EXPERIMENT 5

LIPIDS
I. OBJECTIVES
ISOLATION OF LIPIDS:

a. Aims to extract cholesterol, phospholipids, and cerebrosides from pig’s brain

tissues.

b. To know and understand the solubility of the lipids by utilizing different organic
solvents such as alcohol, ether, and acetone.

c. To hypothseize the identity of the following isolated precipitates

QUANTITATIVE ANALYSIS OF LIPDS

d. To know and understand the qualitative properties of lipids based on the

extracted precipitates

e. To distinguish properties based on their reactivity towards certain tests.

QUESTIONS:
1. In molecular terms, describe how emulsifiers are able to mix polar and non-
polar substances together.
- Emulsifiers can be attracted to both polar and nonpolar compounds because they
are amphipathic, which means it is hydrophilic (polar) at one end and hydrophobic
(nonpolar) at the other end. In an oil and water mixture, the hydrophobic ends/ tails of the
emulsifier will surround the tiny droplets of oil, which will then leave the hydrophilic heads
to be exposed. Due to this, the hydrophilic heads will be attracted to the water molecules,
thus, enables the oil and water to mix.

2. Correlate the structure of lecithin with its use as an edible emulsifying agent.
-Lecithins are emulsifier, which means they are amphipathic in nature. Lecithin has
a glycerol backbone in its structure, and with that glycerol backbone, there are three
molecules bonded to it: two fatty acids and phosphoric acid with choline attached to it.
The two fatty acids are the hydrophobic ends of the structure whilst the phosphoric acid
is the hydrophilic. Lecithin makes a good emulsifier because it likes to be at the edge of
the oil droplet. The hydrophobic end of the lecithin will surround the oil droplet and will be
dissolve in it while the hydrophilic end will be exposed to be dissolve in water. Due to this,
the oil droplets in water are protected and stabilize, and the interfacial tension between
the oil and water phases is lowered, which prevents the two from coalescing with each
other.

-DON’T FORGET TO DRAW THE STRUCTURE OF LECITHIN

3. Cite food products that contain lecithin as one of its ingredients.

- eggs, chocolates, soybeans, sunflower seeds, organ meats, red meat, seafoods,
cooked green vegetables such as broccoli and brussel sprouts

4. What is the molecular basis of how tocopherols contribute to the keeping

qualities of fats?
- Tocopherols (α, β, δ and γ isomers), which are abundant and effective isomers
of vitamin E, are oil soluble compounds. They function as antioxidant ingredient used for
the stabilization of food products that contain lipids such as fats and oils, which are
suspectible to oxidative rancidity. (Moreover, γ-Tocopherol acts as an antioxidant, and
prevents oxidative cleavage in unsaturated fats.) Tocopherols, being the natural
antioxidants as they are, protect the food against oxidation reaction that is caused by free
radicals, which then helps preserve the taste and nutritional value of the food.

-MAKE SURE TO DRAW A STRUCTURE OF TOCOPHEROL

5. Predict and justify the reaction of the following to the Carr-Price reagent:
a. Lard- expected to have negative result because it may have low to non- existent of
vitamin A, which is required to form the characteristic blue-green color from its reaction
with SbCl3.

b. Butter- expected to have positive result due to its high content of vitamin A

II. DEFINITION OF TERMS

1. Fatty acids- molecules that have carboxylic acids at the polar end and long hydrocarbon
chain at the nonpolar tail

2. Simple lipid- organic compounds which are esters of fatty acids with various alcohols

3. Compound lipid- esters of fatty acids that contains additional group aside from alcohols
and fatty acids

4. Derive lipid- derivatives obtained from hydrolysis of simple lipids and compound lipids

5. Hydrophobic- water- hating

6. Hydrophilic- water- loving

7. Glycerol- simple three- carbon compound that contains three hydroxyl groups

8. Amphipathic- molecule that has polar, water- soluble group at one end, and nonpolar,
hydrocarbon group at the other end

