohydrin is also known as the lime test? What is its purpose
Ruhemann’s complex which is - Pupose: test for amino and imino group.
responsible for the resulting blue-purple - Principle involved in the test:
color of the solution. deamination. Deamination is the removal
5. What group of lipid was identified by this of an amino group from a molecule.
test? 2. What is the purpose of the reagent used?
- Phospholipid: Cephalins - The Soda-Lime acts as the Deaminating
(Phosphatidylethanolamine ) are found in agent. It absorbs CO2 and H2O vapor.
most cell membranes, particularly in - To detect N-containing lipids or amino
brain tissues. They are also important in group.
 5. Record your observation.
3. Show the equation involved in the
RESULT
reaction between soda lime and your
sample/s which gave a positive result. SAMPLE OBSERVATION
Residue B Yellow with a yellowish solid /
Yellow precipitate formed

Lecithin Clear yellow with a yellowish

brown / Dark yellow precipitate
formed

GUIDE QUESTIONS
4. What is the compound responsible for
1. Dehydration and Double Composition
your observation?
- When lipids containing phosphate groups
- The compound responsible for producing
in their structures are added to a strong
the expected result is the ammonium
acid solution, the lipid hydrolyses,
hydroxide (NH OH) vapor. It would turn
producing free phosphate.
the red litmus paper into blue since it is a
- To determine the presence of phosphate
weak base.
group.
5. What group of lipids was identified by this
2. WHAT IS THE PURPOSE OF THE
test?
REAGENT USED?
- The group identified is the Sphingolipids
- HNO3 (NITRIC ACID)
which is a class of lipid found in biological
o it serves as the dehydrating
membranes. Sphingolipid, any member
agent.
of a class of lipids (fat-soluble
- To break the bonds in order to reveal the
constituents of living cells) containing the
free phosphate group
organic aliphatic amino alcohol
sphingosine or a substance structurally
3. SHOW THE EQUATION INVOLVED IN THE
similar to it. Among the most simple
REACTION BETWEEN AMMONIUM
sphingolipids are the ceramides
MOLYBDATE AND YOUR SAMPLE/S
(sphingosine plus a fatty acid), widely
WHICH GAVE A POSISTIVE RESULT.
distributed in small amounts in plant and
animal tissues. The other sphingolipids
are derivatives of ceramides.
AMMONIUM MOLYBDATE TEST
PROCEDURE
1. Add 1.0 ml of 6N nitric acid to a portion of
Residue B.
2. Heat the mixture in a boiling water bath for
five minutes and add 1.0 ml of the
ammonium molybdate solution. Continue
heating for another five minutes.
- When a solution containing phosphate
3. Note the change in color of the solution.
ions is heated with a solution of
Record your observations
ammonium molybdate [(NH4)2MoO4]
4. Repeat the test using the contents of a and dilute nitric acid a bright yellow
lecithin soft gel capsule instead. precipitate of ammonium
phosphomolybdate [(NH4)3PO4
 12MoO4] is formed. The yellow a light green then very dark green
precipitate is extremely insoluble in nitric / blue green color
acid.
VEGETABLE The appearance of light green
4. WHAT IS THE COMPOUND OIL (TUBE 2) was observed
RESPONSIBLE FOR YOUR
OBSERVATION? GUIDE QUESTIONS:
- Ammonium Phosphomolybdate 1. What is the principle involved in the
- The precipitation is usually used to Leibermann- Burchard Test?
identify phosphate. The precipitation is - The Liebermann–Burchard or acetic
the formation of yellow ammonium anhydride test is used for the detection of
molybdophosphate from ammonium cholesterol. The formation of a green or
molybdate in acidic solution green-blue color after a few minutes is
positive
5. WHAT GROUP OF LIPIDS WAS 2. What is the purpose of the reagent used?
IDENTIFIED BY THIS TEST? a. Concentrated Sulfuric Acid
- Phospholipids. - When (H2SO4) is added to cholesterol, a
- Phospholipids are found in high water molecule is removed from
concentrations in the lining of practically (hydroxyl group) C3 of cholesterol
every cell of the body, including brain molecule, and it is
cells. They help brain cells communicate oxidized to form 3,5 – cholestadiene. This
and influence how well receptors product is then converted to a green
function. coloration indicating a positive result.
b. Acetic Anhydride
LEIBERMANN- BURCHARD TEST - (CH3CO) reacts with cholesterol in a
chloroform solution to produce a blue
PROCEDURES
green solution.
REMINDER: all equipment and containers
to be used for this test must be COMPLETELY 3. Show the equation involved in the
DRY reaction between Molisch’s test and your
sample’s which give a positive result?
1. Prepare two clean and dry test tube
2. In the 1st tube, dissolve residue C in a 1.0 ml of
methylene chloride
3. In the 2nd tube, place 1.0 ml of vegetable oil
4. Add 5 drop of acetic anhydride, followed by three
drops of concentrated sulfuric acid into both tubes.
Mix thoroughly
5. Note the color of the solution and compare the
resulting intensity
CAUTION: ACETIC ANHYDRIDE and SULFURIC
ACID are very corrosive
RESULT
SAMPLE OBSERVATION
RESIDUE C The appearance of color that ▪ Concentrated Sulfuric acid used in
(TUBE 1) begins as a orange, to pink then
Molisch Test and Leibermann-
purple and progresses through to
Burchard Test has the same fuction
during the chemical reaction and that
 is to removed –OH group in a form of
water.

4. Compound responsible for your

observation?
- HYDROXYL GROUP OF
CHOLESTEROL
- The observation in tt1 was the
appearance of color that begins as a
purplish, pink color and progresses
-
through to a light green then very dark
green color, while in tt2 the appearance
of light green was observed. This color is
due to the hydroxyl group (-OH) of
cholesterol reacting with the reagents
(acetic anhydride).

5. What group of lipids was identified by this

test?
- CHOLESTEROL/ STEROIDS
- Steroids are lipids because they are
hydrophobic and insoluble in water, but
they do not resemble lipids since they
have a structure composed of four fused
rings.
- Steroids are found in the brain and alter
electrical activity in the brain.
- Cholesterol is the most common steroid
and is the precursor to vitamin D,
testosterone, estrogen, progesterone,
aldosterone, cortisol, and bile salts.
- Cholesterol is a component of the
phospholipid bilayer and plays a role in
the structure and function of membranes.

6. Why must one take the precaution of

using only very dry containers and pieces
of equipment?
- Because water might alter the outcome of
the given experiment. It may react with
the corrosive reagents used therefore the
result may not be accurate.

“I CAN DO ALL THINGS THROUGH CHRIST

WHO STRENTHENS ME”
PHILIPPIANS 4:13