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Lipids are a group of water-insoluble compounds that are divided into three categories, the simple lipids (fats, oils and waxes), complex lipids (phospholipids and glycolipids), and the precursor and derived lipids (steroids, ketone bodies and lipidsoluble vitamins). They contain elements such as carbon, oxygen, hydrogen and some may contain phosphorus and nitrogen (Sackheim and Lehman, 1998). Lipids are one of the four major classes of biomolecules (along with carbohydrates, proteins and nucleic ands). They store energy in the body for use when food is not readily available. They are integral for cell membranes to separate compartments within the cell, and can act as chemical messengers in the endocrine system and throughout the body (Nucum, 2007). Fatty acids are constituents of lipids except for derived lipids such as steroids. Generally, they occur in nature having even numbers of carbon atoms in the chain. They may be saturated or unsaturated (Calano et al., 2011). Unsaturated fatty acids contain at least one double bond between two carbons. Since carbon atoms are joined by a double bond, there is no rotation about that bond. This restriction allows the cis- (groups are on the same side of the double bond) and trans- (on opposite sides) forms of the acid. According to Apps et al. (1992), in general, the unsaturated fatty acids have the cis- configuration since chains containing cis- double bonds take up greater space than would the trans- forms (answer to Question 2). The cis- configuration is particularly important for the membrane structure.

According to Zaballa et al. (2010), fatty acids do not dissolve in water because they form dimers. The partially negative oxygen atom of one molecule of fatty acid and partially hydrogen atom of the carboxyl group of the other molecule of fatty acid are capable of forming a hydrogen-bond. Thus, no groups are available for H-bonding with water molecules (answer to Question 1).

Objectives: 1. To determine the reactions of lipids to the different reagent, 2. To learn the different properties of lipids, 3. To prepare a soap from common lipid sources, and 4. To examine the interaction of soap and detergent solutions with different metal ions solutions

Results and Discussion:

Solubility Test

Molecules in which there is unequal or uneven distribution of charges are called polar molecules. Water and ethyl are polar molecules. Sodium chloride, sodium hydroxide and hydrochloric acid are polar compounds because of the uneven distribution of charges, Na+Cl-, Na+OH- and H+Cl-, respectively. On the other hand, ether, chloroform and benzene have no separation of charges so they are nonpolar liquids.

Table 1.a. Solubility of Cottonseed Oil in Different Solvents Sample Used Observation H2O Ethyl alcohol Ether Chloroform Benzene 5% HCl 5% NaOH Did not dissolve, formation of micelles which return on top after shaking Did not dissolve, formation of micelles which return on top after shaking 10 seconds 10 seconds 10 seconds Did not dissolve, formation of micelles which return on top after shaking Did not dissolve, formation of micelles which return on top after shaking

As presented in Table 1.a., cottonseed oil does not dissolve in polar solvents like water, ethyl alcohol, hydrochloric acid and sodium hydroxide. On the other hand, it is miscible with ether, chloroform and benzene which are all nonpolar solvents. It can be said that cottonseed oil is a nonpolar compound, since nonpolar compounds dissolves in nonpolar solvents.

Table 1.a. Solubility of cottonseed oil in ethyl alcohol and ether Cottonseed-ethyl alcohol mixture Cottonseed-ether mixture

Table 1.b. shows the formation of a grease spot in the bond paper formed by the mixture of cottonseed oil with ethyl alcohol and ether. It can be noted that the spot produced with ethyl alcohol is more translucent than with ether. This clearly shows the miscibility of cottonseed oil in ether and its immiscibility with ethyl alcohol.

Tests for Unsaturated of Fatty Acids Fatty acids can be either saturated or unsaturated. Saturated fatty acids contain only single bonds between carbon atoms. Unsaturated fatty acids contain a few double bonds between carbon atoms (Sackheim and Lehman, 1998). The presence of double bonds in fatty acids is easily tested by using a solution of bromine (Br2) in carbon tetrachloride (CCl4). If the sample fatty acid is unsaturated then the bromine reacts quickly with it by adding to the carbon- carbon double bonds (C=C) of the fatty acid. Since, bromine is orange in color and the products are colorless, the reaction is easily observed - the orange color of the bromine solution disappears when it is added to the unsaturated fatty acid. However, after just enough bromine has been added to completely react with all of the double bonds present in the fatty acid sample, the next drop of bromine solution no longer can react and the orange color persists. Therefore, the number of drops necessary to reach this point can be used as a gauge of the level of unsaturation in the fatty acid.

