DEPARTMENT OF PURE AND APPLIED CHEMISTRY

Visayas State University, Baybay, Leyte

CHEM31a Biochemistry
Laboratory Report

Name : Mark Ryan R. Tripole Date Performed : 05/11/2015

Course/Yr : BS Chemistry II Date Submitted : 05/18/2015
Group No : 6 Score

Experiment No. 4
Lipids

OBJECTIVES
 Observe some physical and chemical properties of lipids
 Distinguish between saturated and unsaturated fats.
 Observe saponification of triacylglycerol in the preparation of soap
 Test for reactions of soap with soft water, oil and CaCl2
I. Results

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II. Discussion

INTRODUCTION

Lipids are biological molecules that contain hydrocarbons and essentially make up the
building blocks of the structure and the function of living cells. This particular class of compounds
are naturally occurring molecules that can be broken down into various classifications, the common
of which are fats, waxes, phospholipids, the mono- di- and triglycerides and a lot more. Lipids have a
whole range of functions, but the main biological functions of these lipids include serving as the
structural components of cell membranes, signaling, and storing energy. Aside from these biological
functions, lipids have also found their use in industries like in the food and cosmetics industry,
recently also more or less finding ground in the emerging lipid nanotechnology as well through the
development of what are known as solid lipid nanoparticles (SLN) that has seen use as a relatively
new pharmaceutical delivery system.
Explicitly speaking, lipids are a family of biomolecules that have the common property of
being soluble in organic solvents but not in water. The word lipid actually comes from the Greek
word lipos, meaning “fat” or “lard”. Typically, the lipid content of a cell can be extracted using a
nonpolar solvent such as ether or chloroform. Lipids are an important feature in cell membranes and
steroid hormones. Aside from the solubility of these compounds, there are also a few important
reactions lipids are involved in that are actually of great commercial value. One of these is the
saponification reaction, where triglycerides in fat react in heat with strong bases to form glycerol
and the metallic salts of the fatty acids, which is soap. This experimental procedure was basically
geared towards understanding the physical and chemical properties of lipids and how to differentiate
them based on their different properties. The final part of the experiment tasked the group to
produce soap through the saponification process using lard and run it through various tests in
comparison to a sample using a commercial brand of soap. These will all be discussed in turn in the
following sections of the laboratory report.

PART A – The Formation and Hydrolysis of a Triacylglycerol

The triglycerides or triacylglycerides are actually considered as blood lipids that enable the
two way transfer of adipose fat and blood glucose from the liver. Moreover, the fatty acids are
stored in the body as triglycerides. Shown below is a simple illustration of the general formula for a
triglyceride:

As shown in the figure drawn above, triglycerides are essentially triesters of glycerol (a
trihydroxy alcohol) and fatty acids. Looking closer at the structure of a triglyceride, the three

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hydroxyl groups of glycerol form ester bonds with the carboxyl groups of three fatty acids. For
generic purposes, the diagram in the previous page is draw showing three different R groups to
denote three different fatty acid chains, but the three groups can either all come from the same
fatty acid or a mixture of different ones. For example, glycerol and three molecules of lauric acid
form a triacylglycerol. Taking a closer look at the name, glycerol is named glyceryl and the fatty
acids are named as carboxylates. For example, the lauric acid is named laureate, which gives the
name glyceryl trilaurate or commonly known as trilaurin. The triglycerides can be formed in a
dehydration synthesis reaction (can also be considered as an esterification reaction) between the
glycerol and the fatty acids, and a simple illustration of this reaction is shown below:

Taking bodily functions as a focus example, the triglyceride formed are actually stored in the
body and only broken down when required. When the need arises, the triglycerides are transported
through the blood to the appropriate tissues (muscles, adipose, etc.) by what are known as
lipoproteins (these enable fats and cholesterol to move within the water-based solution of the
bloodstream). The triglycerides then undergo a process known as lipolysis which essentially breaks
them down into glycerol and free fatty acids. The lipolysis reaction is essentially a hydrolysis
reaction and is induced by various hormones in the body like epinephrine, norepinephrine, cortisol
and testosterone. If the formation of a triglyceride is considered as an esterification reaction, then
the lipolysis of a triglyceride into its individual components is the complete reverse. A simple
illustration of this is shown in the diagram below:

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 Given that the two reactions are reversible, a better way of illustrating the reaction would be
the one as shown in the diagram below:

PART B – Solubility of Some Lipids

Many of the biochemicals fall into categories based on their molecular structures. For
example, proteins are essentially compounds that are made up from smaller units called amino acids.
So by the same token, carbohydrates are to saccharides. Lipids, on the other hand, don’t have any
sort of common building blocks because by themselves they’re already quite varied in their chemical
composition. Instead, they are more or less defined based on their solubility. By definition, lipids are
compounds that are considered to possess poor water solubility and the reason for this will be
discussed in this section of the laboratory report.
For this experimental procedure, six different lipid samples were tested for their solubility in
water and in hexane. While all the samples failed to dissolve in water, they did however adequately
dissolve in the hexane solution. To understand the idea behind this, the basic idea of solubility must
be adequately understood first. Solubility is basically the characteristic of a particular solid, liquid
or gaseous chemical substance (which are know as “solutes”) to form a homogeneous solution of the
solute in the solvent. In short, it is a measure of the ability of a substance to dissolve in a given
amount of solution. This solubility is defined by the intermolecular activity that occurs between the
molecules of the solute and the molecules of the solvent when the two are mixed. Looking at lipids
in general, they are pretty non-polar in nature. Shown below are the structures of typical lipids:

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 The whole concept of solubility comes from the fact that like dissolves like, meaning that
polar substances will dissolve much better in polar solvents like water, while non polar substances
like lipids can be dissolved quite readily in nonpolar solvents like hexane. Looking at the structures
of lipids, despite possessing some functional groups that could actually contribute to forming
hydrogen bonds in aqueous solution (or the hydrophilic portion), this is simply dwarfed in degree by
the larger hydrocarbon portion of the molecule (or the hydrophobic portion). The water in this case
is termed as a lipophobic substance (or simply meaning a substance that doesn’t absorb fats).
Lipophilicity, on the other hand, is a measure of a compound’s ability to dissolve either in fats, oils,
or in non-polar solvents. The intermolecular bond that exists between the lipids amongst themselves
and with other substance is primarily through the weaker Van der Waal’s forces and they have little
to no capacity of forming any hydrogen bonds. Because of this, lipids are hydrophobic substances
that form a separate layer when mixed with water. Looking at the case with hexane, hexane has a
much better chance to be able to dissolve the lipids because it is non-polar in nature.
The solubility concept with lipids can be looked at in terms of thermodynamics as well by
observing the change in free energy (∆G). The mixing of a lipid in a solvent can be split down into
three separate steps. First, the lipid particles must separate from one another. Second, the solvent
particles must separate enough to make enough space for the lipid molecules to come between them.
And then third, the lipid and solvent particles must interact to form the solution. Keeping this in
mind, the addition of vegetable oil to water will be used as an example. The nonpolar oil molecules
are held together by the weak Van der Waal’s forces. Since they are weak, the ∆H for this step is
small. Looking at the next step, water is held together by strong hydrogen bonds and the ∆H required
to break them apart is large. The nonpolar lipids do not form strong interactions with the polar water
molecules, and so the negative ∆H for the third step isn’t enough to compensate for the ∆H of the
second step. So in total, the overall ∆H is large and positive and the entropy change ∆S, though
positive, won’t be enough to compensate as well. As a result, ∆G is positive (from ∆G = ∆H - T∆S) for
this situation and the mixing doesn’t occur spontaneously. The same goes for the nonpolar solute to
nonpolar solvent, except ∆H for the second step is smaller and thus the total ∆H is smaller. This
results in a ∆G that is negative and this is a mathematical representation that shows the mixing a non
polar solute in a non polar solvent is something that is thermodynamically favored.
Shown below is a simple table giving a description of the lipids that were used in the
laboratory based on their type and classification:

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 Ultimately, the compounds as shown in the table on the previous page are all lipids based on
their solubility in water. By the basic rule of thumb, lipids are generally insoluble in water and do
not form a solution in it. They fare better in non polar solvents like acetone or in hexane. So if ever
one needed to remove an oil spot from an item of clothing for example, it would be better to remove
it by adding in some of a non polar solvent like acetone to the affected area.