III. THEORY BEHIND THE EXPERIMENT

Lipids are chemical compounds which are frequently found in nature and are
considered to be diverse as they can be present in various conditions such as in egg yolk
and even in the nervous system of the humans. Lipids are hydrophobic, which means
that they are insoluble in water. The reason for this is because when we tend to look at
the structure of lipids, its ends are not charged, making them nonpolar molecules. Water,
on the other hand, are polar molecules. Thus, if we are going to consider the principle,
“like dissolves like”, there will be no sharing of electrons that will happen between lipid
molecules and water molecules. However, despite being insoluble in water, lipids are
highly soluble in organic solvents such as alcohol, ether, chloroform, and acetone. In
accordance to the aforementioned facts, lipids are also an important component in living
cells and microbial membranes.

There are three main classification of lipids: (1) simple lipids, which consist of fats
and oils, triacylglycerol, and waxes; (2) compound lipids, which consist of phospholipid,
glycolipid, and lipoproteins; and lastly, (3) derived lipids, which consist of steroids, fatty
acids, and lipid-soluble vitamins.

Simple lipids are organic compounds which are esters of fatty acids with various
alcohols. One particular example of simple lipid is triacylglycerol or triglyceride, a type of
lipid that is formed when three fatty acids undergoes esterification to a glycerol.
Triacylglycerols functions are to accumulate in adipose tissue (fat cells), provide means
of storing fatty acids in animals, and also serve as concentrated stores for metabolic
energy.

Compound lipids or complex lipids are organic compounds which are esters of fatty
acids that contains additional group apart from alcohols and fatty acids.
Phosphoacylglycerol, a type of compound lipid, is formed when phosphoric acid moiety
undergoes esterification to a phosphatidic acid with another alcohol.
Phosphoacylglycerols are amphipathic, which means they possess long, nonpolar,
hydrophobic tails and polar, highly hydrophilic head groups. Furthermore,
Phosphoacylglycerol are considered to be major components of the cell membrane.

Derive lipids, which are derivatives obtained from hydrolysis of simple lipids and
compound lipids. This third classification of lipid includes fatty acids, steroids, and lipid-
soluble vitamins. Fatty acids are molecules that have carboxylic acids at the polar end
and long hydrocarbon chain at the nonpolar tail, thus, making it an amphipathic molecule.
Fatty acids can either be saturated or unsaturated. Saturated fatty acids only have single
bonds present in the hydrocarbon chain while unsaturated fatty acids have carbon-
carbon double bond present in its hydrocarbon chain. Steroids are lipids that have a fused
ring system, which consist of three six-membered rings and one five-membered ring.
Steroids are hydrophobic and insoluble in water, and they are also an important
component of biological membrane in animal cells.

Lipids are considered to be the most abundant compounds that can be found in
the brain. In fact, the brain is considered to be the second highest lipid content next to
adipose tissue. The brain contains a variety of lipids such as complex lipids
(phosphatides, cerebrosides, and sphingosides), cholesterol, fatty acids, triglycerides,
and so on and so forth. The brain has three main lipids, namely: cerebroside, cholesterol,
and phospholipid, each possesses diverse properties that can describe for their qualities.

Cerebrosides are type of glycosphingolipid that is important to nerve cell

membrane. Cerebrosides contain a sugar residue, usually glucose or galactose, having
been glycosidically bonded to the primary alcohol of ceramide. Ceramides are actually
the parent compounds of glycolipids.

Cholesterols are most well- known type of steroids that occurs in the cell
membrane. Its general structure is having three fused six- membered ring and one five-
membered ring. Cholesterols are highly hydrophobic because the only hydrophilic group
it has is the single hydroxyl group. Cholesterols are important in many biological functions
especially as a precursor of other steroids and vitamin D3.

