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BIOCHEMISTRY
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Biochemistry Laboratory – CH600 (2008-2009) Experiment 7

Characterization of Saponifiable Lipids

*Michelle Dy Sim, Gellina Ann Ram Suderio, Jonnah Kristina Chua Teope
Department of Biology, 3Biology-6, Group # 7, College of Science
University of Santo Tomas, España Street, Manila 1008

January 30, 2009

Abstract:

A saponifiable lipid is one with an ester functional group that can be hydrolyzed under basic conditions. It undergoes
chemical hydrolysis when reacting with a strong base to produce salts of fatty acids called soaps. This experiment aims to
characterize and to subject a fat or an oil sample to alkaline hydrolysis and to test the degree of unsaturation using iodine. For
alkaline hydrolysis, 1g of NaOH was added with 2mL of butter / oil and 5mL of 95% ethanol then heated in a water bath at
750C for 15 minutes. Once cooled, a mongo size piece from the product was taken and submerged in 3mL distilled water.
Using a straw, air was blown into it until bubbles started to form. The bubbles were able to persist for 1 minute and 35
seconds for the coconut oil, 29 seconds for the vegetable oil and 1 minute for the butter. The remaining product was acidified
with a few drops of concentrated H2SO4. The oily layer formed was acidic. For the acrolein test, 1g of KHSO4 was placed in a
test tube together with 5 drops of the butter sample then, heated on a hot plate, after cooling, the mixture showed a black
precipitate that smelled like burnt wood. For the iodine test, 5mL of butter / oil sample was placed in a test tube. 5mL of
water was placed in another test tube, this served as the control tube. To each test tube, iodine solution was carefully and
slowly added, shaking it after each addition. The total number of drops added to each tube for the coconut oil was 100 drops,
for the vegetable oil, 110 drops was used and for the butter, 32 drops.

Keywords:
• Lipids
• Saponification
• Alkaline Hydrolysis
• Acrolein Test
• Fats

I. Introduction

Lipids are a variety of organic molecules grouped together on the basis of their solubility in non -
polar solvents. Their varied biological functions include energy source, energy storage, cell membrane
structural components, hormones, vitamins, vitamin adsorption, protection, and insulation.
 The four main groups of lipids include fatty acids (saturated and unsaturated), glycerides
(glycerol-containing lipids), nonglyceride lipids (sphingolipids, steroids, waxes) and complex lipids
(lipoproteins, glycolipids).

Figure 1: Classification of common phospholipids, glycolipids, and triacylglyerides

Lipids can also be broadly subdivided as either saponifiable or nonsaponifiable. Saponifiable

lipids are esters of fatty acids that can undergo saponification. This includes the triglycerides,
glycolipids, sphingolipids, some waxes and phospholipids. Nonsaponifiable lipids are lipids that do not
have fatty acids or ester linkages. This includes the steroids, prostaglandins, leukotrienes and terpenes.
Waxes are esters of fatty acids and long – chain alcohols. They act as protective coatings for
hair, skin, and feathers. Common waxes include beeswax, carnauba wax and spermaceti.
The carboxylic acid products found in the saponifiable lipids are referred to as fatty acids. The
fatty acids are long, unbranched monocarboxylic acids containing 10 to 22 carbon atoms. They typically
have an even number of carbon atoms due to their biosynthetic pathway. The Fatty acids can be
classified into families based on chain length and on the number of C=C double bonds present. Saturated
fatty acids contain no C=C double bonds. Unsaturated fatty acids contain C=C double bonds. The
double bonds are typically cis.
Fatty acids undergo the reactions typical of any carboxylic acid. When fatty acids react with
alcohols they form esters and water. This type of reaction is called esterification. The reverse of
esterification is called acid hydrolysis. In saponification or base hydrolysis, the sodium or potassium salt
resulting from the base hydrolysis of an ester of a fatty acid is referred to as soap.
 Figure 3: Esterification

Figure 4: Acid Hydrolysis

Figure 5: Alkaline Hydrolysis

Hydrolysis of triglycerides occurs in the presence of strong acids or by lipases. The product of
the reaction is glycerol and three fatty acids.

