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EXPERIMENT 1: EXTRACTION, FRACTIONIZATION AND IDENTIFICATIONOF BRAIN LIPIDS The brain has a high content of lipid compared to all other tissues of the body. It contains a variety of lipids. Complex lipids such as the phosphatides, cerebrosides, and sphingosides contain fatty acid components of the C-16 to C-24 carbon chains, chiefly in the form of stearic, oleic, linoleic and arachidonic acids are found in the brain. The steroids alcohol, cholesterol, occurs abundantly in the brain. This experiment may be divided into four major parts: 1. Extraction of the total lipids. 2. Isolation of the triglycerides and saponificaiton of the triglycerides make way for the study of soaps, fatty acid and glycerol. 3. Isolation and tests on lecithin 4. isolation and tests on cholesterol Objectives: 1. To be able to study the properties of various lipids. 2. To be able to classify and identify the lipids. Procedure: 30 grams of pigs brain (homogenized in ether-alcohol solution) TOTAL LIPID EXTRACT

2/3 portion Isolation of Triglycerides

1/3 portion Lecithin-Cholesterol Isolation

20 ml portion Test on Soaps 1. Salting Out 2. Insoluble Salts 3. Surface Tension

30 ml portion A. Test on Fatty Acids 1. Solubility 2. Translucent Spot 3. Iodine Absorption B. Test on Glycerol 1. Solubility 2. Translucent Spot 3. Acrolein 4. Benedicts Test

A. Test on Lecithin 1. Acrolein 2. Iodine Absorption 3. Phosphorus B. Test on Cholesterol 1. LiebermannBurchard Test 2. Salkowski Test

20 ml Portion Test on Soap

Salting Out 5 ml of Soap Solution

Insoluble Soaps 5 ml 5 ml Distilled Water

Surface Tension 5 ml 5 ml Soap 5 ml Solution Solution 2% NaCl

Add NaCl

Add CaCl2

Add MgSO4

Add Sulfur Powder




Add Water

Add Water

Add Water





Results and Observations Test Salting Out Insoluble salts Obtained Results A white precipitate was formed, and dissolved upon addition of water. CaCl2 and MgSO4 formed white precipitates and upon contact with water, it did not dissolve. In water: The sulfur powder stayed at the water surface (floated). In soap solution: The sulfur powder went at the bottom of the tube (sank). In 2% NaCl: The sulfur powder stayed at the water surface (floated). Discussion: Lipids that contain fatty acid ester linkages can undergo hydrolysis, a reaction that is catalyzed by strong acid or base. Alkaline hydrolysis of fatty acid esters is known as saponification. Saponification of triglycerides produces glycerol and fatty acids salts. High molecular mass of fatty acid salts are commonly referred to as soap. The chemical reaction between a triglyceride and a base to form glycerol and fatty acids salts is shown below. Expected Results White precipitate which dissolves in water. White precipitates which are insoluble in water In Water: The sulfur powder floats. In soap solution: The sulfur powder sinks. In 2% NaCl: The sulfur powder floats.

Surface Tension

When the hydrolyzed solution is saturated with NaCl, the soap separates. This principle is used in manufacturing soaps. Insoluble soaps are formed when calcium and magnesium react with the higher fatty acids of soaps and forms an insoluble white curd like precipitate of magnesium or calcium salts (scum).

Surface tension expresses the force with which the surface molecules attract each other. It is also a property of liquids arising from unbalanced molecular cohesive forces at or near the surface, as a result of which the surface tends to contract and has properties resembling those of a stretched elastic membrane. Water has a high surface tension because of its hydrogen bonding as shown below.

Substances that break hydrogen bonding of water that causes to lower its surface tension are called surfactants. Soap is an example of surfactant. Soaps have an unusual behavior in water because it comprises both hydrophilic and hydrophobic regions. The cleaning action of soaps is due to the arrangement of the hydrophilic (polar) heads, which are the carboxyl groups; and hydrophobic (nonpolar) tails are the hydrocarbon chains. When in water the surfactant molecules reversibly assemble into polymolecular aggregates called micelles. By gathering the

hydrophobic chains together in the center of the micelle, the grease or oil present in the solution will be emulsified by the hydrophobic tail inside of the micelles and it is rinsed away. Hydrophilic heads Hydrophobic tails

Hydrophobic (nonpolar) tail

Hydrophilic (polar) head

30 ml Portion A. Test on Fatty Acids

Solubility Heat the solution

Translucent Spot Test Small amount of precipitate

Iodine Absorption Test Small amount of precipitate

Add HCl

Ordinary writing paper

Add 5 ml of CHCl3



Add Hubls iodine

Place in water

Place in CHCl3



30 ml Portion B. Test on Glycerol

Solubility Precipitate

Acrolein Test 0.5 grams of powdered KHSO4

Translucent Spot Test Precipitate

Benedicts Test Litmus paper If acidic



Add 10 drops of Glycerol solution

Ordinary writing paper

Add dilute Na2CO3


in 5 ml Benedicts Reagent


Note the odor


Add 5 drops of Glycerol solution Boil for 2-3 mins.