Phospholipids are lipids that are the main component of the cell membrane.
Phospholipids is made up of phosphoric acid being esterified to both glycerol and some
other alcohol.

Before analysis of lipids must be done, a quantitative isolation of lipids must be

accomplished. Quantitative isolation of lipids involves a tedious process in which it is
highly time- consuming, and most probably be a relatively uninteresting thing to do.
However, any slackness or mistakes will result to certain loss of specific components.
There are many solvents or combination of solvents that will be used to extract lipids from
tissues, and so to ensure that other enzymes are deactivated, and recovery is completed,
care must be taken in handling these solvents. Nonlipid components that are co-extracted
after the initial extraction must then be eliminated from the recovered lipids. A particular
way to do this is by simple washing procedures or other solvent partition procedures such
as using aqueous salt solution. During the quantitative isolation of lipids, lipids are
susceptible in undergoing oxidation or peroxidation, which may result to changes in the
lipids’ structure and biological properties, thus, precautions must be observe or taken in
order to minimize the risk of occurrence of these particular reactions.

When quantitative isolation of lipids was done, qualitative analysis of lipids is to be

followed. In qualitative analysis of lipids, there are some preliminary tests and specific
tests that are done in order to detect the presence or absence of lipids on the basis of
color change, and also to classify the certain characteristic groups of lipids based on their
reactivity with certain reagents. There are several methods or tests used in qualitative
analysis of lipids: (1) Acrolein’s test: used to detect the presence of glycerol and fat by
heating the substance to be tested with a dehydrating agent; (2) Test for unsaturation:
used to detect the presence of unsaturated fatty acid or the amount of double bonds
present in that certain lipid sample by undergoing addition reaction with iodine; (3) Test
for phosphate is done when lecithin is hydrolyzed in acidic medium, which then results to
breaking of both fatty ester bonds and phosphate ester bonds, and releasing free fatty
acids and inorganic phosphate. By using molybdate test, we can detect the presence of
phosphate in the hydrolysate; (4) Emulsification test: used to detect the presence of polar
and nonpolar groups in lipids, specifically in bile and lecithin. An addition of emulsifying
agents such as bile salts, soaps, and etc. will enable the formation of a separate layer or
a homogenous water- lipid mixture; (5) Liberman- Burchard test: used to detect the
presence of cholesterol by using strong concentrated acids such as sulfuric acid and
acetic anhydride, which will then act as dehydrating and oxidizing agent; (6) Carr-Price
reaction: used to detect the presence of vitamin A and related carotenoids. The use of
chloroform is required to dissolve vitamin A, and antimony trichloride will react with
vitamin A, which will then result to the appearance of blue color, which indicates a positive
result; (7) Modified Furter- Meyer test: used to determine tocopherols, which are the most
active form of vitamin E. The result of this test would be bronze- red solution; lastly, (8)
Molisch test: used to detect the presence of carbohydrates.

IV. DATA
V. DISCUSSION OF RESULTS
A. Isolation of Lipids from Brain Tissue
In quantitative analysis, adding cold acetone to the brain tissue was the very first
thing that we did. After which, we left it for a night in order for the brain tissue to settle.
Once the brain tissue has settled, it will undergo into the process of decantation, and the
extract that will be obtained from the process per se will be labeled as acetone extract
whilst the other one that is left (the brain tissue) is the residue 1. By utilizing the steam
bath, the acetone extract was evaporated, leaving a dry residue behind. The dry residue
will then be added with a minimal hot 95% ethanol. After that, the dried residue with hot
ethanol will be evaporated, which then yields us with white dried crystals that are soluble
in both acetone and hot alcohol but insoluble in ether.

After obtaining the data for the precipitate 1, we then added 15 mL cold ether to
the residue 1. After which, we then decanted and evaporated the residue 1, and then
added it with 20 ml acetone to ensure the purity of precipitate 2. This yields us crystal-
like, yellow precipitates, which are soluble in both ether and hot alcohol but is insoluble in
acetone.