PART C – TEST FOR UNSATURATION

Due to the ever prevalent dangers in the handling of bromine in the laboratory, this portion of
the experimental procedure was not performed. The basis of this test is basically the fact that the
carbon to carbon double bonds are the reactive portions of the molecule, or are areas that are more
electron dense compared to the other areas of the structure. Because of this, it is fairly easy for
them to react and in the reaction with bromine, bromine is added eventually added to the double
bond and making the molecule fully saturated. This addition reaction uses up the free floating
bromine in solution, which turns from the typical brown color to colorless. The rate at which the
bromine is used up is an indication of the degree of saturation that the lipid sample possesses. Shown
below are the structures of the fatty acids and fatty acid content of the oils that were intended to
be used in the experiment:

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 Looking first at the fatty acids shown above, the most unsaturated fatty acid would be the
Linoleic acid, because it has two double bonds at positions 9 and 12 along the hydrocarbon chain.
The most saturated fatty acid would be the stearic acid because of the absence of the double bonds
in its structure. Oleic acid just rests smack dab in the middle having only one carbon to carbon
double bond located at carbon number 9. In reaction to the bromine test, the rule of thumb is that
the more unsaturated the sample to be tested is, then the more drops of bromine needed just to be
able to saturate the hydrocarbon chain. By that token, then the oleic acid will take fewer drops to
completely use up the bromine because it only has one double bond to add across. The linoleic acid
on the other hand would take up more drops because there is unsaturation present at two positions
in the structure. The stearic acid most likely won’t use up any of the bromine added since each
carbon in the structure of stearic acid has already been saturated with hydrogen.
Looking at the samples of the oil that were intended to be used in the experiment, it is quite
obvious that oils and fats are very diverse in their structure, containing various proportions of
different fatty acid chains. But given the physical states of the compounds, there is bound to be
more of the unsaturated fatty acid chains with the oils than compared to butter, which is somewhat
solid at room temperature. So based on this observation, then it is to be expected that the oils would
decolorize bromine to a much greater extent than compared to butter, which shows a fine line
between both the saturated and saturated fatty acids.
A simple diagram of the bromination across the double bond is shown below:

PART D – Saponification: Formation of Soap

Saponification is just one of the reactions that lipids (fats in particular) undergo given specific
conditions. This process essentially happens when a fat is heat with a strong base such as NaOH to
form glycerol and the sodium salts of the fatty acids, which is basically soap. Of course, the resulting
soap is ultimately defined by the base that is used. When NaOH is used in saponification, the soap
obtained is a solid soap that can be molded into a desired shape, while the use of KOH actually
produces a softer, liquid soap. By the same token, oils that a polyunsaturated produces softer types
of soap. In commercial product, the names that accompany the soap such as “coconut” or “avocado
shampoo” give an idea of the sources of the oil that are used in the production of the product.
Shown below is a general reaction in the formation of soap:
Heat
Fat or oil + strong base glycerol + salts of fatty acids (soaps)

Taking the saponification of tristearin or glyceryl tristearate as an example:

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PART E – Reactions of Soaps
As has already been defined previously, a soap is the salt of a fatty acid. Their main usage
falls under utilization as surfactants for washing, bathing, and cleaning, but they are also used in
textile spinning and are important components of lubricants. Soaps are key components of most
lubricating greases, which are usually emulsions of calcium soap or lithium soaps and mineral oil. The
main theory to consider when looking at soaps is the fact their properties as such arise from their
possession of the hydrophilic carboxylate head and the hydrophobic hydrocarbon tail. For the
experimental procedure, the group was tasked to test for the various reactions and observe the
characteristics of soaps, having one sample made in the laboratory through saponification and one
sample made from a commercial brand of laundry soap. Each of the tests shall be discussed in turn
because both of the soaps ended up with the same results anyway.
E-1: Shaking
After shaking separate test tubes filled with a small amount of soap solution from both
samples, both showed a foamy lather that persisted for some time before disappearing. This is
something that is common with all soaps: they can all make bubbles. When these bubbles are all put
together cumulatively, it is called a foam or a lather. The relative proportions or the type of soap
doesn’t matter, the same thing essentially happens when they are mixed in air and water. The
simplest explanation for this phenomenon is that these soaps contain components that are known as
foaming agents. But this explanation doesn’t exactly fit the bill for defining exactly what happens
behind the scenes. Taking a glass of water as an example, the molecules are grouped in water
because they’re equally attracted to each other. The molecules above, however, are at the edge and
since they don’t have any molecules above that they can hold, they tend to form stronger bonds with
what’s around them, and this phenomenon is what contributes to surface tension. Soap molecules
are essentially the same but they have a striking difference from water. One end (the carboxylate
end) is attracted to water, while the other end (the hydrophobic hydrocarbon portion) isn’t. So
essentially when soap is added to water, the carboxylate ends swoop in and are attracted to the
water molecules while the hydrophobic ends stick up into the air. Any soap molecules that are
trapped underneath will fight their way to the surface and eventually what is formed are three
separate layers; a layer of soap molecules, a layer of water and another layer of soap molecules that
are trying to push themselves away from the main body of water underneath. These three layers
form the wall of a bubble. A simple diagram illustrating this concept is shown below:

Though this may seem quite small as just a simple unit, combining a whole lot of these
together will result in the foam that is typical of soap.

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E-2: pH

Looking at the results for both soap samples, when tested with pH paper, both showed a pH
level of around 11 based on the scale that came with the box of the pH paper. This shows that soap
solutions are quite basic in nature and it is this characteristic that helps them neutralize acids, such
as those in dirt for example. A possible explanation for this high pH could be the simple fact that the
saponification process uses quite a strong base as one of the reactants, as well as the fact that the
fatty acids are pretty basic themselves in solution. Another simple explanation could be through
using the hydrolysis concept to see how these fatty acid salts behave in water. A simple equation for
this is shown below (using sodium stearate as an example):

In the equation above, the sodium stearate dissolves in water and splits it into hydrogen ions
and hydroxide ions. The carboxylate portion of the stearate is protonated to form stearic acid. This is
a relatively weak acid that more or less prefers to stay in its molecular form in solution (low K a). This
leaves the sodium cations and the hydroxide ions in solution, and these two have no affinity for each
other whatsoever. The presence of the hydroxide ions in solution is what contributes to the high pH
of soaps in general.
E-3: Addition of Oil
This section of the experiment was all about observing the action of soaps on substances such
as oils when in solution. It is already common knowledge that oil and water don’t mix because of the
concept of like dissolving like. The nonpolar oil can’t dissolve in the polar water and so the two form
separate layers when mixed. The presence of soap, however, acts as a sort of bridge that allows oil
to technically dissolve in water. All this arises from what happens to the soaps when they’re in the
water. Soaps are made up of fatty acid salts, and the conjugate base of these salts has two
important portions that have been mentioned before, the hydrophilic head and the hydrophobic tail.
When oil is droppered into a soap solution, the soap particles act fast and the hydrophobic tail (non
polar) sections quickly surround the molecule of oil (also non polar). On the other hand, the polar
carboxylate ends are attracted to the water molecules and face them. What happens is that the oil
molecule is eventually surrounded by these soap molecules up to a point where they are enclosed in
a sphere that is known as a micelle. These micelles, having the negatively charged carboxylate heads
on the outer portion of the structure, are soluble in water. And because of this, the trapped oil
molecules inside can be dissolved and rinsed away. A simple diagram illustrating this concept is
shown below:

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 The dissolving of fat inside the hydrophobic interior of micelles is also more popularly known
as the process of emulsification. The soap is essentially pulling non-polar molecules into an aqueous
solution where it would not normally be soluble. A solution that contains micelles can be referred to
as an emulsion and molecules that can form such are also referred to as emulsifying agents, wetting
agents or surfactants.