Results and Observations: Test Solubility test H2O CHCl3 Translucent Spot test FATTY ACID Obtained Results insoluble soluble It formed a spot and remained even after long standing Expected Results insoluble soluble There is a presence of translucent spot and remains even after long standing

Iodine Absorption test

The iodine solution was The iodine solution decolorized decolorized GLYCEROL Obtained Results

Test Solubility test H2O CHCl3 Translucent Spot test

Expected Results

soluble soluble insoluble insoluble It formed a spot but it did There is a presence of not remain even after long translucent spot and standing remains even after long standing pungent odor negative Pungent odor positive

Acrolein Test Benedicts test

Discussion: The degree of solubility in water and in nonpolar solvents of fatty acids will depend on whether it is a saturated or unsaturated and if it is saturated whether it is short-chain, medium-

chain or long-chain fatty acids. Below is a table showing the solubility of a short chain saturated fatty acid, long-chain saturated fatty acids and unsaturated fatty acids in water Short-chain saturated fatty acids Limited solubility Long-chain saturated fatty acids Insoluble Unsaturated fatty acids Insoluble

Solubility in Water

However, glycerol behaves differently in water from fatty acids. Glycerol is soluble in water but insoluble in nonpolar solvents. Glycerol is soluble in water because it contains three hydroxyl or alcohol groups. These hydroxyl or alcohol groups are hydrophilic and are responsible the solubility of glycerol in water. Another test for fatty acids and glycerol is the translucent spot test, which is a simpler test for the lipids wherein marks on an unglazed paper shows the presence of triglycerides (fats/glycerol). Below is a figure of a translucent spot test. On the left is a negative result while on the right shows a spot which is a positive result.

NEGATIVE (No spot formed)

POSITIVE (A translucent spot is formed)

The volatility of fatty acids will also depend whether the fatty acids are saturated or unsaturated and if saturated whether it is a short-chain, medium-chain or long-chain fatty acids. Below shows the difference of short-chain, long-chain fatty acid and unsaturated fatty acid. Short-chain saturated fatty acids Volatile Long-chain saturated fatty acids Nonvolatile Unsaturated fatty acids Nonvolatile


Acrolein test is a test for the presence of glycerin or fat. Acrolein (CH2=CH-CHO), 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. This unsaturated aldehyde is produced by the loss of water by the glycerol portion of the fat molecule. In biological membrane this will also result in the formation of free radicals, a process known as lipid peroxidation.


Acrolein (pungent odor)

Another reaction exhibit by fatty acids is halogenation reaction which is the basis of the chemical procedure known as Iodine Absorption test. It is use for determination of unsaturation of fats. Halogens ( I, Br ) will add across the double bonds and thus the decolorization of an iodine or bromine solution will indicate the presence of unsaturated fatty acids. Iodine from Huble's solution adds up across the unsaturation sites of oil or fat giving a colourless product and a little more of iodine gives a permanent violet colour to the solution.

H3C(CH2)16COOH + I2 H H

no re a cion t

Benedict's test identifies reducing sugars based on their ability to reduce the cupric (Cu2+) ions to cuprous oxide at basic (high) pH. Cuprous oxide is green to reddish orange. In this carbohydrate test, a green solution indicates a small amount of reducing sugars, and reddish orange indicates an abundance of reducing sugars. Nonreducing sugars such as sucrose produce no change in color (the solution remains blue). Monosaccharides such as glucose and fructose are reducing sugars, meaning that they possess free aldehyde (-CHO) or ketone (-C=0) groups that reduce weak oxidizing agents such as the copper in Benedict's reagent. Benedict's reagent contains cupric (copper) ion complexes with citrate in alkaline solution.

H3C(CH2)7 C =C (C H2)7CO O H + I2


1/3 Portion A. Test on Lecithin 1/3 portion Heat until syrupy consistency

Add 15 ml of ether


Add 15 ml acetone


Precipitate (lecithin)

Filtrate (cholesterol)

Precipitate (lecithin) Acrolein test 0.5 grams of powdered KHSO4 Iodine Absorption Test Small amount of Precipitate Test for Phosphorus Small amount of Precipitate

Add 10 drops of glycerol Solution Heat

Ordinary writing paper

Add 5 ml of 10% HCl

Boil for 10 mins.

Note the odor


Add equal volume of Conc. HNO3 and ammonium molybdate solution Warm

Yellow crystals

Filtrate (cholesterol) Evaporate the ether-acetone filtrate Cool

Add CHCl3 Filter

Clear solution

Salkowski test 2 ml

Liebermann-Burchard test 2 ml

Add equal volume of H2SO4 and CHCL3

Add 10 drops acetic anhydride


Add 2 drops of conc. Sulfuric acid Stand for 15 mins.

