Experiment 24:
SYNTHESIS AND GC ANALYSIS OF
FATTY ACID METHYL ESTERS
O
CH2O C R
O CH2OH RCO2CH3
KOH
CHO C R' CHOH + R'CO2CH3
CH3OH
CH2OH R"CO 2CH3
O
Glycerol Fatty acid methyl esters
CH2O C R" forms CH3O-K+ (FAMES)
and H2O
Vegetable oil
Objectives:
To synthesize fatty acid methyl esters
through transesterification of an unknown
vegetable oil.
To purify your product using column
chromatography.
To identify and analyze the purity of your
compound using TLC and GC analysis.
To identify the unknown oil based on fatty
acid methyl ester composition.
CHEMICAL EQUATION
(Hydrolysis of a fat)
O
CH2O C R
O O
CH2OH R C O R C OH
O
-
OH O O
CHO C R' CHOH + H3O
R' C O R' C OH
CH2OH O O
O
Glycerol
R" C O R" C OH
CH2O C R"
Fatty Acid salts Fatty Acids
fat
CHEMICAL EQUATION
(Transesterification of vegetable oil)
O
CH2O C R
O
CH2OH R C O CH3
O
O
KOH
CHO C R' CHOH +
CH3OH R' C O CH3
CH2OH O
O
Glycerol
forms CH3O-K+ R" C O CH3
CH2O C R"
and H2O Fatty acid methyl esters
Vegetable oil (FAMES)
COMMON FATTY ACIDS
Saturated=
NO double bonds!
O
14 Myristic Acid (saturated) 16 O Palmitic Acid (saturated)
OH 14:0 16:0
OH
18 O Stearic Acid (saturated)
18:0
OH
18
18
OH Oleic Acid (monounsaturated)
9 18:1 9 12
O
OH Linoleic Acid (polyunsaturated)
9 18:2 9,12
Monounsaturated= O
ONE double bond! Polyunsaturated=
>one double bond!
SHAPES OF FATTY ACIDS
More cis double bonds = more “bent” shape
EXPERIMENTAL PROCEDURE
(Synthesis)
Add 10 drops of unknown
oil and methanol to vial
with stir bar.
Add 1M KOH/methanol.
Stir in heat block.
Attach reflux condenser.
Heat to reflux. Maintain
temperature b/n 70-80oC
for 30 minutes.
Cool flask.
EXPERIMENTAL PROCEDURE
(Purification and Isolation)
DO NOT add Saturated NaCl
to vial.
MODIFICATION
Extract product
2X with hexane
1X with 9:1 hexane: diethyl
ether.
After each extraction step,
pass organic layer through SiO2
column into a small test tube.
Proceed to PRODUCT
ANALYSIS.
EXPERIMENTAL PROCEDURE
(Product Analysis)
TLC Analysis
Perform analysis of product along with provided
standards of the unknown oil and FAMEs solutions.
Must use 10% H2SO4 stain with heat in order to
visualize spots.
Have stained TLC plate inspected by lab instructor.
GC Analysis
With instructor permission, prepare sample for GC
analysis.
GC ANALYSIS OF FAMES
STANDARD SOLUTION
• Oncereturned, identify and quantify FAMEs
present in your unknown oil.
• Compare these results to table on p. 201 in
order to identify your unknown oil.
Table 24.1
Be sure to give
CENTIMETER
measurements for
ALL spots and give
measurement for
SOLVENT FRONT
on this diagram!
Table 24.2
GC Retention Times (min) Area Adjusted
Compound Percent Area %
Standard Sample
XXX
pentane
Methyl Palmitate
Methyl Oleate
Methyl Linolate
Methyl Stearate
RELATIVE COMPOSITION OF OILS
Saturated Monounsaturated Polyunsaturated
Fatty Acids Fatty Acids Fatty Acids
Total % SFUs Total %
Name of Oil PUFAs
Myristic Palmitic Stearic Oleic Linoleic
C14 C16 C18 C18 C18
(14:0) (16:0) (18:0) (18:19) (18:19,12)
Corn 0 11 2 13 28 58 59
Olive 0 13 3 16 71 10 11
Peanut 0 11 2 13 48 32 32
Soybean 0 11 4 15 24 54 61
Safflower 0 7 2 9 13 78 78
Cottonseed 1 22 3 26 19 54 55
Palm 1 45 4 50 40 10 10
Table 24.3
Did you use UNKNOWN A, B, or C?
Unknown Letter
Once you have a tentative identification
% SFA of your unknown vegetable oil based on
your comparison of the calculated values
in Table 24.2 to the table on p. 201 of
%MUFA your lab manual, complete this table by
filling in the EXPECTED % for your oil
using the values provided in the table on p.
201.
%PUFA
These values may not match your
calculated values EXACTLY, but should be
close enough for you to have verification
of the identity of your unknown oil!
Give the name of your unknown oil here!
Unknown Identity
SAFETY CONCERNS
CAUTION:
SULFURIC ACID and POTASSIUM
HYDROXIDE are very corrosive!
HEXANE and DIETHYL ETHER are
extremely flammable!
METHANOL is very flammable and toxic!
WASTE MANAGEMENT
• All liquid waste in container labeled “ORGANIC
WASTE (FAMEs).
• Leave TLC plates with instructor.
• Place filter papers in the yellow SOLID WASTE
can.
• Place used TLC capillaries, SiO2 columns, and
glass Pasteur pipets in broken glass container.
CLEANING
Rinse all glassware with wash acetone
ONLY!
DO NOT return any glassware to lab
drawer dirty or wet!