Table 2.a. Reaction of Oleic acid with Bromine water in CCl4 Sample Used Oleic acid Observation A faint orange color formed after addition of 52 drops of bromine water in CCl4

As shown in Table 2.a., 52 drops of bromine water is needed to react with the oleic acid to produce a faint orange colored solution. It can be noted that oleic acid contains a carbon-carbon double bond, thus, an unsaturated fatty acid. According to Nucum (2007), oleic acid is a 18-carbon fatty acid with one double bond. Oleic acid can be found in olive oil. Hanus iodine test is also performed to determine the degree of unsaturation of the fatty acids. In this test, Hanus iodine solution which contains iodine reacts with the carbon-carbon double bonds of fatty acid. The iodine will be decolorized by unsaturated fatty acids. No discoloration of the solution will indicate presence of a saturated fatty acid. If the substance reacted and the color disappeared in a shorter time, this means that the fatty acid contain lesser double bonds, thus, the degree of unsaturation is lesser. While, if it is requires higher amount of time before the color disappeared, this means that the substance contains a high degree of unsaturation.

Table 2.b. Reaction of various lipids in Hanus Iodine solution Test tube palmitic acid oleic acid cottonseed oil Observation No discoloration Color disappear in 10 seconds Color disappear in more than two minutes

Table 2.b. shows that palmitic acid does not react with the Hanus iodine solution. It can be said that palmitic acid contains no double bond, thus, a saturated fatty acid. Palmitic acid is a 16-carbon saturated fatty acid (Sackheim and Lehman, 1998). Oleic acid decolorized the iodine solution in 10 seconds. This shows that

oleic acid has a carbon-carbon double bond and therefore an unsaturated fatty acid. This supports the observation that oleic acid reacts with bromine water in CCl4. It is also presented in Table 2.b. that cottonseed oil gives the longest time before the iodine solution decolorizes. This means that cottonseed oil has double bonds and the number of double bonds is greater than the double bonds in oleic acid. It can be said that cottonseed oil is more unsaturated than oleic acid. Acccording to Sackheim and Lehman (1998), oils are composed of long chain of fatty acids that contains unsaturated sites. Cottonseed oil has the unsaturated fatty acids, oleic acid and linoleic acid. Linoleic acid is a 18-carbon fatty acid with two double bonds.

Acrolein Test According to Sackheim and Lehman (1998), the acrolein test, which is a test for the presence of glycerol, is used as a general test for fats and oils, since all fats and oils contain glycerol (answer to Question 3). When glycerol is heated to a high temperature, especially in the presence of a dehydrating agent such as potassium bisulfate (KHSO4), a product acrolein results. This substance is easily recognized by its strong, pungent odor.

Table 3. Result of Acrolein Test Sample Used 2 drops of Glycerol 2 drops of cottonseed oil Observation unpleasant odor/burned odor Used oil odor/ burned odor

As seen in Table 3, the product acrolein is formed and its formation is indicated by the production of a pungent odor. Cottonseed oil also produces the same odor. When fats or oils are heated to a high temperature or are burned, the unpleasant odor is that of acrolein.

Test for Rancidity

Fats and oils develop an unpleasant odor and taste when allowed to stand at room temperature for a short period of time. That is, they become rancid. They are oxidized by the oxygen in the air which produces short-chain acids or aldehydes.

Table 4. Result of Test for Rancidity Observation Test Indicator Fresh oil Phenolphthalein Methyl orange pH paper Clear solution Orange color produced pH - 7 Rancid oil Clear solution Red color formed pH 1

Presented in Table 4 are the comparison with fresh oil and rancid oil with regards to their acidity using different indicators. With phenolphthalein indicator, no change in color is obtained. Both rancid oil and fresh oil are in pH range of 1-8.5. In methyl orange, fresh oil has not change in color while rancid oil gives a red color. This shows that fresh oil is within the pH range 4-9 whereas rancid oil is within 1-4. With the more accurate pH paper, rancid oil is found to be strongly acidic while fresh oil is around neutrality. Hence, it can be said that oxidation of oils accompanies a

change in acidity - from neutral to acidic. This can be due to the degradation products, acids. Oxidation is the type of rancidity occurs in vegetable shortenings. This can be prevented by the addition of antioxidants, substances that can inhibit oxidation. Two naturally occurring antioxidants are vitamin C and vitamin E.

Saponification The chemical reaction of an ester with a strong base such as NaOH to form soap and glycerol is called saponification. It is the process used in making soaps. Soaps have been made from vegetable and animal oils and fats cooked in alkali bases like sodium hydroxide and potassium hydroxide (Zaballa et al., 2010).

Table 5. Making of soap Sample Used Coconut oil + alcoholic KOH Observation Soft solid/ yellow

Table 5 shows that saponification of coconut oil with KOH will produce a soft and yellow soap. According to Sackheim and Lehman (1998), potassium soaps are soft or liquid soaps.

Properties of Soap Soap solution in saturation with NaCl forms curds or granular solid (Table 6.a.). With the addition of NaCl, excess Na ions are produced which reacts with esters anions to form precipitate. This is called as salting out.

