Experiment No. 5
Title: Lipids
Group No. 3
Date performed:
April 26, 2021
Group Members:
Patricia Gamboa
Year / Section :
BSBI – 2B
Date Submitted:
May 4, 2021
Salve Mae Gallardo
Shaira Kee
Objectives
To study some properties of lipids and reactions used for their identification.
Materials & Methods
Materials:
Test tubes, Bunsen burner, test tube holder, transfer pipets, pH sticks, bond paper,
vegetable oil, oil of wintergreen, distilled water, alcohol, dilute HCl, dilute NaOH, ether,
chloroform, acetone, glycerol, lecithin, oleic acid, stearic acid, olive oil, coconut oil,
Hubl’s solution, bile salts
Methods:
A. Properties of Lipids
1. Solubility
Determine the solubility of vegetable oil in 1 mL of the following solvent: Water, Dilute
NaOH, Chloroform, Dilute HCl, Ether and Acetone. Observe and tabulate the results.
2. Formation of a Translucent Spot
Place one drop of vegetable oil on a piece of ordinary bond paper. Note the formation of
a semi-transparent spot. Allow the oil to evaporate spontaneously. Does the translucent
spot disappear? Repeat the same procedure using the oil of wintergreen instead of
vegetable oil. Note the difference in the results.
3. Reaction to Litmus Paper
Test the reaction of fresh vegetable oil with red and blue litmus papers previously
moistened with water. What is the reaction? Allow the oil to stand uncovered until the
next laboratory period and test again with litmus paper. Is there any change in the
reaction?
B. Reactions of Lipids
1. Test for Unsaturation
Use dry test tubes. In each of four test tubes, place separately two drops of the sample
(olive oil, oleic acid, stearic acid, and coconut oil), and add 1 mL of chloroform. Then
add a drop of Hubl’s solution (iodine in mercuric chloride solution). Cover the test tube
and mix well. If the mixture decolorizes, continue adding Hubl’s solution drop by drop,
shaking well after each drop, until it is no longer decolorized. Use the same dropper for
all samples. Record the number of drops used.
2. Lieberman-Burchard or Acetic Anhydride Reaction
In a clean, dry test tube, dissolve a pinch of the sample separately (cholesterol and bile
salts) in 1 mL of chloroform. Then add five drops of acetic anhydride and a drop of
concentrated H2SO4. Mix. Note the change in color.
Results & Observations
Solvent
Solubility
Water
Not Soluble
Dilute NaOH
Not Soluble
Chloroform
Soluble
Dilute HCl
Not Soluble
Ether
Soluble
Acetone
Soluble
Table 1. Results for Solubility Experiment of Vegetable Oil in Varying Solvents
Samples in Test tubes
Number of drops
Olive Oil
several drops
Oleic Acid
1 drop
Stearic Acid
1 drop
Coconut Oil
1 drop
Table 2. Results for the Test of Unsaturation
Sample in Test Tube
Appearance of Color
Bile Salt
Yellow or Golden
Cholesterol
Deep Green
Table 3. Results for the Lieberman-Burchard or Acetic Anhydride Reaction
Discussion & Conclusion
Lipids are a large and diverse group of naturally occurring organic compounds
that have different properties. These characteristics are related to their solubility in
nonpolar organic solvents like ether, chloroform and acetone. Solubility is a property of
a solute to readily dissolve in a solvent to form a solution. The chemical and physical
characteristics of the solute and the solvent play a major role in the level of solubility. It
is important to note that there’s a limit to the amount of solutes a particular solvent could
hold in a solution and beyond this limit solutes would start to precipitate at the bottom.
But in this experiment, the rule that is evident is the characteristic of polarity. Generally,
the rule of thumb in solubility is that of “like dissolves like”. Meaning, polar compounds
have a greater tendency to dissolve in polar solvents and vice versa. This explain why
oil which is a nonpolar substance is not miscible in water which is a polar substance.