[Phospholipids are lipids that are made up of phosphoric acid being esterified to both
glycerol and some other alcohol. We observed in the experiment that phospholipids are
soluble in both ether and hot alcohol but is insoluble in acetone. Acetones are considered
to be fairly polar, and to react chemically with lipids, the solvent that should be used must
not be so polar.]

The last residue, the residue 3, was then extracted twice and was added with 10
ml boiling 95% alcohol. Under certain temperatures, some moieties are capable of
undergoing crystallization, thus, recrystallization of the product that hasn’t been
unprocessed is necessary to obtain a product that is already purified. By doing the
procedures needed in residue 3, we obtained precipitate which can be described as
creamy, light yellow precipitate, which are soluble in both ether and alcohol.

[Cholesterol’s structure contains three fused six- membered ring and one five-
membered ring and are highly hydrophobic because the only hydrophilic group it has is
the single hydroxyl group. Cholesterols, as what was observed in the experiment, are
soluble in both ether and alcohol.]

After doing the quantitative isolation of lipids, all three precipitates were all ready
for the qualitative test. All three precipitates were then going to be subjected to different
tests, which have unique properties that are able to help us determine which of the three
precipitates are cerebroside, phospholipid, and cholesterol.
 Acrolein test is used to detect the presence of glycerol and fat by heating the
substance to be tested with a dehydrating agent. Here, brain precipitate 1 gives a faint
rotten smell of an egg; brain precipitate 2 gives strong smell of a Sampaloc; and lastly,
brain precipitate 3 exhibits a very faint smell of white egg. All three precipitates have
yielded negative result. Acrolein test is based on dehydration reaction wherein the water
molecules are removed from the glycerol by adding a reagent, which is potassium
hydrogen sulphate and the reaction between glycerol and potassium hydrogen sulphate
results in the formation of Acrolein, which is characterized by its odor.

Test for unsaturation is used to detect the presence of amount of double bonds
present in that certain lipid sample by undergoing addition reaction with iodine. In this
test, we utilize Hanus reagent. Both brain precipitate 1 and 2 only need one drop of Hanus
reagent to decolorize while brain precipitate 3 needs two drops of the said reagent to
decolorize. Iodine reacts with the double bonds found in a lipid structure, and if the lipid
is more unsaturated, the more iodine it will take up.

Test for phosphorous is used to detect the presence of phosphates by utilizing

molybdate test. Here, only brain precipitate 2 had exhibited a yellow precipitate, thus,
yielding a positive result. On the other hand, both brain precipitate 1 and 3 exhibited white
precipitate, therefore, they both have negative results. The color yellow of the precipitate
came from the phosphate ions in the solution that was heated with ammonium molybdate.

Liebermann- Burchard Test is used to detect the presence of cholesterol. All three
brain precipitates have exhibited some shades of green with both brain precipitate 1 and
2 exhibiting a light green mixture while brain precipitate 3 exhibited a blue- green colored
mixture. The reason for having a green colored solution as positive result it’s because of
the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the
conjugation of the unsaturation in the adjacent fused ring.

Emulsification Test is used to detect the presence of polar and nonpolar groups in
lipids, specifically in bile and lecithin. Furthermore, in emulsification test, the lipid or oil in
water appears on the top of the water because of the high surface tension of water which
gets together to form a separate layer. An addition of emulsifying agents such as bile
salts, soaps, and etc. will enable the formation of a separate layer or a homogenous
water- lipid mixture

Lastly, Molisch test is used to detect the presence of carbohydrates. Here, only the
brai precipitate 2 had yielded a positive result because it is the only one among the three
brain precipitates to exhibit a violet interphase. The other two (brain precipitate 1 and 3)
only exhibited a yellow colored solution.

