Solubility test

The first test is the solubility test in which it aims to detect the presence and solubility of
lipids in the given samples/ to see if they would mix together with the reagents added. In
this case, the test sample which is the cottonseed oil is tested with different reagents
with different polarities. Consequently, the best way to tell if something will dissolve is to
look at the polarities of the solvent and the solute. If the polarities of the solvent and
solute match, then the solute will probably dissolve. If the polarities of the solvent and
solute are different, the solute probably won’t dissolve.

Table

2 drops of cottonseed oil was added to each of the 1 mL solvents distilled water, ethyl
alcohol, ether, 5% hydrochloric acid, and 5% sodium hydroxide.

For the polar reagents, we have the distilled water, 5% HCI (hydrochloric acid) solution,
and 5% sodium hydroxide solution which are all polar solvents that resulted to two
distinct layers that are present in the sample and the sample was immiscible. This is
because of the polarity of the samples does not mix well together as you can see from
table ,cottonseed oil contains nonpolar bonds and the other containing polar bonds
which explains the two distinct layers present in the sample. If you will notice, the NaOH
(sodium hydroxide) solution produced a cloudy appearance, this is because the
cloudiness produced in the 5% sodium hydroxide could be related in the ability of the
sodium hydroxide to dissolve oils and turn them into soap through a process called
saponification. The use of the amount of sodium hydroxide that is lacking in the
saponification reaction will cause the formation of residual fatty acids (oil) after the
reaction/ which explains the cloudiness of the solution.

For ether, the sample produced a uniformed face which indicated that ether is miscible
to the cottonseed oil. This is because ether is nonpolar and cottonseed oil is nonpolar,
thus, creating a uniformed face.

For the 95% ethyl alcohol, the case is different, this is because ethanol is both polar and
nonpolar. The observation for ethyl alcohol and cottonseed oil has something to do with
the miscibility, molecular similarity, and polarity of both samples Since both samples
have parts with no charge, they are similarly enough to not repel each other and evenly
mix together because molecules of oil and alcohol have similar enough polarities that
does not repel each other enough to separate /which explains why ethyl alcohol formed
a uniformed face appearance with the cottonseed oil.

Result

As you can see cottonseed oil is immiscible to the given polar substance like
distilled water, 5% Hydrochloric acid and 5% Sodium hydroxide since cottonseed oil is
nonpolar. While cotton seed oil is miscible to both ether and 95% ethyl alcohol since
their polarities match. But for the 95% ethyl alcohol, the case is different, this is because
ethanol is both polar and nonpolar.

Second table

The second table is still related to the solubility test but only 95% ethyl alcohol and ether
were used as reagents to clearly distinct their polarity components of the reagents to the
cottonseed oil.

The same concept earlier about ethyl alcohol and ether may be applied in this table in
which the molecules of oil and alcohol have similar enough polarities that does not repel
each other enough to separate/ and ether is nonpolar which easily dissolved in the
cottonseed oil. The results of the observations are related with the miscibility properties
of the cottonseed oil. Cottonseed oil is slightly miscible in ethanol (95%) and miscible in
ether. When ethyl alcohol was added to the cottonseed oil, wet oily segment in the
middle of the layer was observed because the oil did not incorporate well with the
alcohol which indicated that ethyl alcohol is slightly soluble in cottonseed oil. Ether on
the other hand when added to the cottonseed oil resulted in drying up the majority of the
area where the sample was set. This indicates that ether is soluble to the cottonseed oil.
Result

Cottonseed oil is slightly miscible in ethanol (95%) and miscible in ether. The
same concept about ethyl alcohol and ether may be applied in this, in which the
molecules of oil and alcohol have similar enough polarities that does not repel each
other enough to separate, and ether is nonpolar which easily dissolved in the
cottonseed oil.

Test for Unsaturation

The test for unsaturation aims to detect the amount of presence of double bonds in the
lipid samples.

There are two types of reagents added in the different test samples to see if the
presence of double bonds is present in the lipid sample.

Table

The first reagent is the bromine in carbon tetrachloride (Br2 in CCI4) which is the
reagent used in the bromine’s test in which a colorless solution would result to a positive
result because of the presence of unsaturated compound in the sample (www.django-
wiki.org, 2021).

