BIOCHEMISTRY - Laboratory Practical - NAU 2015

MODULE IV

METABOLISM OF MAJOR CLASSES OF BIOMOLECULES

Laboratory Work 12

EVIDENCE OF STRUCTURE AND PROPERTIES OF LIPIDS

Objective: to investigate the solubility of Lipids, master Methods for lipid detection, prove their unsaturation, and isolate the constituent components of Simple Lipids.

Basic Theoretical Background

Lipids are bioorganic molecules that vary in Structure and function; all are insoluble in polar Solvents, such as Water, but soluble in non-polar organic solvents (acetone, chloroform, diethyl ether, etc.). When fats are mixed with water, emulsions are formed, the stability of which depends on the medium in which they are created. The presence of emulsifying agents in water (soaps, Bile acids, carbonates, etc.) increases emulsion stability. Emulsion formation is due to surface-active particles of bile acids, soap, or carbonate migrating into the surface water layer surrounding fat droplets, coating them and preventing their coalescence.

Lipids are predominantly esters of alcohols and Higher Fatty acids (HFAs). Higher fatty acids contain more than 10 carbon atoms and can be saturated or contain double bonds. Natural HFAs have an even number of carbon atoms and a zig-zag conformation of the radical; for unsaturated ones, the double bonds are methylene-interrupted. Vitamin F is a complex of polyunsaturated HFAs. Arachidonic acid is a polyunsaturated HFA from which tissue Hormones are synthesized in the body.

Simple lipids are composed solely of alcohol and HFAs. Complex Lipids contain additional components such as phosphates, CARBOHYDRATES, amino alcohols, Amino Acids, etc. Phospholipids are the primary molecules that make up Introduction/36.html">Biological Membranes. Lipids are an important source of energy and accumulate in the body. Reserve lipids are neutral lipids (triglycerides (TGs), triacylglycerols). They are composed of the trihydric alcohol glycerol and HFAs.

Equipment: test tube rack, pipettes, dropper bottles, volumetric flasks, beakers, funnels, water bath, gas burner.

12.1. Solubility of lipids and emulsion formation

Materials and Reagents: vegetable oil, alcohol, benzene, chloroform, 1% Na₂CО₃ solution.

Procedure

Pour 0.2–0.3 ml of vegetable oil into four test tubes, then add 5 ml of water to the first, 5 ml of alcohol to the second, 5 ml of benzene to the third, and 5 ml of chloroform to the fourth. Vigorously shake the contents of all test tubes. In the first test tube, the oil and water quickly separate into two layers; In the second test tube, a turbid solution forms due to the insufficient solubility of oil in alcohol; in the third and fourth, the solutions are clear.

Add a few drops of oil to two test tubes. Add 2 ml of water to one of them, and 2 ml of Na₂CО₃ solution to the second. Intensely shake the Contents of the test tubes and observe The formation of an emulsion. Note the different Stability of the emulsions in the two test tubes.

12.2. Detection of lipid unsaturation

Materials and reagents: solution of butter in chloroform (0.2–0.25 g of butter in 1 ml of chloroform), solution of vegetable oil in chloroform (the same amount of oil in 1 ml of chloroform), saturated bromine water (5 g of bromine with 100 ml of water in a Glass-stoppered flask, shaken in a fume hood, occasionally opening the stopper to release bromine vapors).

Procedure

Fats of plant origin contain a higher proportion of unsaturated fatty acid residues than fats of animal origin. The varying degree of lipid unsaturation can be demonstrated by bromination of butter or vegetable oil.

Place 1 ml of the vegetable oil solution in chloroform into one test tube, and 1 ml of the butter solution in chloroform into the second. Then add bromine water dropwise to each test tube until decolorization ceases. Record The amount of bromine water added to each test tube.

12.3. Saponification of fat, formation of insoluble soap
and isolation of free fatty acids

Materials and reagents: vegetable oil, 50% alcoholic KOH or NaOH solution, 5% СаС1₂ solution, concentrated HCl.

