Organic chemistry practical course (OCP1) WS 04/05 529-0229-00 Assistant: Fabio Di Lena Isolation of cholesterol, lecithin and fatty acids from egg yolks HO CH 3 CH 3 CH 3 CH 3 CH 3 H H Presented by Moreno Wichert Student of Interdisciplinary Natural Sciences (Biochem Phys.) D-CHAB, ETH Zurich Nuolen SZ January 29, 2005 H 2 C HC H 2 C O C R 2 O C R 1 O O O P O O - O CH 2 CH 2 N(CH 3 ) 3 Choline R COOH 1. Method Isolation of cholesterol from egg yolks. Many other compounds are also present in egg yolks, including lecithins and fats, which will also be isolated. Cholesterol, lecithins and fats are all soluble in a combination of ether and alcohol. Extraction of pulverized egg yolks with ether and alcohol therefore seperates these compounds and some others from all the insoluble compounds in egg yolks. H 2 C HC H 2 C O C R 2 O C R 1 O O O C R 3 O 3 KOH H 2 C HC H 2 C OH OH OH - O C R 2 - O C R 1 O O - O C R 3 O K + K + K + 2. Outline of the procedure page 2 Egg yolks extraction with ether and alcohol Insoluble compounds (separate) Solution of cholesterol, lecithins, fats and other compounds evaporation of solvent Residue of cholesterol, lecithins, fats and other compounds ether Solution of cholesterol, lecithins, fats and other compounds acetone Precipitation of lecithins (seperate) Solution of cholesterol, lecithins, fats and other compounds evaporation of solvent Residue of cholesterol, lecithins, fats and other compounds KOH, ethanol, reflux Solution of cholesterol, glycerol, soap and other compounds 1. add water 2. extract with water Aqueous solution of soap and glycerol (seperate) Ether solution of cholesterol and other compounds (seperate) evaporation of solvent Residue of cholesterol and other compounds recrystallization Cholesterol HCl Glycerol and precipitation of fatty acids extract with ether Ether solution of fatty acids evaporation Fatty acids page 3 4. Experimental accomplishment Removal of insoluble material Two eggs were heated in boiling water for 10 minutes. The rest of the eggs was seperated from the yolks which then were pulverized and weight. The pulverized yolks were placed into a 250 ml beaker and 40 ml of methanol 1 and 50 ml of diethyl ether 2 were added. After mixing well for 5 minutes, the substance was gravity fi ltered into another 250 ml beaker while keeping the insoluble material in the fi rst beaker. Another 20 ml of ether were added to the insoluble material in the fi rst beaker and fi ltered once again. The alcohol-ether solution was evaporated to dryness with a rotation evaporator. A thin yellow residue appeared. Isolation of lecithins 25 ml of ether were added to the residue and it was slightly warmed to dissolve all the ma- terial. Acetone 3 was added slowly with constant stirring until the lecithins precipitated as a gummy bulk. As much as 150 ml of acetone were needed. The solution was gravity fi ltered into a 250 ml round-bottom fl ask using a loose wad of cot- ton. The lecithins were washed with 10 ml of acetone and fi ltered into a round-bottom fl ask using the same cotton wad. The lecithins were thoroughly dried, weight and the percentage yield was determined. Isolation of cholesterol The acetone-ether solution was distilled to obtain a liquid oil residue. 25 ml of a 15 % potas- sium hydroxide 4 solution in ethanol were added to the distillate. The mixture was refl uxed for 25 minutes. The solution was cooled to room temperature and it was poured into a 250 ml seperatory funnel. 