Figure 2: Hydrolysis of triglycerides

II. Methodology

A. Alkaline Hydrolysis:
A gram of NaOH was carefully weighed into a small Erlenmeyer flask. Then, 2mL of butter / oil
sample was added together with 5mL of 95% ethanol. The mixture was heated in a water bath at 75 oC
for at least 15 minutes. The solution was cooled in a beaker of cold water. A small mongo sized piece of
the solid product was removed using a glass stirring rod and placed in a test tube filled with 3mL of
distilled water. A soda straw was placed inside the test tube and air was slowly blown into it until
bubbles were formed. Observations were described and noted.
B. Acrolein Test for Fats:
A gram of KHSO4 was placed in a test tube. Then, 5 drops of the butter / oil sample was added.
The mixture was heated over the hot plate for a few minutes. The tube was held by a test tube holder.
After heating, the mixture was allowed to cool, and the appearance and odor of the acrolein was
described.

C. Test for Unsaturation of Fatty Acids:

Two tubes were readied. The 5mL butter / oil sample was placed in one tube. In another tube,
5mL of water was placed. The tube containing water served as the control tube. To each tube, iodine
solution was added slowly and carefully just until it failed to be decolorized, shaking it after each
addition. The number of drops used was recorded.

III. Results and Discussion

Saponification is the hydrolysis of an ester under basic conditions to form an alcohol and the salt
of a carboxylic acid (carboxylates). Saponification is commonly used to refer to the reaction of a
metallic alkali (base) with a fat or oil to form soap. Saponifiable substances are those that can be
converted into soap.

Sodium hydroxide (NaOH) is a caustic base. If NaOH is used, a hard soap is formed, whereas
when potassium hydroxide (KOH) is used, a soft soap is formed. Vegetable oils and animal fats are fatty
esters in the form of triglycerides. The alkali breaks the ester bond and releases the fatty acid and
glycerol. If necessary, soaps may be precipitated by salting it out with saturated sodium chloride.
Saponification value represents the number of milligrams of potassium hydroxide or sodium
hydroxide required to saponify 1g of fat under the conditions specified. It is a measure of the average
molecular weight (or chain length) of all the fatty acids present. As most of the mass of a fat / triester is
in the 3 fatty acids, it allows for comparison of the average fatty acid chain length.
Acrolein is the simplest unsaturated aldehyde. It is produced widely but is most often
immediately reacted with other products due to its instability and toxicity.
Acrolein test is a test for the presence of glycerin or fats. A sample is heated with potassium
bisulfate, and acrolein is released if the test is positive. When a fat is heated strongly in the presence of a
dehydrating agent such as KHSO4, the glycerol portion of the molecule is dehydrated to form the
unsaturated aldehyde, acrolein (CH2=CH-CHO), which has the peculiar odor of burnt grease.
Oils, fats and lipids are relatively interchangeable names for a variety of chemical compounds
that share common solubilities in organic solvents such as ether, chloroform, and methanol.
The fatty acid residues may also differ according to the extent of present unsaturated bonds in the
hydrocarbon chain. The extent of unsaturated bonds can be demonstrated by the degree of decolorization
of a halogen solution. This is usually measured by the iodine number. Iodine adds less readily than
bromine across the double bonds in an unsaturated bond. The iodine number of a fat is the number of
grams of iodine that will be consumed by 100g of fat or oil. This value is used to measure the relative
degree of unsaturation in fats. The more double bonds a fat contains, the more iodine is required for the
addition reaction; thus, a high iodine number means a high degree of unsaturation. As a general rule
animal fats are saturated and vegetable fats are unsaturated with some exceptions. The typical iodine
numbers for some fats is shown in the table below:

Table 1: typical iodine numbers for some fats

TYPICAL IODINE #'s
Coconut oil 8 – 10
Butter 25 – 40
Beef tallow 30 – 45
Palm oil 37 – 54
Lard 45 – 70
Olive oil 75 – 95
Peanut oil 85 – 100
Cottonseed oil 100 – 117
Corn oil 115 – 130
Fish oils 120 – 180
Soybean oil 125 – 140
Safflower oil 130 – 140
Sunflower oil 130 – 145
Linseed oil 170 – 205
 The observations and data gathered from this experiment are shown in the table below:
Table 2: Results from the experiment performed
A. Saponification
Formation of Oily B. Acrolein C. Iodine Test
Group # Sample (brand) bubbles layer Test
Odor:
Burning
Lauric Oil The bubble synthetic
1 (Minola Premium
Coconut Oil)
persisted for 1
minute and 35
acidic material 100 drops

seconds. Appearance:
Black in color

Odor:
The bubble did Burning oil
Vegetable Oil not persist for 1 acidic 110 drops
4 (Spring Cooking Oil) minute. It lasted
only for 29
Appearance:
Formation of
seconds. black residue

