Activity 4

Simple Lipids

AGANAN, Jeremiah; AMBAT, Mark Clien;

BAGA, Mark Jusen Nicole; FLORES, Ethan Kriel; CACHO, Raphael

Saint Louis University

School of Natural Sciences

Department of Pharmacy

Abstract:

A lipid is a fat-soluble molecule. To put it another way, lipids are insoluble in water
but soluble in at least one organic solvent. The other major classes of organic compounds
(nucleic acids, proteins, and carbohydrates) are much more soluble in water than in an
organic solvent. Lipids are hydrocarbons (molecules consisting of hydrogen and oxygen),
but they do not share a common molecule structure. Lipids thus include fats, oils, waxes
and related compounds. There are two major lipid classes. Simple lipids are esters of
fatty acids and alcohols. Triacylglycerol’s/ glycerides and waxes are examples.
Triglycerides acts as energy reserve and structural padding/ support to organs whereas
waxes finds their use in manufacturing of polishes, candles and varnishes. While
compound lipods are esters of fatty acids with alcohols and other groups, such as
phosphate, carbohydrate, and nitrogenous compounds. Example of compound lipids are
Cephalins, Lecithins, and Glycoproteins. In this experiment the objective was to
determine the lipids prescence in the following test which is Solubility test, Spot test,
Iodine absorption test, Acrolein test, and Formation of insoluble Test for soap, which
included: Surface tension, Salting out soaps, and Formation of insoluble soap test. On
the experiment there were samples given to use, which is Lanolin, Cottonseed oil, Lard
and test on the following procedures on determining the presence of lipids and also
observing their chemical properties. After doing the following procedures, there were
chemical tests, chemicals that yielded negative result and others yielded positive, This
means that the following samples that yield positive have lipids.

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Introduction:

Lipids can be more formally defined as substances such as a fat, oil or wax that
dissolves in alcohol but not in water. Lipids contain carbon, hydrogen and oxygen but
have far less oxygen proportionally than carbohydrates. Lipids are an important part of
living cells. Together with carbohydrates and proteins, lipids are the main constituents of
plant and animal cells. Cholesterol and triglycerides are lipids. Lipids are easily stored in
the body. They serve as a source of fuel and are an important constituent of the structure
of cells. Lipids, include fatty acids, neutral fats, waxes and steroids like cortisone.
Compound lipids (lipids complexed with another type of chemical compound) comprise
the lipoproteins, glycolipids and phospholipids.

Structure of Lipids:

According to Bailey (2018) Lipids are made of the elements Carbon, Hydrogen and
Oxygen, but have a much lower proportion of water than other molecules such as
carbohydrates. Unlike polysaccharides and proteins, lipids are not polymers, they lack a
repeating momomeric unit. They are made from two molecules, Glycerol and Fatty Acids.
A glycerol molecule is made up from three carbon atoms with a hydroxyl group attached
to it and hydrogen atoms occupying the remaining positions. Fatty acids consist of an
acid group at one end of the molecule and a hydrocarbon chain, which is usually denoted
by the letter R.

They may be saturated or unsaturated. A fatty acid is saturated if every possible

bond is made with a Hydrogen atom, such that there exist no C=C bonds. Saturated fatty
acids on the other hand do contain C=C bonds. Monounsaturated fatty acids have one
C=C bond, and polyunsaturated have more than one C=C bond.

Classification of Lipids:

Lipids can be classified according to their hydrolysis products and according to

similarities in their molecular structures. Three major subclasses are recognized simple
lipids, Fats and oils which yield fatty acids and glycerol upon hydrolysis, Waxes, which
yield fatty acids and long-chain alcohols upon hydrolysis. Fats and Oils are compound
lipids (Aryal, 2018).

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 Phospholipids, which yield fatty acids, glycerol, amino alcohol sphingosine,
phosphoric acid and a nitrogen-containing alcohol upon hydrolysis. They may be
glycerolphospholipids or sphingophospholipids depending upon the alcohol group
present (glycerol or sphingosine). Glycolipids, which yield fatty acids, sphingosine or
glycerol, and a carbo-hydrate upon hydrolysis. They may also be glycerolglycolipids or
sphingoglycolipids depending upon the alcohol group present (glycerol or sphingosine).

