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
/>sheets which our assistant handed out (no source)
page 5
IR reference spectrum for cholesterol
/>(January 29, 2005)