When oleic acid was tested with the bromine in carbon tetrachloride reagent, it
produced a colorless solution which indicates that it tested positive for the bromine’s
test. This is because oleic acid is an example of a monounsaturated fatty acid that
contains only 1 double bond which is why it tested positive in the bromine’s test.

Reaction
Oleic acid molecules have an unsaturated double bond with the chemical properties of
single unsaturated carboxylic acid. It is capable for having addition reaction bromine.
Bromine atoms in the Br2 molecules are capable of adding across the double bonds.
This type of addition is known as halogen-addition or halogenation. When this occurs,
the orange-red colour of the bromine solution is decolourised. In this case, oleic acid
(unsaturated fatty acid) undergoes addition with bromine solution.

For the second test

For the other 3 samples in the test for unsaturation, it was tested with Chloroform
(CHCI3) in Hanus Iodine to see if the given samples contain double bonds/unsaturated
bonds.

The indicator for the iodine test is that when the sample remained in pink color, it
means that there are no unsaturated bonds present in the sample and only saturated
bond is present. On the other hand, when the sample’s pink color faded away, it
indicates that there are unsaturated bonds present in the sample. The three sample
resulted in a pink color which means that they all contain saturated bond when with
Chloroform (CHCI3)and Hanus Iodine was added.

As you noticed, the oleic acid turned into saturated bonds compared to the bromine test
wherein it contains unsaturated bonds. This has something to do with the reagent
added which is the iodine. This is because a greater amount of iodine will bind to a
sample that has a greater proportion of unsaturated fatty acid (5m Editor, 2011). This
would result to the unsaturated fatty acid to become saturated fatty acid by taking up
molecules of iodine, which explains why the three samples tested negative in the iodine
test.

It is also good to take note that palmitic acid and coconut oil are already a saturated
bond, so there would be no effect when iodine is added. Hence, it would still remain
saturated which explains the pink color of the sample that indicated the presence of
saturated bond which tells that the samples tested negative for the unsaturation test.
Additional info: Coconut Oil has almost no double bonds, making it nearly 95%
saturated fats (that's almost as close to 100% that the natural earth wants to be).
General Reaction
Fatty acids react with a halogen [iodine]  resulting  in the addition of the halogen
at the C=C double bond site.  In this reaction, iodine monochloride  reacts with the
unsaturated bonds to produce a di-halogenated single bond, of which one carbon has
bound an atom of iodine.

Chemical reaction of triglyceride

An example of triglyceride occurring in the saponifiable fraction of oils with
a saturated fatty acid residue, a monounsaturated fatty acid residue and a triple
unsaturated (polyunsaturated) fatty acid residue. The triple esterified glycerol (marked
black) can be seen in the center of the structure. Such a triglyceride has a high iodine
value (approx. 119). Below, the reaction product after the addition of four equivalents of
iodine or bromine to the four C = C double bonds of the unsaturated fatty acid residues.

NEXT SLIDE PLEASE

Emulsifying Action

Emulsification, or to emulsify something, is defined as the mixing of two liquids

that usually are unmixable together to form an emulsion.

Emulsifying agents emulsify the lipid, after which the lipids appear as tiny
droplets suspended in the solution. A positive result would give a precipitate and will
form an emulsion.
Table

In each of 2 test tubes, 5 drops of 2% albumin solution was placed. In test tube 1
a pinch of cholesterol was added. So more particles of cholesterol are visible and
formed an insoluble solution. The clumps or tiny dispersed droplets or globules
suspended in the sample is an example of emulsion which means that it tested positive
for the emulsifying action test and lipids are present in the sample.

For the 2% albumin solution mixed with cholesterol and lecithin, the color of the
sample turned into yellow and lesser particles are present in the sample because the
cholesterol incorporated with the albumin solution which indicates that the sample
tested negative in the emulsifying action test because no emulsion was formed. The
color yellow came from the lecithin which would not have any effect to the sample.
Lecithin is a good emulsifier that helped the 2% albumin solution and
cholesterol+lecithin reagents come together. Lecithin is a combination of phospholipids
and other minor substances like triglycerides and carbohydrates which are often used
for emulsifying, smoothing food textures, homogenizing liquid mixtures and repelling
sticking material.

