Lab 7 &8 lipids

Lipids are Biomolecules that contain fatty acids or a steroid nucleus. Soluble in organic solvents but not in water. Named for the Greek word lipos, which means “fat.” Extracted from cells using organic solvents.

Definition: Lipids are a group of naturally occurring substances consisting of the higher fatty acids, their naturally occurring compounds and substances with different structures, Organic substances relatively insoluble in water but soluble in organic solvents like chloroform, ether and benzene.

Storage form of energy Structural component of cell membrane Precursor of many steroid hormones, vitamin D Protection of internal organs

Types of lipids Lipids with fatty acids Waxes Fats and oils (trigycerides) Phospholipids Sphingolipids Lipids without fatty acids Steroids.

1. Simple Lipids : fatty acid with alcohol (GLYCEROL or other than GLYCEROL) If alcohol is glycerol …….Fats (triglyceride) and Oils Oils: Triglycerides rich in unsaturated fatty acids are generally liquid at room. Fats: Triglycerides rich in saturated fatty acids are generally semisolids or solids at room temperature. If alcohol other than glycerol …….. Waxes Result of hydrolysis gives FA with alcohol and containing additional[prosthetic] groups. 2. Conjugated Lipids (polar lipids): Result of hydrolysis gives FA with alcohol and containing additional[prosthetic] groups. A. Phospholipids - contain a phosphoric acid molecule and a fat molecule. FA + ALCOHOL + PHOSPHORIC ACID B. Glycolipid- contain a carbohydrate and a fat molecule. Glycolipids FA + ALCOHOL[SPINGOSINE] +CARBOHYDRATE WITH NITROGEN BASE C. Sulfolipids - contain a sulfate radical. D. lipoprotein ( Lipid with prosthetic group PROTEIN ) Classification of Lipids

Fatty acids Fatty acids are saturated with all single C–C bonds. are solids at room temperature unsaturated with one or more double C=C bonds. are liquids at room temperature.

Lipoproteins Combine lipids with proteins and phospholipids. Are soluble in water because the surface consists of polar lipids.

Types of Lipoproteins Lipoproteins Differ in density, composition, and function. Include low-density lipoprotein (LDLs) and high- density lipoprotein (HDLs).

Transport of Lipoproteins in the Body Copyright © 2007 by Pearson Education, Inc. Publishing as Benjamin Cummings

Lipoproteins consist of core (triacyglycerol and or cholostylester)and surface monolayer(phospholipid unesterified cholestrol and specific proteins) VLDL transport triglycerides from liver to adipose tissue and mucsle LDL transport cholesterol from liver to tissue HDL transport the cholesterol from tissues back to liver

High-density lipoprotein (HDL) Because HDL particles remove excess cholesterol from the body, they are considered protective. That’s why HDL cholesterol is often referred to as ― good cholesterol.

Low-density lipoprotein (LDL) When the cells have all the cholesterol they need, they reduce the number of receptors, or gateways, for cholesterol to enter. As a result, cholesterol contained in the LDL particles (LDL cholesterol) starts to build up in the blood stream.

Over time, circulating LDL cholesterol undergoes chemical changes (becomes oxidized) and also reduces in size. These smaller particles more easily enter the blood vessel wall and start to build up under the vessel lining. Deposits of LDL cholesterol particles in vessel walls are called plaques and can lead to inflammation, bleeding into the area, and calcium buildup (calcification). Eventually, the buildup of plaques can start to crowd the space within the blood vessel and obstruct the blood flow. This process of plaque accumulation is called atherosclerosis.

When atherosclerosis affects the arteries leading to the heart (coronary arteries), one may have chest pain and other symptoms of coronary artery disease. If plaques tear or rupture, a blood clot may form — blocking the flow of blood or breaking free and plugging an artery downstream. If blood flow to part of the heart stops, a heart attack will occur. If blood flow to the brain stops, a stroke occurs. This is why LDL cholesterol is often referred to as ―bad cholesterol.

3.Derived Lipids A. Fatty acids B. Glycerol C. Cholesterol and other steroid (Vit. D) D. Vitamins A, E, K

It include a wide variety of substances with different structures:

Cholesterol Is the most abundant steroid in the body. is a waxy substance found in all of the body cells. Has methyl CH 3 - groups, alkyl chain, and -OH attached to the steroid nucleus.

Functions help and maintain cell membranes. protect and insulate nerve fibers. involved in the formation of sex hormones, such as estrogen. production of bile salts, which help digesting fats.

