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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 TeopeDepartment of Biology, 3Biology-6, Group # 7, College of Science

University of Santo Tomas, España Street, Manila 1008

January 30, 2009Abstract:

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 #'sCoconut oil  8 – 10Butter 25 – 40Beef tallow 30 – 45Palm oil 37 – 54Lard 45 – 70Olive oil 75 – 95Peanut oil 85 – 100Cottonseed oil 100 – 117Corn oil 115 – 130Fish oils 120 – 180Soybean oil 125 – 140Safflower oil 130 – 140Sunflower oil 130 – 145Linseed 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

Group # Sample (brand)B. Acrolein

TestC. Iodine TestFormation of

bubblesOilylayer

1 Lauric Oil(Minola Premium

Coconut Oil)

The bubble persisted for 1 minute and 35

seconds.

acidic

Odor:Burning synthetic material

Appearance:Black in color

100 drops

4 Vegetable Oil(Spring Cooking Oil)

The bubble did not persist for 1 minute. It lasted

only for 29 seconds.

acidic

Odor:Burning oil

Appearance:Formation of black residue

110 drops

7 Butter(Dari Creme)

The bubbles persisted for 1

minute.acidic

Odor:Burnt oil

Appearance:Formation of black residue

32 drops

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.

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[9] Walker, S., and David McMahon. Biochemistry Demystified. New York: McGraw-Hill, 2008.Internet Sources[1] Lipids – Retrieved January 23, 2009http://www.chem.latech.edu/~deddy/chem121/Lipids.htm

[2] Saponification – Retrieved January 25, 2009http://en.wikipedia.org/wiki/Saponification

[3] Hydrolysis – Retrieved January 27, 2009http://en.wikipedia.org/wiki/Alkaline_hydrolysis

[4] Acrolein Test – Retrieved January 27, 2009http://www.answers.com/topic/acrolein-test

[5] Fats – Retrieved January 29, 2009http://www.chemcases.com/olestra/olestra05.htm

[6] Iodine Test – Retrieved January 29, 2009http://www.chemhelper.com/alkeneunsaturation.html