Table 6.a. Salting Out Sample Used Soap solution + saturated NaCl Observation Formation of curds, granular solid

Addition of NaCl in the manufacture of soaps is sometimes done in order to increase yield and to make the soap harder since it allows the separation of the glycerol and the soap. This may be the reason why soaps do not form suds in seawater.

Table 6.b. Formation of Fatty acids Sample Used Soap solution + HCl Observation Formation of a white precipitate

The soap produced with saponification of coconut oil with alcoholic KOH is a potassium ester. This means that the soap contains positive and negative charges. With the addition of excess HCl, the K+ ions of soap react with the Cl- ions of the acid to form the white precipitate (Table 6.b.). Furthermore, the ester ions of soap react with H+ ions of the acid to produce the fatty acid. Coconut oil contains caprylic acid (Sackheim and Lehman, 1998), thus it can be said that this is the fatty acid formed when the potassium soap is reacted with HCl. Calcium and magnesium ions present in hard water react with soap to form insoluble calcium and magnesium soaps. As presented in Table 6.c., soap solution gives the formation of precipitate. The precipitates are the calcium and magnesium soaps.

Table 6.c. Insoluble Salts Observation Ions Solution Soap Solution 5% Calcium chloride 5% Magnesium chloride Formation of a precipitate Formation of a precipitate Detergent Solution Clear solution/ no precipitate Clear solution/ no precipitate

On the other hand, detergent solution does not allow any formation of precipitate with the addition of calcium chloride and magnesium chloride 6.c.). This shows the cleansing power of detergents even in hard water. Detergents like soaps have a nonpolar part and a polar part. Since most dirt are held on skin and clothing by a thin layer of grease or oil, these as nonpolar will dissolve in nonpolar part of the detergent. Mechanical washing causes the oil and grease to break up into small drops. The detergent then emulsifies that oil and grease, which can be easily washed away (answer to Question 5). Detergents also act as a surfactant; it lowers surface tension of the water, making emulsification easier. (Table

Liebarmann-Burchard Test for Cholesterol Cholesterol which is found in animal fats is the most common sterol. Sterols are those steroids containing one or more OH groups and no C=O groups. Steroids are high molecular mass tetracyclic (four-ring) compounds. This four-ring structure is a parent compound of steroids like cholesterol (answer to Question 6).

Parent Compound

Table 7. Result of Liebermann-Burchard Test Sample Used Observation From clear solution-yellow color solution -brick red color - dark red brown color to violet green color


LiebermanBurchard test is a colorimetric test to detect cholesterol, which gives a deep green color (Table 7). The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the conjugation of the unsaturation in the adjacent fused ring. This test uses chloroform, acetic anhydride and sulfuric acid as reagents. According to Sackheim and Lehman (1998), the deposition of excess lipids, primarily cholesterol from the bloodstream results to atherosclerosis. One way of combating heart disease and atherosclerosis is to reduce concentration of cholesterol in the bloodstream. In a study conducted by Weggeman et al. (2001), they supported the advice to reduce consumption of eggs in order to limit cholesterol intake. They also validated studies that dietary cholesterol (from eggs) raises the ratio of total cholesterol to HDL cholesterol, thereby affecting the cholesterol profile.

Emulsifying Action of Lecithin Emulsions are colloidal solutions that are not clear, homogeneous, do not settle and do not pass through a filter paper. As shown in Table 8, the mixture albumin solution + cholesterol + lecithin produces a homogeneous mixture, therefore the one which forms emulsion. Whereas, albumin solution and cholesterol forms a heterogeneous mixture. Cholesterol is not soluble to albumin solution, but with the presence of lecithin, the cholesterol dissolves in albumin.

Table 8. Result of Emulsification Test Sample Used Albumin solution + cholesterol Albumin solution + cholesterol + lecithin Observation Solution is slightly turbid: with traces of undissolved cholesterol Solution is yellow-colored: formation of an emulsion

In this test, it can be observed the emulsifying action of lecithin. For lipids to become a good emulsifier, it must contain a nonpolar region that attracts the nonpolar substances and a polar region to attract the polar molecules. Lecithin is a fatty acid that acts as a good emulsifier because it contains a nonpolar and a polar portion by which it helps the two immiscible substances to be mix or to lessen their surface tension. When it is mixed with albumin, a solution is formed which is capable of dissolving the cholesterol (answer to Question 7).

Conclusion: Lipids are insoluble to water, hence they are nonpolar compounds. They can be saturated or unsaturated. Fats and oils give positive results to the Acrolein test since are capable of producing glycerol when they are hydrolyzed. Lipids can be oxidized or hydrolyzed to form fatty acids. This causes rancidity as indicated by becoming acidic.