Cholesterol in the body Is obtained from meats, milk, and eggs. Is synthesized in the liver. Is needed for cell membranes, brain and nerve tissue, steroid hormones, and Vitamin D. Clogs arteries when high levels form plaque. Considered elevated if plasma cholesterol exceeds 200 mg/dL.

Chemical tests for: 1. Solubility in polar and nonpolar solvent 2. Emulsification 3. Oxidation of unsaturation FA 4. Qualitative analysis of detection in natural products ( salkowski reaction)

Solubility in polar and nonpolar solvent (like dissolve like ) The solubility depend on polarity Reagents : L IPIDS : Olive oil (vegetable oil), butter (animal fat), stearic acid (saturated fatty acid), oleic acid (unsaturated fatty acid), Solvents: dilute acid and alkali solutions, acetone,cold alcohol, hot alcohol, benzene, chloroform, ether and carbon tetrachloride.

Solubility in polar and nonpolar solvent Procedure 1. Add a 2drops of the liquid fat or 0.1 g of the solid fat in labeled test tubes. 2. To each test tube add a 1 ml of solvent and write down your observations. 3. Repeat the experiment with a different solvent and make your observations.

An emulsion is a mixture of two or more liquids that are normally immiscible (no mixable).mixtureliquidsimmiscible Bile Salts Synthesized from cholesterol and stored in the gallbladder When you eat fat, the bile salts act as soap and help emulsify the large globules of fat.

A. Two immiscible liquids, not yet emulsified B. An emulsion of Phase II dispersed in Phase I C. The unstable emulsion progressively separates D. The surfactant (outline around particles) positions itself on the interfaces between Phase II and Phase I, stabilizing the emulsionsurfactant Emulsification types: Temporary and permanently Permanently more free fatty acids and no more bond found Temporary few fatty acids and few bond broken

Emulsification Reagents Neutral olive oil(partially oxidation, produce few fatty acids), 0.5 % Na 2 C0 3 (emulsifying agent), Rancid olive oil(complete oxidation many of free fatty acids) Prepare neutral olive oil: shaking ordinary oil with 10% solution of sodium bicarbonate this mixture should then be extracted with ether and the ether should be removed by evaporation Rancid olive oil: to prepare add 5 drops of oleic acid to 10 ml of olive oil and mix well

Rancidity is the development of unpleasant smells in fats and oils, which are often accompanied by changes in their texture and appearance.

Procedure 1.Shake up a drop of neutral olive oil with a little water in a test tube, the fat becomes finally divided forming an emulsion, upon standing fat separates and rises to the top. 2.To 5 ml water in a test tube add 2 to 3 drops of 0.5 percent Na 2 CO 3. Introduce a drop of neutral olive oil and shake, the emulsion is not permanent and is not so transitory. 3.Repeat step 2 using rancid olive oil. In this case the alkali combines with the free fatty acids to form soap and the soap being an emulsifying agent, it emulsifies the fat.

Oxidation of un saturation FA unsaturated fatty acids have one or more double bonds Into test tube place one drop of oleic oil and 3 ml of Na 2 CO 3 Warm it slightly and drop solution KMnO4 (oxidizing agent).After each drop the violet color disappear The end of the reaction recognized by ppt brown solid MnO2

Qualitative analysis of cholesterol detection in natural products salkowski reaction Concentrated sulfuric 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. Simulatineously the sulfuric acid sulfonates the molecule of bi cholestadien at position 7,7 of aromatic ring and as a final product, red color bi sulfonic acid of bicholestadien is formed

B) Bisulfonic acid of bi-cholestadiene

Salkowski reaction Reagents: 2% pure cholesterol, extract of yellow hen egg, lecithin and oil Chloroform(Inhalation: Inhalation of high concentrations may cause central nervous system effects characterized by nausea, headache, dizziness, unconsciousness and coma) Conc sulfuric acid

Procedure Into the dry test tubes the reaction is very sensitive for even traces of water add 1ml o chloroform solution of 2% of pure cholestrol an extract of yellow hen egg Add carfully concentrated sulfuric acid keeping the tube under 45 angle don’t mix In the presence of cholesterol, the upper chloroform layer turns red whereas a lower acidic become yellow green

Isolation of cholesterol from hen egg Hen eggEgg whiteAlbuminEgg yolk Lipids + glycoproteins

Egg yolk 5% Cholesterol 28% phospholipids 66% glycerides

Egg yolk is rich in lipids of which the most two prominent (are cholesterol and phosphatidyl choline (lectithin Phosphatidylcholine (once given the trivial name 'lecithin') is usually the most abundant phospholipid in animals and plants, often amounting to almost 50% of the total, and as such it is obviously a key building block of membrane bilayers. In particular, it makes up a very high proportion of the outer leaflet of the plasma membrane. Phosphatidylcholine is also the principal phospholipid circulating in plasma, where it is an integral component of the lipoproteins, especially the HDL.lipoproteins On the other hand, it is less often found in bacterial membranes, perhaps 10% of species, but there is none in the 'model bacteria', Escherichia coli and Bacillus subtilis.