Properties of Lipids:

Lipids may be either liquids or non-crystalline solids at room temperature. Pure

fats and oils are colorless, odorless, and tasteless. They are energy rich organic
molecules and insoluble in water and soluble in organic solvents like alcohol, chloroform,
acetone, benzene. No ionic charges, solid triglycerols (Fats) have high proportions of
saturated fatty acids. Liquid triglycerols (Oils) have high proportions of unsaturated fatty
acids. In this experiment the analysts must observe and determine the presence of lipids
in the following procedures using samples to test them, which is cottonseed oil, lard,
linseed oil, and soap solution which is used to test for soaps. At the end of the experiment
the analyst must able to obtain information and results regarding on the reaction of certain
samples to the chemical tests conducted to test for the presence of lipids.

Experimental Procedures:

For this activity, we use the following reaents which is ethyl alcohol, chloroform,
Hubl’s solution (2.6 grams of iodine (I2) and 3 grams of mercuric chloride (HgCl2) in 100
milliliter of 95% ethanol). Potassium bisulfites, saturated sodium chloride solution, 10%
hydrochloric acid (HCl), calcium chloride test solution and magnesium sulfate.

The samples that was used are coconut oil, lard, cottonseed oil, glycerol, linseed
oil, lanolin and commercial soap with the use of the following materials which is pipette,
spot plate, filter paper and bunsen burner.

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 The test that was conducted is five, this includes the following which is Solubility,
Spot Test, Iodine Absorption Test, Acrolein Test and Test for soap (Surface Tension,
Salting-out of soaps and Formation of Insoluble soap).

A. Solubility Test

The first test is solubility test wherein the sample given was Coconut oil; the group
took three dry test tubes. To the first test tube the group added 2 mL of distilled water, to
the second the group added 2 mL of ethyl alcohol and to the third was 2 mL of chloroform.
To the each of the three test tubes the group added 3 groups of coconut oil. The
observation of the solution was noted.

B. Spot Test

Next is the spot test with the given sample of Lead, Cottonseed and Glycerol; the
group placed a drop of the material on a piece of paper and allowed to dry in every
sample. The observation of the transparency was noted.

C. Iodine Absorption Test

Then the Iodine Absorption Test use a sample of Cottonseed oil, Coconut oil and
Linseed oil; the group dissolved the test material in 1mL of chloroform, then added Hubl’s
solution of 2.g I2 and 3 g HgCl2 in 100mL of 95% ethanol drop by drop, shaking the test
tube after each addition until the Hubl’s solution was no longer decolorized. The
observation was recorded according to the number of drops used for each sample of the
solution was noted.

D. Acrolein Test

In Acrolein Test, the sample given was Lanolin, cottonseed oil and Lard; the group took
a clear test tube and added four drops of the sample. Then added a pinch of potassium
bisulfite and heated vigorously. After, the group noted the smell of the fumes of the gas
which came from one of the test tubes. The observation was noted.

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 E. Test for Soap

For the Test for Soap, there were three tests conducted, test for surface tension,
salting-out of soap and formation of insoluble soap.

E.1. Surface Tension

For surface tension, wi will be using a clean and dry one mL pipette and recorded the
number of drops that formed from 1 mL of pure distilled water. Then, they rinsed the
pipette with the prepared soap solution. The observation was recorded according to the
number of drops from 1mL of the soap solution. Then, without rinsing the pipette, it was
filled with pure distilled water. The observation was recorded again according to the
number of drops.

E.2. Salting-out of Soaps

For the salting-out of soaps, the researcher took twenty mL of the soap solution in a
large test tube then added 10 mL of the saturated solution sodium chloride solution. The
precipitation formed was noted. The group collected the precipitate on a filter paper,
drained and pressed out the liquid. The frothing was dissolved then it was acidified with
10% of hydrochloric acid (HCl). The observation of the precipitation was noted.

E.3. Formation of Insoluble Soap

For the formation of the insoluble soap, the researcher took 5mL of the soap solution
the added a few drops of calcium chloride test solution, an insoluble soap was precipitated
out. In another test tube, the researcher took 5mL of the soap solution the added a few
drops of magnesium sulphate solution, an insoluble soap were precipitated out.

Results and Discussion:

1. Solubility Test

Lipids are nonpolar organic compounds including fats, oils, hormones, and certain
components of membranes that are grouped together because they do not interact
appreciably with water. Solubility of a substance depends on a simple rule “like dissolves

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like” this statement indicates that a solute will dissolve best in a solvent that has a similar
chemical structure to itself. The overall salvation capacity of a solvent depends primarily
on its polarity. Solubility test is based on the property of lipid to dissolve in different
solvents. Lipids are readily miscible in non-polar solvents like chloroform, partially soluble
in a polar solvent like ethanol and immiscible in a polar solvent like water. The sample
given was cottonseed oil which was dissolved in different solvents such as distilled water,
ethyl alcohol, and chloroform. The cottonseed oil is immiscible with distilled water.
Cottonseed oil is partially miscible with the second solvent which is ethyl alcohol.
Cottonseed oil is miscible with the last solvent which is chloroform. Therefore both the
ethyl alcohol and chloroform yielded a positive result while distilled water yielded a
negative result.