After subjecting all the three brain precipitates in qualitative analysis of lipids, we
now had identified the three brain precipitates per se. The brain precipitate 1 is
cerebroside. Cerebrosides are glycosphingolipids that contain a sugar residue, usually
glucose or galactose, having been glycosidically bonded to the primary alcohol of
ceramide. We’ve observed here that cerebrosides were actually soluble in both acetone
and hot alcohol, but insoluble in ether. As what was observed, precipitate 1 has white
dried crystals. Apart from these facts, if we’re going to talk about the molecular structure
of cerebrosides

B. Qualitative Analysis of Lipids

In qualitative analysis of lipids, there are some preliminary tests and specific tests
that are done in order to detect the presence or absence of lipids on the basis of color
change, and also to classify the certain characteristic groups of lipids based on their
reactivity with certain reagents.

Acrolein test is used to detect the presence of glycerol and fat in the samples. By
doing Acrolein test, we observed that all of our samples exhibit a pungent smell. For an
instance, in our observations with the following sample: Glycerol smells like rotten egg
white; coconut oil smells like a used oil; olive oil has a sweet smell; lecithin has this smell
which can be compared to a Sampaloc due to the sourness of the smell; and lastly,
cholesterol, which exhibits a strong pungent smell of alcohol. Acrolein test is based on
what is called dehydration reaction wherein the water molecules are removed from the
glycerol by adding a reagent, which in this case is potassium hydrogen sulphate. The
reaction between glycerol and potassium hydrogen sulphate results in the formation of
“Acrolein”. Acrolein is characterized by exhibiting pungent smell. Thus, since all of our
samples exhibit pungent smell or some kind of an irritating odor, we could say that glycerol
was being detected in all of our samples.

Test for unsaturation is used to detect the presence of unsaturated fatty acid or the
amount of double bonds present in that certain lipid sample by undergoing addition
reaction with iodine. To do detect the presence of unsaturated fatty acids or double bonds,
we utilized Hanus reagent in this experiment. In our observations, we noticed that
glycerol, coconut oil, and cholesterol only need 2 drops to decolorized while cod liver
needs 3 drops to decolorize, olive oil needs 4 drops, and lastly, tocopherol needs 5 drops
to decolorize. This just means that iodine reacts with the double bond/s found in the
structure of fatty acids. When iodine is taken up by a double bond of unsaturated fatty
acids, the unsaturated fatty acid per se becomes a saturated one. So, if the lipid contains
more unsaturated fatty acids or more double bonds that means, it will take more iodine.

Test for phosphorous is used to detect the presence of phosphates in the acidic
solution. The presence of phosphate in the hydrolysate can be detected by molybdate
test. In our experiment, we observed that only lecithin yielded a yellow precipitate while
the rest samples (glycerol, coconut oil, 0.1% bile, cholesterol, cod liver oil, tocopherol,
precipitates 1,2, and 3) yielded a white precipitate. This means that among the samples,
only lecithin had yielded a positive result for the test for phosphorous. The test for
phosphorous is done when lecithin is hydrolyzed in acidic medium, which then results to
breaking of both fatty ester bonds and phosphate ester bonds and releasing free fatty
acids and inorganic phosphate.

Liberman- Burchard test is used to detect the presence of cholesterol by using

strong concentrated acids such as sulfuric acid and acetic anhydride, which will then act
as dehydrating and oxidizing agent. In our experiment, we observed each sample has
different colors but still shows some shades of green. For olive oil, the color was yellow
green; for lecithin, the color was brownish green; for cholesterol, the color was dark green;
for brain precipitate 1, the color was green; for brain precipitate 2, the color was very light
green; for brain precipitate 3, the color was light green; lastly, only 0.1% bile is immiscible,
meaning there was no changes that was observed. From these observations, we can say
that aside from 0.1% bile, all the samples that was mentioned have yielded a positive
result since they all exhibit green colored solution. The reason for having a green colored
solution as positive result it’s because of the hydroxyl group (-OH) of cholesterol reacting
with the reagents and increasing the conjugation of the unsaturation in the adjacent fused
ring.