Odor:
Butter The bubbles Burnt oil
(Dari Creme) persisted for 1 acidic 32 drops
7 minute. Appearance:
Formation of
black residue

Soaps are the salts of strong bases and weak acids; therefore, they should be slightly basic
because if soap is too basic it could cause damage to skin, surfaces to be cleaned, or clothes.
Saturated oil forms less bubbles in froth tests than unsaturated oils. The unsaturated oils form
more bubbles because of the presence of numerous double bonds. H2SO4 was added in the mixture in
order to separate glycerol from the oil. Saturated oil will require less drops of iodine than the unsaturated
ones.

IV. Conclusion

Lipids are biomolecules that are insoluble in water and highly soluble in non – polar organic
solvents such as ether, chloroform and benzene. Lipids perform many important roles. They are the
highly concentrated fuel reserves that reside mostly in our adipose tissues. They constitute the bulk of
biological membranes, forming an impermeable yet flexible barrier for all of our cells. As certain
hormones and hormone receptors, they are signal carriers. They also form certain vitamins.
There are two general types of lipids. Complex lipids include fats, oils, and waxes that contain
ester linkages that can be hydrolyzed to smaller molecules. Simple lipids such as cholesterol and other
steroids do not have the ester linkages and cannot be hydrolyzed.
Fatty acids are carboxylic acids and are therefore weak acids. For fatty acids, the value of pKa is
around 4.5. Therefore, generally speaking, fatty acids are neutral below pH 4.5 and charged above pH
4.5. Fatty acids do not dissolve in water; instead they are dispersed by the formation of micelles in
water. As the fatty acid carbon chain length increases, the melting point and boiling point increases.
Unsaturated fatty acids have lower melting points and boiling points than saturated fatty acids. This is
due to the fact that the cis C=C bond causes "kinks" in the hydrocarbon chains, which reduces the extent
of association of the molecules. Less energy is required to separate the molecules.
The animal fats have a larger percent of saturated fatty acids while the vegetable oils have a
greater portion of unsaturated fatty acids. The higher melting points of the solid animal fats are due to
the fact that the saturated fats have straight chains and the molecules can pack together more closely.

V. References
Books
[1] Brink, M.F. and David Kritchevsky. Symposium : Dairy Lipids and Lipid Metabolism. Westport,
Conn.: Avi Pub. Co., 1968.

[2] Gunstone, F.D. Fatty Acid and Lipid Chemistry. London: Blackie Academic and Professional, 1996.

[3] Hames, D. and Nigel Hooper. Biochemistry. New York: Taylor and Francis, 2005.

[4] Holman, R. Progress In the Chemistry of Fats and Other Lipids. New York : Academic Press, 1958.

[5] Mckee, T. and James R. Mckee. Biochemistry: The Molecular Basis of Life. Boston: McGraw-Hill,
2003.

[6] Murray, R., Daryl K. Garner and Victor W. Rodwell. Harper’s Illustrated Biochemistry. New York:
McGraw-Hill, 2006.

[7] Tyman, H.J.P. and M.H. Gordon. Developments In the Analysis of Lipids. Cambridge: The Royal
Society of Chemistry, 1994.

[8] Voet, D. and Judith G. Voet. Biochemistry. New York: John Wiley and Sons, 2004.

[9] Walker, S., and David McMahon. Biochemistry Demystified. New York: McGraw-Hill, 2008.
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http://en.wikipedia.org/wiki/Saponification

[3] Hydrolysis – Retrieved January 27, 2009

http://en.wikipedia.org/wiki/Alkaline_hydrolysis

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http://www.answers.com/topic/acrolein-test

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http://www.chemcases.com/olestra/olestra05.htm

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http://www.chemhelper.com/alkeneunsaturation.html