This also applies to other polar substances like diluted NaOH, and HCl. With that, this
further explains why oil is miscible in nonpolar substances like ether (Ophardt, 2003) ,
chloroform (Drijber & Jeske, 2019) and acetone (Monakhova et al., 2014). As for
Chloroform, it can either be polar or nonpolar which explains why oil is also miscible
with it. Chloroform does not have much of an asymmetrical charge, thus it is not
strongly polar. That is why in this experiment chloroform acts as a nonpolar solvent. To
continue, Acetone acts as a good solvent for a lot of organic chemicals due to the
presence of nonpolar C–C and C–H bonds as well as a highly polar C=O bond.
Moreover, Acetone also has a polar C=O bond that can form hydrogen bonds with the
dipoles of water. Acetone also contains two methyl groups that interact with oil through
dispersion forces and both the methyl groups and oil are nonpolar. With that, Acetone
has both polar and nonpolar properties which allows it to interact with polar and
nonpolar compounds via the dipole-dipole and dispersion intermolecular forces
(Monakhova et al., 2014). Basically, the polarity of the solute and solvent plays a major
role in solubility.
Another notable property of lipids is its ability to form translucent spots. This has
something to do with the relationship between light and matter. When light strikes the
surface of paper, it bounces right back, which is why a piece of paper looks ‘normal’.
However, when the same piece of paper is smeared with oil, it appears more
translucent, because light doesn’t have to do all that bouncing and scattering (diffuse
reflection). It simply must pass from air through grease. Paper, for instance, has a
refractive index of 1.5. Oil’s refractive index is similar to that of paper, or at least, more
similar than the refractive index of air (which is 1). When this material and the object in
question are brought close together, light passes through from one to another without
reflection or refraction (Ashish, 2019). The spot will not disappear, like water, the lipids
soak into the paper fibres, but the lipid spot evaporates much more slowly than water
(because the forces that hold the lipid molecules together are stronger than those in
water molecules) (Saunders, N. 2005). So the spot remains. The same will occur with
the oil of wintergreen, the spot will not disappear.
Lastly, lipids were tested to see its reaction to a Litmus paper. Acids and bases
refer only to aqueous (water-based) solutions, that is why the pH paper won't change its
color in non-aqueous liquids such as vegetable oil (Helmenstine, A.M., 2020). This does
not indicate that the oil is neither acidic or alkaline, but instead the litmus paper only
works with aqueous solutions. These indicator papers work by measuring the balance of
positive hydrogen ions, which are predominant in acids, and the negative hydroxide
ions, which are predominant in alkalines, that float around in ionic aqueous solutions.
Since oil is not an ionic solution, the paper does not react to it. This would only result in
the paper becoming translucent. In relation to this, it is noted that most vegetable oils
are weak acids. Since they are not soluble in water, their acidity cannot be measured in
terms of pH levels. It is usually estimated in percent free acidity. When oil is left for
some time and exposed to the air, Peroxidation (oxidative deterioration) can occur
wherein oxygen ion replaces a hydrogen ion within a fatty acid molecule and higher
numbers of double bonds within the fatty acid increase the possibility of oxidative
rancidity, theoretically, oil will turn acidic (Feiner, G., 2016). The breaking of these
carbon-carbon double bonds by the oxygen in the air is a relatively slow process and
depending on the duration, the litmus paper result will most likely be the same.
In this experiment, the reactions of lipids were also observed. First, the test for
unsaturation was conducted. Unsaturated fatty acids like the sample that was being
tested can react with halogens like iodine due to the presence of double bonds carbon.
To determine if the samples are unsaturated, Huble’s reagent was being utilized. This
reagent reacts with an alcoholic plan of iodine that contains some mercuric chloride
(Raza, 2019). The principle of this test is to know if all the neutral fat that contains
glycerides fatty acid is unsaturated. Double bonds are found in the structure of
unsaturated fatty acids, which becomes saturated via the of iodine. During the reaction,
the violet shade of iodine stayed with only 1 drop per sample to oleic acid, stearic acid
and coconut oil which means that they are strongly saturated (Supriya, 2019). However,
for the olive oil it takes several drops of huble's reagent for the color to appear that only
meant that it is unsaturated. It is worth to take note that the violet shade of iodine
doesn't vanish even after mixing the sample. If more iodine is attached, then iodine's
value is higher compared to the sample which can indicate that it is more reactive, less
stable, softer, and more susceptible to oxidation and rancidification. (Iodine value.