35 ml of water were added to the round-bottom fl ask and after swirling the wash water was added to the seperatory funnel. This procedure was repeated with another 35 ml of water. The water-alcohol solution was extracted twice with 35 ml portions of ether. The two extracts were combined, then the ether was washed with 10 ml of water. This water was also added to the alcohol-water solution and it was saved for the isolation of the fatty acids. The ether solution was shaken with 15 ml of saturated salt solution to lower the water content. The solutions were seperated and further the ether solution was dried over a small amount of calcium chloride 5 . The solution was fi ltered into a tared 150 ml beaker and it was evaporated to dryness with the rotation evaporater. The cholesterol was recrystallized from methanol using decolorizing carbon 6 . The purifi ed product was dried thoroughly and the weight and the percentage yield was determined. Isolation of the fatty acids The aqueous solution containing the soap was acidifi ed by carefully adding concentrated hydrochloric acid 7 with stirring until a drop of the solution turned blue litmus paper red and the fatty acids had precipitated. Then the entire mixture was placed into a 250 ml seperatory funnel and extracted with 25 ml portions of ether. The ether extracts were combined, shaken with 15 ml of saturated salt solution to reduce the water content, seperated, then further dried over a small amount of calcium chloride. The dried ether was fi ltered into a 125 ml Erlenmeyer fl ask and the solvent was evaporated with a rotation evaporater. The yield and the percentage yield were then determined. page 4 Physical and safety data of the substances 1) CH 4 O ; M = 32.04 g mol -1 ; ρ = 0.791 g cm -3 ; melting point = -98 °C ; boiling point = 64.7 °C ; poison class 3 ; WGK 1 ; risk phrases: R11-23-24-25-39 ; safety phrases: S7-16-36-37-45 ; MAK: 270 mg m -3 2) C 2 H 5 OC 2 H 5 ; M = 74.12 g mol -1 ; ρ = 0.714 g cm -3 ; melting point = -116 °C ; boiling point = 35 °C ; WGK 1 ; risk phrases: R12-19-22 ; safety phrases: S9-16-29-33 3) C 3 H 6 O ; M = 58.08 g mol -1 ; ρ = 0.79 g cm -3 ; melting point = -94 °C ; boiling point = 56.2 °C ; poison class 5 ; WGK 1 ; risk phrases: R11-36-66-67 ; safety phrases: S9-16-26 ; MAK: 1200 mg m 3 4) KOH ; M = 56.10 g mol -1 ; ρ = 2.04 g cm -3 ; melting point = 360 °C ; boiling point = 1320 °C ; poison class 2 ; WGK 1 ; risk phrases: R11-22-34-35 ; safety phrases: S7-16-26-36-37-39-45 5) CaCl 2 ; M = 110.99 g mol -1 ; ρ = 2.15 g cm -3 ; melting point = 782 °C ; boiling point > 1600 °C ; poison class 5 ; WGK 1 ; risk phrases: R36 ; safety phrases: S22-23-24 6) C ; M = 12.01 g mol -1 ; melting point > 2000 °C ; WGK 0 ; risk phrases: R11 ; safety phrases: S2-16 7) HCl ; M = 36.46 g mol -1 ; ρ = 1.06 g cm -3 ; melting point = - 40 °C ; boiling point = 84 °C ; poison class 3 ; WGK 1 ; risk phrases: R34-36-37-38 ; safety phrases: S26 ; MAK = 8 mg m -3 5. Analytical results and characterization Yield: lecithin: 4.1 g cholesterol: 0.2 g fatty acids: 1.3 g IR bands [cm -1 ]: lecithin: cholesterol: fatty acids: 3352 (-NH or crystal water) 3607 (O-H stretch) 2855 (C-H stretch) 2939 (C-H stretch) 2928 (C-H stretch) 1731 (keton group) 1602 (aromatic system) 1710 (keton group) 1209 (-N + R 3 ) 1467-1100 (methyl groups) 1214 (0=C-O-C=O) 6. Literature http://www.aist.go.jp/RIODB/SDBS/sdbs/owa/sdbs_sea.cre_frame_disp?sdbsno=887 sheets which our assistant handed out (no source) page 5 IR reference spectrum for cholesterol http://www.aist.go.jp/RIODB/SDBS/sdbs/owa/sdbs_sea.cre_frame_disp?sdbsno=887 (January 29, 2005) . alcohol Insoluble compounds (separate) Solution of cholesterol, lecithins, fats and other compounds evaporation of solvent Residue of cholesterol, lecithins, fats and other compounds ether Solution of cholesterol, lecithins, fats. other compounds acetone Precipitation of lecithins (seperate) Solution of cholesterol, lecithins, fats and other compounds evaporation of solvent Residue of cholesterol, lecithins, fats and other compounds KOH, ethanol, reflux Solution. Method Isolation of cholesterol from egg yolks. Many other compounds are also present in egg yolks, including lecithins and fats, which will also be isolated. Cholesterol, lecithins and fats