E-4: Addition of MgCl2 and CaCl2

Soaps by themselves are basically efficient at what they are intended to do, which is mainly
to clean (whether the application be laundry, or dishes, or whatever they may be). However, the
water they are in isn’t always that pure and there might be impurities in the water that could
potentially react with the soap molecules. This actually does happen and this contributes to a
condition of water known as hardness. When a particular water source is considered as “hard”, this
just means that it has compounds within it that will hinder the emulsification of soap, and so there’s
going to be less lather or less foam. Basing off of the results from the experimental procedure, both
of the samples gave the same results on the addition of magnesium chloride and calcium chloride,
which was observed through the formation of a precipitate. This precipitate is commonly known as
scum, and is something that can easily be observed after tub baths. A representative equation for
each is shown below:

Sodium stearate + magnesium chloride

2C17H35COONa & MgCl2 (aq) → (C17H35COO)2Mg (s) & 2NaCl (aq)

Sodium stearate + calcium chloride

2C17H35COONa(aq) + CaCl2(aq) → (C17H35COO)2Ca(s) + 2NaCl (aq)

III. Conclusion
Lipids are an essential part of the biomolecules and their study has led to some significant
advancements in the understanding of the inner workings some biological processes at the molecular
level. As has been shown in the report, lipids are compounds that are more or less characterized
based on the solubility rather than by some other chemical properties. By virtue of their definition,
lipids don’t generally have an affinity for water because of their non-polar nature. Rather, they
would do well to dissolve in non-polar solvents like acetone or hexane for example. The basics that
have led to the understanding of this has led to the understanding of the mechanisms behind the
action of soap, and how micelles are (added to this is the knowledge gained of the micelles that can
be found in the body during the digestion of fats). Through the experimental procedure, a better
understanding of lipids has been obtained, the various reactions that they can potentially go through
(like saponification), and overall what causes them to act the way that they do given any particular
chemical circumstance.

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IV. References
 "Solid Lipid Nanoparticle"
http://en.wikipedia.org/wiki/Solid_lipid_nanoparticle
 "Fats, Oils, Waxes & Phospholipids"
http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/lipids.htm
 "Lipolysis"
http://en.wikipedia.org/wiki/Lipolysis
 "Lipid Hydrolysis"
http://www.biotopics.co.uk/as/lipidhydrolysis.html
 "Factors Affecting Lipid Solubility"
http://faculty.etsu.edu/currie/New_Folder2/study/lipidsol.htm
 "Molecular Basis for Water Solubility and Fat Solubility"
http://www.chemistry.wustl.edu/~edudev/LabTutorials/CourseTutorials/Tutorials/Vitamins/
molecularbasis.htm
 "Fatty Acid Content of Common Oils"
http://www.chempro.in/fattyacid.htm
 "What makes soap foam?"
http://health.howstuffworks.com/skin-care/cleansing/products/soap-foam.htm
 "Why does soap form bubbles?"
http://blog.sciencegeekgirl.com/2009/05/02/why-does-soap-form-bubbles/
 "Foam"
http://en.wikipedia.org/wiki/Foam
 "Why does soap form bubbles?"
http://physics.stackexchange.com/questions/83368/why-does-water-sometimes-form-
bubbles-when-i-pour-it-into-a-glass
 "Lipids"
 en.wikipedia.org/wiki/Lipid
 "Micelle Formation"
www.mpikg.mpg.de/886719/MicelleFormation.pdf
 "What are micelles?"
www.wisegeek.com/what-are-micelles.htm
 "Why oil and water don't mix."
humantouchofchemistry.com/why-oil-and-water-do-not-mix.htm
 "Soap scum"
en.wikipedia.org/wiki/Soap_scum
 "Soap pH: Potential of Hydrogen"
www.thesoapbox.co.nz/soap-ph.htm

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