Carr- price is used to detect the presence of vitamin A and related carotenoids.
Since the group 7 is the one who is the assigned reporter, they are the only ones who
performed the experiment itself. According to their observations, the coconut oil has a
semi-orange to light red color; the olive oil has a dark to blue-violet color, and the cod
liver oil has a light blue to pinkish-red color. This means that only olive oil and cod liver
oil yielded a positive result. The use of chloroform is required to dissolve vitamin A, and
antimony trichloride will react with vitamin A, which will then result to the appearance of
blue color, which indicates a positive result

Modified- Furter Meyer test is used to detect vitamin E and tocopherols. In our
experiment, we observed that lecithin yielded a yellow mixture while the 0.1% bile yielded
a pale-yellow solution. On the other hand, cholesterol yielded a very light- yellow mixture
while cod liver oil yielded a golden- yellow colored mixture. Lastly, tocopherol has no color
and only yielded a clear solution. This means that with the exception of tocopherol,
lecithin, 0.1% bile, cholesterol, and cod liver oil yielded a positive result for carotenoids.
If tocopherols were detected to any of the samples, the result of this test would be bronze-
red solution.

Molisch test is used to detect the presence of carbohydrates. In our experiment, it

was observed that in lecithin, 0.1% bile, and brain precipitate 1, the color of the mixture
is yellow; in cod liver, it was observed that the color of the mixture was reddish brown; in
tocopherol, the color of the mixture is very light green; in brain precipitate 2, the color of
the mixture that was observed was orange; in brain precipitate 3, the color of the mixture
exhibits is very light yellow; and lastly, the 0.1% was observed to have a colorless mixture.
This means that none of the samples yielded a positive result for this test since the
positive result should have purple product at the interface of the two layers. The
compound responsible for this is carbohydrate since dehydration of the carbohydrate by
sulfuric acid to produce aldehyde, which then condenses with the phenolic structure.

Lastly, emulsification test is used to detect the presence of polar and nonpolar
groups in lipids, specifically in bile and lecithin. In this test, we observed that 0.1% bile
salt solution has a clear solution with small patches of bubbles and a few huge patches
of bubbles when viewed under a 40x magnification in the microscope. Tiny crystal of
cholesterol, on the other hand, has huge patches of bubbles, and the solution was clear;
1% aqueous solution of lecithin has a lot of small patches of bubbles and few quite huge
patches of bubbles too; precipitate 1 was observed to have a formation of residues
because the precipitate didn’t dissolve in olive oil; the precipitate 2 was observed to have
its precipitate settled at the bottom; and lastly, precipitate 3 was observed to have a
suspension of precipitate in the solution. The process of Emulsification stabilizes the
water and oil emulsion by the help of emulsifying agents. The lipid or oil in water appears
on the top of the water because of the high surface tension of water which gets together
to form a separate layer. An addition of emulsifying agents such as bile salts, soaps, and
etc. will enable the formation of a separate layer or a homogenous water- lipid mixture.

VI. CONCLUSION
ISOLATION OF LIPIDS:
In conclusion:
- Organic solvets such as acetone, ether, and hot alcohol are used to isolate lipids such
as cerebroside, phospholipid, and cholesterol from brain tissue.

- Precipitate 1 is Cerebroside because cerebrosides are soluble in both acetone and hot
alcohol but is insoluble in ether
- Precipitate 2 is Phospholipid because phospholipids are soluble in both ether and hot
alcohol but is insoluble in acetone

- Precipitate 3 is cholesterol because it is soluble in both ether and alcohol.

QUANTITATIVE ANALYSIS OF LIPIDS

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https://www.khanacademy.org/science/biology/macromolecules/lipids/a/lipids

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https://www.ams.usda.gov/sites/default/files/media/tocopherols%20report%202015.pdf

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