Encyclopedia Britannica, 1998).
Next, the Lieberman-Burchard or Acetic Anhydride Reaction was also tested.
Liebermann-Burchard Test is a chemical estimation or reaction that is predominantly
occurring to pentaenylic compound such as cholesterol (Bangash, 2017).The
cholesterol is a lipidic waxy alcohol that is mainly found in the cell membrane and is
readily soluble in acetone. In this test, acetic anhydride was being utilized as a solvents
and dehydrating agents while sulfuric acid was being applied as dehydrating at the
same time a oxidizing agents (Buffin, 2014). During the addition of concentrated sulfuric
to cholesterol, the water molecule was removed from the C3 of the cholesterol and
oxidation that occurred formed a 3,5-cholestadiene. The end product was being
converted into a polymer which contains a chromophore that is the reason behind why
results to the coloration of the cholesterol of a deep green that relatively indicates that
the test is positive. The colour reactions of cholesterol produce a number of products
with varying absorptivities. This is sometimes being monitored under strict control
condition as it is undesirable in quantitative analysis. Furthermore, the reaction may not
be very nonspecific and require some degree of purification of the analyte before it can
be applied successfully to the quantitative measurement of data in research (De la
Huerga, J. and Sherrick, J. C, 1972).
In conclusion, lipids are structurally diverse inorganic compounds that have
hydrocarbon chains or rings as a major part of their chemical structure. Lipids possess
different properties which in turn result in varying positive or negative reactions to
certain types of tests like the litmus paper, Lieberman-Burchard and Huble’s reagent.
These properties are brought about by their chemical structure which in turn gives lipids
unique characteristics that enables their identification through conducting certain tests.
Answers to Questions
1. What common characteristics do lipids possess?
Lipids are a group of structurally diverse inorganic compounds that have hydrocarbon
chains or rings as a major part of its chemical structure, with the primary types of
hydrocarbons being fatty acids and steroids (Kerr,
characteristic, lipids display a wide variety of
et. al 2015). Beyond its basic
features. For such, a very common
characteristic of lipids is that it is water insoluble and also it is organic solvent soluble
compounds.This means that lipids have an inability to dissolve in water. However, it will
or can be dissolved in a variety of organic solvents, such as benzene, acetone, alcohol,
carbon tetrachloride,and chloroform ("What are the common characteristics of all lipids",
n.d.). Lipids insolubility is often referred to as hydrophobic, or “water-fearing.”
Furthermore, water bonding between the oxygen and hydrogen atoms results in a polar
covalent bond which creates a negative charge at the oxygen end of the water molecule
while having a positive charge at the hydrogen end. This is the reason why water
doesn't have equal bonds. On the other hand the bonding between carbon and
hydrogen atoms in lipids is not polar, that means that it shares equal bonds (Chandler,
2017). Molecules with only the same polarity can dissolve each other. This implies that
nonpolar bonds will normally dissolve in nonpolar solvents, it also holds true with polar
bonds that will dissolve in polar solvents via the attraction of same bonds (Lents, et. al
n.d). For this reason, lipids can be easily distinguished and it can also use to generally
classify them.
2. What is an emulsion?
An emulsion is a colloid of two or more immiscible liquids where one liquid contains a
dispersion of the other liquids. In other words, an emulsion is a special type of mixture
made by combining two liquids that normally don't mix. Emulsions usually appear cloudy
or white because light is scattered off the phase interfaces between the components in
the mixture. If all of the light is scattered equally, the emulsion will appear white. Dilute
emulsions may appear slightly blue because low wavelength light is scattered more.
This is called the Tyndall effect. Two liquids can form different types of emulsions. For
example, oil and water can form an oil in water emulsion, where the oil droplets are
dispersed in water, or they can form a water in oil emulsion, with water dispersed in oil.