SAMPLE AND SOLVENT PICTURES OBSERVATION

Cottonseed oil in distilled Immiscible
water

Cottonseed oil in ethyl Partially miscible

alcohol

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 Cottonseed oil in Miscible
chloroform

2. Spot Test

Spot test is done to differentiate between fixed oils and volatile oils. However,
compared to volatile oils, which are also liquid at room temperature, they are non-volatile.
Thus, the paper remained stained with a fatty substance that have come in contact with
it. A spot test is also a preliminary test for the lipids which can be detected by the
appearance of a translucent and greasy spot. Most grease or fat have a high boiling point.
So, they are non-volatile. In room temperature, the spot of water can absorb enough heat
from the air and evaporate. But the spot of grease can never absorb enough heat to
evaporate.

SAMPLE PICTURES OBSERVATION

Lard Translucent spot is
observed
Cottonseed oil Translucent spot is
observed
Gycerol Translucent spot is
observed

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 3. Iodine Absorption Test

This test aims to analyze the amount that was absorbed by the iodine at the double
bonds until all the double bonds are saturated with iodine. In addition to that, the greater
the value of the sample means that it is more susceptible to oxidation. According to T.H.
Sanders, the amount of iodine that was required for each sample to impart the color from
Hubl’s solution (mixture of iodine, ethanol, and mercuric chloride) thus enables the
researcher to measure the degree of unsaturation for fatty acids. The fatty acid will react
with a halogen due the presence of double bonds and the reaction is called addition
reaction. In this procedure, the iodine that was absorbed will be determined by the drops
that were introduced after the solution has no longer decolorized. This reaction is the
basis for the analytical parameter, iodine number which is official in the USP.

For this test, only two samples are used due to unavailability of coconut oil. The
cottonseed and linseed have shown a distinct color (Orange) after a few drops. The
cottonseed produced a yellow cloudy solution after 100gtts of Hubl’s solution while the
linseed produced a yellow solution after putting 150gtts of Hubl’s solution. We therefore
conclude that between the two samples, the linseed has a higher iodine value (IV)
therefore the degree of unsaturation is higher. Thus the linseed is more susceptible to
oxidation, compared to linseed which has an iodine value of 100.

SAMPLE PICTURE OBSERVATION

COTTONSEED 100gtts of Hubl’s solution
produced a yellow
coloration in a cloudy
solution.

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 LINSEED 150gtts of Hubl’s solution
produced a yellow
coloration in a cloudy
solution.

4. Acrolein Test

The test is based on dehydration reaction where the water molecules removed from
the glycerol by the addition of reagent potassium hydrogen sulfate. The reaction between
glycerol and potassium hydrogen results in the formation of acrolein that is characterized
by a distinct smell. Acrolein test is used to detect the presence of glycerol or fat. When
fat is treated strongly in the presence of a dehydrating agent such as potassium bisulfate
(KHSO4), the glycerol portion of the molecule is dehydrated to form an unsaturated
aldehyde, acrolein that has a pungent irritating odour (Malubay, N. D). Acrolein is a
volatile, highly reactive α,β-unsaturated aldehyde that is produced by many mechanisms
relevant to human exposure. Acrolein also is known to elicit several different biochemical
responses when exposed to cells from transcription factor activation to cell death
(Zemsky, 2017). The test is conducted by taking pure glycerol in a dry test tube, adding
it with a few crystals of potassium bisulphate and heating it vigorously, and then it
produces a very pungent odour of acrolein. The removal of water from glycerol by
potassium bisulphate is what causes the acrolein to form (Kumar, n.d.). In this
experiment, a dehydrating agent was added to the sample oils which are lanolin, lard,
and cottonseed oil are then heated. The samples that have been subject to testing, all
resulted to pungent odor which yield a positive result, meaning there is a presence of
glycerol, or fat in the sample. If no pungent smell was produced, glycerol or fat is absent
in the sample (Supriya, N. D.).