Experiment Principle: Based on the solubility in different organic solvent, lipids can be separated from each other. Cholestrol is readily soluble in acetone, while most complex lipids are insoluble in this solvent. Reagents and instruments A hard boiled hen egg Acetone, Ice, Water bath, Erlenmeyer flask, Funnel, Filter paper, 250 ml beaker

Reagents and instruments A hard boiled hen egg Acetone, Ice, Water bath, Erlenmeyer flask, Funnel, Filter paper, 250 ml beaker

Acetone (systematically named propanone) is the organic compound with the formula (CH 3 ) 2 CO. It is a colorless, volatile, flammable liquid, and is the simplest ketone. organic compoundformulaketone

1. Extraction 1. Peal the hard boiled egg and remove the egg white. 2.In a 250 ml beaker weigh a ½ egg yolk and mash it. 3. To the mashed egg yolk add a 25 ml of acetone and keep stirring for 5 min. 4. Allow the solid to settle and carefully remove the acetone (the above liquid phase) and transfer it to a clean 100 ml Erlenmeyer flask and cover it tightly. 5. To the remained solid phase add a new 25 ml of acetone and repeat step 4. Now you have collected twice the 25 ml of acetone extract (it contains the cholesterol). 1. Peal the hard boiled egg and remove the egg white. 2.In a 250 ml beaker weigh a ½ egg yolk and mash it. 3. To the mashed egg yolk add a 25 ml of acetone and keep stirring for 5 min. 4. Allow the solid to settle and carefully remove the acetone (the above liquid phase) and transfer it to a clean 100 ml Erlenmeyer flask and cover it tightly. 5. To the remained solid phase add a new 25 ml of acetone and repeat step 4. Now you have collected twice the 25 ml of acetone extract (it contains the cholesterol).

2. Isolation 1. Using a funnel and a filter paper, filter the 50 ml of acetone extract into a clean 100 ml beaker. 2.Evaporate the acetone by placing the beaker on the steam bath in the hood until about 10 ml of extract is left. 3.Cover the warm acetone extract and immediately transfer it on ice and let it stand for 20 min. A white precipitate will form (this is the crude cholesterol). Remove the flask from ice. Get rid of the liquid phase. 1. Using a funnel and a filter paper, filter the 50 ml of acetone extract into a clean 100 ml beaker. 2.Evaporate the acetone by placing the beaker on the steam bath in the hood until about 10 ml of extract is left. 3.Cover the warm acetone extract and immediately transfer it on ice and let it stand for 20 min. A white precipitate will form (this is the crude cholesterol). Remove the flask from ice. Get rid of the liquid phase.

4.To the cholesterol precipitate add 15 ml of acetone and stir at room temp. Note that not all precipitate will dissolve (the contaminating phospholipids remain insoluble). 5. Weigh a clean, dry watch glass. Filter the dissolved cholesterol solution into the pre- weighed watch glass. Allow the acetone to evaporate in the hood. The remained crystals is the cholesterol. 6. Weigh the watch glass with cholesterol. Calculate the yield of cholesterol. 4.To the cholesterol precipitate add 15 ml of acetone and stir at room temp. Note that not all precipitate will dissolve (the contaminating phospholipids remain insoluble). 5. Weigh a clean, dry watch glass. Filter the dissolved cholesterol solution into the pre- weighed watch glass. Allow the acetone to evaporate in the hood. The remained crystals is the cholesterol. 6. Weigh the watch glass with cholesterol. Calculate the yield of cholesterol. 3. Purification

Solubility of cholesterol: Soluble in diethyl ether, acetone. Very slightly soluble in cold water. Solubility in water: 0.2mg/100ml or 0.2% Slightly soluble in alcohol; more soluble in hot alcohol. Soluble in chloroform, pyridine, benzene, petroleum ether, oils, fats, aqueous solutionsof bile salts. Solubility in ether: 1 g/2.8 ml ether. Solubilitiy in chloroform: 1 g/4.5 ml chloroform. Solubility in pyridine: 1 g/1.5 mlpyridine. whereas acetone a carbonyl group with a negatively charged oxygen and a short chain of hydrocarbons, this makes it a more suitable substance to act as a dissolvent for an amphiphilic compound.