Procedure

Under the action of alkali, fats undergo Hydrolysis to form soaps and glycerol:

Class="center"> Tristearin     Glycerol  Sodium stearate

Add 20 mL of an alcoholic KOH (NaOH) solution to a flask containing 1 mL of oil, mix, and boil on a water bath under a reflux condenser for 1 hour. After saponification, dilute the solution with water to a total volume of 20 mL. The resulting soap solution is used for subsequent reactions.

Add 1 mL of a CaCl₂ solution to a test tube containing 2 mL of the soap solution.

When a calcium salt solution is added to a potassium soap solution, insoluble calcium salts of Fatty acids are formed:

An insoluble calcium soap (calcium stearate) is formed.

Add 1 mL of concentrated HCl to a test tube containing 2 mL of the soluble soap solution. The addition of concentrated Hydrochloric acid to the soap yields free fatty acids:

The formation of insoluble free acids is observed, which collect at the top of the test tube.

12.4. Qualitative reactions for lecithin

Materials and reagents: dried egg yolk, alcohol, acetone, saturated solution of cadmium chloride CdCl₂ in ethanol, 5% alcoholic vanillin solution, concentrated sulfuric acid, filter paper.

Procedure

Lecithin (phosphatidylcholine) is one of the phosphoacylglycerols found in biological membranes. Lecithin is insoluble in acetone and forms a stable emulsion with water. Qualitative tests for lecithin include reactions with cadmium chloride, vanillin, and sulfuric acid.

Add 15 mL of hot alcohol to a beaker containing 200–300 mg of ground egg yolk and mix. After
10–15 min, cool the mixture and filter it into a dry test tube. Pour 2–3 mL of acetone into another dry test tube and add the obtained filtrate dropwise. The appearance of turbidity followed by the precipitation of lecithin indicates that lecithin is insoluble in acetone.      Lecithin

Add distilled water dropwise to 2–3 mL of the filtrate to form a stable emulsion.

Add an equal amount of a saturated alcoholic cadmium chloride solution to 2–3 mL of the filtrate. A yellowish-white precipitate is formed.

Add 2 drops of a 5% alcoholic vanillin solution to 2 mL of the filtrate, and carefully layer 1 mL of concentrated sulfuric acid down the side of the tube. A red ring is formed.

Processing of experimental data

Write the condensed and structural formulas for palmitic, stearic, arachidic, oleic, palmitoleic, linoleic, linolenic, and arachidonic acids. Classify these higher fatty acids (HFAs).

Construct a table presenting the formulas of various lipid classes, their biological roles, identification reactions, and chemical properties.

Provide the formulas for various types of soaps. Write the reaction equations for the formation of liquid soap from solid fat, solid soap from liquid fat, and solid insoluble soap from liquid fat.

Selection/5.html">Control Questions and tasks

1. How are lipids classified? Which lipids are stored in fat depots? Write the general structural formula for such lipids. Which lipids serve as the main structural component of biological membranes? Write the formula for any one of them.

2. What reactions are common to The Biosynthesis of neutral lipids and phosphoacylglycerols? Under what conditions does phospholipid synthesis predominate?

3. What transport forms of lipids do you know? What structural components do they consist of? What is the Biological Significance of each?

4. Write the reaction of an unsaturated lipid with bromine water, and Wagner's reaction with oleic acid.

5. WHAT IS A soap? Which soaps are liquid and which are solid? Which soaps are readily soluble in water and which are insoluble? From what substances can soap be obtained? Write the reaction equations. Why are liquid or soluble solid soaps not used for washing in seawater?

6. What metabolic transformations are characteristic of lipids?

7. Write the reactions of anaerobic and aerobic oxidation of glycerol, and calculate its energy yield.

8. Calculate the energy yield of the complete aerobic oxidation of a tristearin molecule.

9. Which Vitamins are essential for Lipid METABOLISM?

References: [1; 2; 4–7].