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 Sample Observation
1. Lanolin Burnt candle odor

2. Cottonseed oil Burnt lard odor

3. Lard Odorless

5. Test for Soap

Saponification or known as soaps s a process by which triglycerides are reacted with

sodium or potassium hydroxide (lye) to produce glycerol and a fatty acid salt, called
"soap." The triglycerides are most often animal fats or vegetable oils. When sodium

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hydroxide is used, a hard soap is produced. Using potassium hydroxide results in a soft
soap. The reaction is used commercially to make soap, lubricants, and fire extinguishers.
Lipids that contain fatty acid ester linkages can undergo hydrolysis. This reaction is
catalyzed by a strong acid or base (Helmenstine, 2019).

Saponification is a process by which triglycerides are reacted with sodium or

potassium hydroxide (lye) to produce glycerol and a fatty acid salt, called "soap." The
triglycerides are most often animal fats or vegetable oils. When sodium hydroxide is used,
a hard soap is produced. Using potassium hydroxide results in a soft soap. Saponification
is the name of the chemical reaction that produces soap. In the process, animal or
vegetable fat is converted into soap (a fatty acid) and alcohol. The reaction requires a
solution of an alkali (e.g., sodium hydroxide or potassium hydroxide) in water and also
heat. The reaction is used commercially to make soap, lubricants, and fire extinguishers
(Helmenstine, 2016).

A. Surface Tension

Water holds unique properties which makes it "sticky" at the surface. Each individual
water molecule has one large oxygen atom and two smaller hydrogen atoms. The
hydrogen atoms hold a slightly positive charge, making the entire water molecule polar.
Like tiny magnets, the hydrogen atoms attract the oxygen atoms from other water
molecules, creating temporary hydrogen bonds within the water.Each water molecule
experiences a pull from other water molecules from every direction, but water molecules
at the surface do not have molecules above the surface to pull at them. These water
molecules have more pull from the water below than the surface above. This difference
in force packs the water molecules at the surface closer together than they are inside the
liquid. The thin, dense layer of molecules produces the phenomenon called surface
tension.

Detergent molecules' two ends make it able to break through the surface tension of
water. The end of the detergent molecule which attaches to fat (grease) repels water
molecules. It is known as hydrophobic, meaning "water fearing." By attempting to move
away from the water molecules, the hydrophobic ends of the detergent molecules push

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up to the surface. This weakens the hydrogen bonds holding the water molecules together
at the surface. The result is a break in the surface tension of the water.

SAMPLES RESULTS
1. Distilled Water

Drops: 20gtts
2.Soap Solution

3. Distilled water with soap solution

Drops : 56gtts

In the results we get on testing the surface area for test for soap, we can see that
distilled water with soap solution has more drops because it is mixed with water and
also , it produces bubbles that makes the soap and water stand up, while the other two
distilled water and soap solution is much lesser than the distilled water with soap
solution, That is because thre was no reaction unlike the distilled water with soap
solution.

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 B. Salting-out of Soaps

Salting out is also known as salt-induced precipitation, salt fractionation, anti-solvent

crystallization, precipitation crystallization, or drowning out. It is an effect based on the
electrolyte and non-electrolyte interaction, in which the non-electrolyte could be less
soluble at high salt concentrations. It is used as a method of purification for proteins, as
well as preventing protein denaturation due to excessively diluted samples during
experiments. The salt concentration needed for the protein to precipitate out of the
solution differs from protein to protein. This process is also used to concentrate dilute
solutions of proteins. Dialysis can be used to remove the salt if needed. The principle in
salting-out of soaps is that salt compounds dissociate in aqueous solutions. This property
is exploited in the process of salting out. When the salt concentration is increased, some
of the water molecules are attracted by the salt ions, which decreases the number of
water molecules available to interact with the charged part of the protein. There are
hydrophobic amino acids and hydrophilic amino acids in protein molecules. After protein
folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic
areas while hydrophilic amino acids interact with the molecules of solvation and allow
proteins to form hydrogen bonds with the surrounding water molecules. If enough of the
protein surface is hydrophilic, the protein can be dissolved in water (Garett, 2013).

SAMPLE RESULT
1. Soap with saturated chloride solution

In the experiment , it formed a white precipitate which means that the soap reacted
with the saturated chloride solution, and also it was acidify on 10% hydrochloric acid
(HCL).