Note the end color Results and Observations: Lecithin TEST Acrolein test Iodine absorption test Obtained Result It produces an unpleasant odor (pungent) Pinkish color of precipitate region upon dropping of iodine (decolorization of iodine) Yellow precipitate formed and formation of crystals upon cooling Cholesterol Test Salkowski test Obtained result CHCl3: cherry red color Acid layer: clear Dark green color Expected result CHCl3: Cherry red color Acid layer: fluorescent green Green color Expected Result Pungent Decolorization of iodine

Test for phosphorus

Yellow precipitate formed and formation of crystals upon cooling

Liebermann-Burchard reaction

Discussion: As aforementioned, Acrolein test is a test for the presence of glycerine or fat. Lecithin or phosphatidyl choline is a phosphoglycerides which, the parent compound is phosphatidic acid and its structural backbone is glycerol. Lecithin produce acrolein and will yield expected result in iodine absorption test because the presence of double bonds. As we stated earlier that lecithin has a phosphatidic acid as its parent compound, it will yield yellow crystal, an expected result in

test for phosphorus. Test for phosphorus detects the presence of free phosphate in acidic solution can be detected by adding a molybdate to the solution. HPO42-(aq) + 12MoO42-(aq)+3NH4+(aq)+23H3O+(aq) (NH4)3[P(Mo3O10) 4](yellow,s) + 35 h20(l) The equation above illustrates the pertinent reaction between phosphate and ammonium molybdate solution in presence of nitric acid. When lipids contains phosphate groups in their structures are added to a strong acid solution such nitric acid, the lipid hydrolyses, producing free phosphate. The free phosphate then reacts as in equation above, forming yellow precipitate which forms crystals upon cooling.

For cholesterol analysis we perform the Salkowski test and the Liebermann-Burchard Test. The Salkowski test is a qualitative analysis of cholesterol detection in natural products. Salkowski reaction is the reaction of cholesterol with concentrated sulphuric acid. Concentrated sulphuric acid is highly hygroscopic and it removes two molecules of water from two molecules of cholesterol, it causes a connection at position 3, forming bi- cholestadien (a). Simultaneously the sulphuric acid sulphonates the molecule of bi-cholestadien at positions 7,7 of aromatic ring and, as a final product, red colour bi-sulphonic acid of bi-cholestadiene is formed (b).

a. bi-cholestadien


bisulfonic acid of bi-cholestadiene

Liebermann-Burchard Test on the other hand is a colourimetric test for unsaturated sterols, notably cholesterol; a blue-green color develops when such substances are added to acetic anhydride and sulphuric acid in chloroform

Some biological importance of lipids Surfactant in lungs is dipalmitoyl lecithin which lowers the surface tension in the alveoli. The result of cohesive forces where molecules pulls each other inward is surface tension. This collapses the alveoli, but because there is a presence of surfactant in our lungs, it lowers the surface tension thus preventing it to collapse during expiration also a study is conducted that surfactant proteins present in the lungs can recognize bacterial, viral and fungal surface oligosaccharides and thus can opsonize these pathogen Bile Acids 1. Primary Bile Acids Cholic Acid and chenodeoxycholic Acid Synthesized in the liver from cholesterol 2. Secondary Bile acids Deoxycholic acid and lithocholic Acid Synthesized in the small intestines from the primary bile acids FUNCTIONS: Aid in fat digestion and absorption Facilitate absorption of fat-soluble vitamins (A,D,E,and K) Help in solubilization of cholesterol in bile C21H30O2 Progesterone Chemical name:Preg-4-ene-3,20-dione FUNCTION Intermediate metabolite in biosynthesis of other steroid hormones Maintain pregnancy during the first trimester Involved in menstrual cycle Drug for threatened abortion Component of oral contraceptives

C21H30O5 Cortisol Chemical name: 11beta,17alpha,21-Trihydroxypegn-4-ene-3,20-dione FUNCTIONS Regulate intermediary metabolism Immunosuppressive and anti-inflammatory action Play an important role in stress response.

C21H28O5 Aldosterone FUNCTIONS Regulates water and sodium metabolism Involved in blood pressure regulation

C19H28O2 Testosterone Chemical name:17beta-Hydroxyandrost-4-en-3one FUNCTIONS Normal male sexual development of the embryo Develops and maintain male secondary sexual characteristics Important for male fertility

C18H24O2 Estradiol Chemical name: estra-1,3,5(10)-triene-3,17beta-diol FUNCTIONS Normal female sexual development Development of female secondary sexual characteristics Induce differentiation of mammary glands Involved in menstrual cycle Affects synthesis of neurotransmitter and receptor protein in the CNS Influences lipid metabolism increase HDL, decrease LDL