Experiment 24: SYNTHESIS AND GC ANALYSIS OF FATTY ACID METHYL ESTERS

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)

CHEMICAL EQUATION (Transesterification of vegetable oil)

COMMON FATTY ACIDS Saturated= NO double bonds! Monounsaturated= 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 a reaction vial with stir bar.  Add 1M KOH/methanol.  Place vial in heat block and begin stirring the solution.  Attach reflux condenser.  Heat to reflux. *Maintain temperature b/n o C for 30 minutes.* (Excessive heat will ruin experiment!)  Cool flask to room temp.

EXPERIMENTAL PROCEDURE (Purification and Isolation)  Extract product 2X with hexane 1X with 9:1 hexane: diethyl ether.  After each extraction step, pass organic layer through SiO 2 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% H 2 SO 4 stain with heat in order to visualize spots. Have stained TLC plate inspected by lab instructor to ensure polar impurities have been completely removed.  GC Analysis With instructor permission, prepare sample for GC analysis.

GC ANALYSIS OF FAMES STANDARD SOLUTION Once returned, identify and quantify FAMEs present in your unknown oil. Compare these results to table on p. 203 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 Compound GC Retention Times (min)Area Percent Adjusted Area % StandardSample pentane XXX Methyl Palmitate Methyl Oleate Methyl Linolate Methyl Stearate

RELATIVE COMPOSITION OF OILS Name of Oil Saturated Fatty Acids Total % SFUs Monounsaturated Fatty Acids Polyunsaturated Fatty Acids Total % PUFAs Myristic C 14 (14:0) Palmitic C 16 (16:0) Stearic C 18 (18:0) Oleic C 18 (18:1  9 ) Linoleic C 18 (18:1  9,12 ) Corn Olive Peanut Soybean Safflower Cottonseed Palm

Table 24.3 Unknown Letter Did you use UNKNOWN A, B, or C? % SFA  Once you have a tentative identification of your unknown vegetable oil based on your comparison of the calculated values in Table 24.2 to the table on p. 201 of your lab manual, complete this table by filling in the EXPECTED % for your oil using the values provided in the table on p  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! %MUFA %PUFA Unknown Identity Give the name of your unknown oil here!

SAFETY CONCERNS CAUTION: n SULFURIC ACID and POTASSIUM HYDROXIDE are very corrosive! n HEXANE and DIETHYL ETHER are extremely flammable! n 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, SiO 2 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!