Further, they can form multiple emulsions, such as water in oil in water. Most emulsions
are unstable, with components that won't mix on their own or remain suspended
indefinitely (Helmenstine, 2020). Emulsifier molecules work by having a hydrophilic end
(water-loving) and hydrophobic end (water-hating). The hydrophilic end of the emulsifier
molecule is attracted to the water and the hydrophobic end is attracted to the fat/oil. By
vigorously mixing the emulsifier with the water and fat/oil, a stable emulsion can be
made (Institute of Food, Science and Technology, 2017).
3. How are emulsions stabilized?
An emulsion is an unstable system because there is a natural tendency for a liquid
system to separate and reduce its interfacial area and hence, its interfacial energy
(Jones et al., 1978). The stability of an emulsion can be defined as its ability to maintain
its properties (Maphosa et al., 2017). In other words, the capability of the phases of the
emulsion to remain mixed together. The extent of this stability is associated with various
factors like particle size, particle size distribution, density between the dispersed and
continuous phases, and chemical integrity of the dispersed phases (Given, 2009).
Mechanisms of destabilization occur due to several factors like pH, ionic strength,
temperature and more (McClements, 2005; Sjöblom, 2006). With that, substances such
as emulsifiers, stabilisers, weighting agents, ripening inhibitors, and texture modifiers
are introduced in order to increase the kinetic stability of emulsion systems for longer
periods (Kerkhofs et al., 2011; Payet & Terentjev, 2008). Emulsions are stabilized by
increasing the repulsion between the dispersed phase by increasing the electrostatic
(long range) or steric (short range) repulsion. When a surface adsorbs on the interface,
the interfacial tension between the two phases decreases. The reduced interfacial
tension depends on the concentration of the surfactant according to the Gibbs isotherm.
Adsorbed surfactants or solid particles stabilize emulsions via two main mechanisms
namely, steric stabilization and electrostatic stabilization. Steric stabilization arises from
a physical barrier to contact and coalesce. Such an instance can be seen from a high
molecular weight polymers that can adsorb on the surface of the dispersed phase
droplets and extend significantly into the continuous phase, providing a volume
restriction or a physical barrier for particle interactions. As polymer coated particles
approach, the polymers are forced into closer proximity and repulsive forces arise which
keeps particles apart. On the other hand, electrostatic stabilization is based on the
mutual repulsive forces that are generated when electrically charged surfaces approach
each other. In an electrostatically stabilized emulsion, an ionic or ionizable surfactant
forms a charged layer at the interface. For an oil-in-water emulsion, this layer is
neutralized by counter ions in the continuous phase. If the counter ions diffuse, the
disperse phase droplets act as charged spheres as they approch each other. If the
repulsive forces are strong enough, the droplets are repelled before making contact and
coalesce. As a result, the emulsion is stabilized. Stabilisers are a group of additives that
have the capability to stabilize emulsions by thickening the aqueous phase while
emulsifiers are surface active molecules that adsorb to the surface of freshly formed
droplets of an oil-water interface during homogenisation, which forms a protective
membrane that prevents the droplets from aggregating (Dickinson, 2009; Weiss, 2005).
With that, emulsifiers act as surface-modifying substances at the interface between
each droplet and the continuous phase (Robins, 2002). Due to their amphiphilic nature,
they possess both hydrophilic portions which align with the aqueous phase and
hydrophobic portions that align with the lipid phase (Cottrell & Van Peij, 2014). In
relation to that, Emulsifiers can be oil or water soluble forming a fluid that has a
close-packed layer at the interface with low interfacial tension. This results in an
emulsion with a small droplet size distribution which is stabilized by the fluid
Gibbs-Marangoni mechanism or the weak electrostatic repulsion (Robins et al., 2002).
In general, electrostatic stabilization is significant in oil-in-water emulsions since the
electric double layer thickness is greater in water than in oil. With that, both electrostatic
and steric forces can prevent aggregation or coalescence and thereby stabilize
emulsions (Urrutia, 2006).
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