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 C. Formation of Insoulble Soap

Soap is a mixture of sodium salts of various naturally occurring fatty acids. Air bubbles
added to a molten soap will decrease the density of the soap and thus it will float on water.
If the fatty acid salt has potassium rather than sodium, a softer lather is the result. Soap
is produced by a saponification or basic hydrolysis reaction of a fat or oil. Currently,
sodium carbonate or sodium hydroxide is used to neutralize the fatty acid and convert it
to the salt (Libretexts, 2019). Formation of insoluble soap test will conclude positive for
both sample calcium hydroxide TS (CaOH), and magnesium sulfate (MgSO4). Thus this
test is specific test for calcium hydroxide TS (CaOH), and magnesium sulfate (MgSO 4).
The calcium hydroxide (CaOH) sample produced a cloudy solution, and the sample
magnesium sulfate (MgSO4) produced a layer solution. Esters can be hydrolyzed by alkali
to yield the parent alcohol and salt. When the fatty acid possesses a long chain the salt
formed is a soap which we commonly use. This process is called saponification. Oils and
fats usually contain long chain fatty acids and are, therefore, the starting materials for the
preparation of soap. Take 1 ml of the oil in a test tube and add an equal amount of
alcoholic KOH solution, mix them thoroughly and keep the mixture during the course of
warming and shake up gently with a little distilled water. Appearance of some oil drops
will indicate the incomplete saponi-fication. After complete saponification no oil drops will
appear (Parul, 2019).

Sample Observation Documentation

1. Soap solution with Became cloudy upon
calcium chloride TS addition of the reagent

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 2. Soap solution with Forms a layer of solution of
magnesium sulfate the reagent

Conclusion:

We therefore conclude that the following experiments that were conducted enabled
us to determine the presence and characteristics, as well as the chemical properties of
soaps as form of lipids and simple lipids. The analyst became more knowledgeable about
the presence of lipids in the following experiments conducted. Lipids are fatty, waxy, or
oily compounds that are truly “hydrophobic” and insoluble in polar solvents such as water,
but soluble in organic solvents. Based on the results of the tests, the following conclusion
were made. For the Solubility test, Cottonseed oil is miscible in Ethyl alcohol and
Chloroform but immiscible in distilled water. Fatty acids are immiscible in distilled water
because they are composed mostly of hydrocarbon chains which are insoluble in water
and the carbon and carbon to hydrogen bonds found in lipids are considered non-polar.
Following the solubility principle which is, “like dissolves like”, cottonseed oil is partially
miscible in Ethyl alcohol and miscible in chloroform because both are non-polar organic
compounds. For the Spot test, all the three samples resulted positive wherein there is an
appearance of translucent and greasy spot which is evident in the filter paper. Most
grease or fat have a high boiling point, so they are non-volatile. The spot of grease can
never absorb enough heat to evaporate. When the liquid is inside the sheet of paper, it
diffracts light. So, light can pass from one side of the paper to another side which results
to a translucent effect. For the Iodine Absorption Test, the samples change in color upon
the addition of the Hubl’s solution and turns to clear/transparent and pale yellow color.
The more unsaturated, multi-bonded the lipid is, the more it absorbs the iodine. The less
iodine it absorbs, the lipid is taken for to be saturated, single bonded. For the Acrolein

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Test, Lanolin, Cottonseed oil and Lard yielded a positive result of burnt cooking oil smell
or pungent smell. This means that these samples contain glycerol. For the Tests for
Soaps, three tests have been performed. The first test for soap which is the Surface
Tension test showed that plain tap water produces a much larger, stable drop of water
than the soapy water does which resulted in lower number of drops to amount into 1 ml.
This is because plain tap water has higher surface tension, so the surface is "stronger"
and can hold together a larger drop. Adding soap lowers the water’s surface tension so
the drop becomes weaker and breaks apart sooner. For the Test for Salting-out of Soaps,
a precipitate is formed upon the addition of Hydrochloric acid. When solutions of sodium
chloride and hydrochloric acid are mixed, sodium chloride precipitates. This is because
hydrochloric acid creates an excess of chloride ions in the solution which takes the sodium
with them. For the Test for Insoluble soaps, both sample which is the Ca2+ and Mg2+
yielded a negative result of not having a precipitate upon the addition of metal cations.
This is due to using synthetic detergents for the soap solutions. Synthetic detergents may
be soluble in both acidic and alkaline solutions and don't form insoluble precipitates in
hard water. Lipids are soluble in non-polar solvents and insoluble in polar solvents, have
high boiling point and low volatility, can either be saturated or unsaturated depending on
the bonds, and they contain glycerol. The metallic salts of fatty acids are called soaps.
They are surface active agents which breaks surface tension. They are produced by the
reaction of fatty acids and Sodium hydroxide or Potassium hydroxide, which is called
saponification. These soaps react with Ca2+ and Mg2+ to form insoluble salts. The
different tests performed in this experiment determined the chemical properties of simple
lipids and of soaps as metallic salts of fatty acids.

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