Result

So as you can see cholesterol and 2% albumin are not well mixed together, more
particles and clumps of powdered cholesterol are still present in the sample and the two
samples are insoluble. It implies that the sample tested positive because it is an
example of emulsion.

On the other hand when cholesterol, lecithin and 2% Albumin was mixed
together there is the color of the sample turned into yellow because of the lecithin,
lesser particles are present and it is slightly soluble it implies that sample tested
negative in the emulsifying action test because there were no clumps present in the
sample and Lecithin is a good emulsifier.
 Reaction
Lecithin (phosphatidylcholine) is, along with cholesterol, the third major lipid
constituent in bile. Lecithin is an amphiphilic compound whose head groups are
hydrophilic and water soluble and whose nonpolar tail groups are hydrophobic and fat
soluble. It is thus capable of forming soluble micelles with cholesterol which is, by itself,
essentially insoluble in water. The LCAT is the gene provides instructions for making an
enzyme called lecithin-cholesterol acyltransferase (LCAT). This enzyme plays a role in
removing cholesterol from the blood and tissues. 

AND NOW FOR THE QUESTIONS

Q1 Why is fatty acids insoluble in water?

Recalling the previous concepts that had been discussed, water is known to be
polar which means that it is capable of dissolving many ionic and polar substances.
However, nonpolar substances cannot be dissolved in water because they lack polar
functional groups which cannot form hydrogen bonds. Since fatty acids are known to be
nonpolar and are mostly composed of hydrocarbon chains, it would not dissolve in
water because water is polar and fatty acids are nonpolar which are incompatible to be
dissolved together. Fatty acid contains long hydrocarbon chains in which they are
described as hydrophobic, meaning that they are water fearing or simply nonpolar.

Q4 Show the structure of the parent compound of cholesterol.

Chemically, cholesterol is an organic compound belonging to the steroid family;

its molecular formula is C27H46O. In its pure state it is a white, crystalline substance that
is odourless and tasteless. Steroids consist of four fused rings (A, B, C, and D).
Chemically, these hydrocarbons are cyclopentano per hydro phenenthrenes. They
contain a five-membered cyclopentane (D) ring and the three rings of phenanthrene.
 Q5 Explain the cooperative effect of lecithin and albumin.

Lecithin is an emulsifier made up of about five smaller molecules that has a

backbone of glycerol that bond up to three other molecules. Lecithin is a molecule with
one end that its hydrophilic end dissolves in water and the hydrophobic end that
dissolves in oil droplets (Gavigan, 2010). It also contains a variety of ingredients which
suspends fats and oils and keeps them from mixing with other substances. Albumins on
the other hand are water-soluble proteins that have a massive polar molecule. Albumins
are made in the liver and its main role is to maintain the osmotic pressure of the blood
compartment, provide nourishment of the tissues, transport hormones, vitamins, and
other substances throughout the body (Cameron et al., 2020). Both albumins and
lecithin acts as good emulsifiers because they both have a polar and non-polar
molecule that aids in reducing the immiscibility of two substances.

AND NOW FOR THE Conclusion

In the solubility test, the polarity of the sample plays a big role in the solubility of the
solution in which the cottonseed oil is nonpolar which means it is only attracted to the
reagents that contain nonpolar bonds that resulted in a soluble mixture like ether and
95% ethyl alcohol while it is insoluble in distilled water, 5% Hydrochloric acid and 5%
Sodium hydroxide.

For the test for unsaturation

The test for unsaturation aims to detect the amount of presence of double bonds in the
lipid samples. For the test for oleic acid and bromine in carbon tetrachloride, it remains
a colorless solution which means that oleic acid contains an unsaturated carbon-carbon
bond.

For the test for palmitic acid, Oleic acid, and cottonseed oil added by chloroform and
Huble’s iodine reagent. The three samples resulted in a pink color which means that
they all contain saturated bond when with Chloroform (CHCI3) Hanus Iodine was
added.

Lastly, the purpose of emulsification is to stabilize emulsion state by preventing break

down. For this test, cholesterol and 2% albumin are mixed together, the two samples
are insoluble this happened because their polarity doesn’t match. On the other hand,
when 2% albumin, cholesterol and lecithin are mixed together it resulted in a slightly
soluble mixture. This happens because lecithin is a good emulsifier that helped the 2%
albumin solution and cholesterol reagents come together.