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 CH3

CH3

CH3 CH3 CH3

H

H

HO

O H2C

O

C

R1

O HC

O

C

R2

O H2C

O

P O-

O

CH2CH2

N(CH3)3

Choline

COOH R

Presented by Moreno Wichert Student of Interdisciplinary Natural Sciences (Biochem.-Phys.) D-CHAB, ETH Zurich Nuolen SZ January 29, 2005

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. 2. Outline of the procedure extraction with ether and alcohol

Egg yolks

Insoluble compounds (separate)

Solution of cholesterol, lecithins, fats and other compounds evaporation of solvent

Solution of cholesterol, lecithins, fats and other compounds

ether

Residue of cholesterol, lecithins, fats and other compounds

acetone Precipitation of lecithins (seperate)

Solution of cholesterol, lecithins, fats and other compounds

Residue of cholesterol, lecithins, fats and other compounds

evaporation of solvent

KOH, ethanol, reflux Aqueous solution of soap and glycerol (seperate)

Ether solution of cholesterol and other compounds (seperate)

Solution of cholesterol, glycerol, soap and other compounds

1. add water 2. extract with water

HCl

evaporation of solvent Residue of cholesterol and other compounds

Glycerol and precipitation of fatty acids

extract with ether

Ether solution of fatty acids evaporation

recrystallization

Fatty acids

Cholesterol O H2C

O

C

O R1

H2C

OH

K+ -O

O HC

O

C

O

C

R1

O R2

3 KOH

HC

OH

K+ -O

O H2C

C

C

R2

O R3

H2C

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OH

K+ -O

C

R3

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 methanol1 and 50 ml of diethyl ether2 were added. After mixing well for 5 minutes, the substance was gravity filtered into another 250 ml beaker while keeping the insoluble material in the first beaker. Another 20 ml of ether were added to the insoluble material in the first beaker and filtered 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 material. Acetone3 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 filtered into a 250 ml round-bottom flask using a loose wad of cotton. The lecithins were washed with 10 ml of acetone and filtered into a round-bottom flask 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 % potassium hydroxide4 solution in ethanol were added to the distillate. The mixture was refluxed 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 flask 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 chloride5. The solution was filtered into a tared 150 ml beaker and it was evaporated to dryness with the rotation evaporater. The cholesterol was recrystallized from methanol using decolorizing carbon6. The purified 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 acidified by carefully adding concentrated hydrochloric acid7 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 filtered into a 125 ml Erlenmeyer flask and the solvent was evaporated with a rotation evaporater. The yield and the percentage yield were then determined.

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Physical and safety data of the substances 1)

CH4O ; 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)

C2H5OC2H5 ; 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)

C3H6O ; 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 m3

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)

CaCl2 ; 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

cholesterol:

fatty acids:

IR bands [cm-1]: lecithin:

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+R3)

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)

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IR reference spectrum for cholesterol http://www.aist.go.jp/RIODB/SDBS/sdbs/owa/sdbs_sea.cre_frame_disp?sdbsno=887 (January 29, 2005)

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