Biochem 386 GC-MS Analysis of Fatty Acids in Oils and Fats Wear safety goggles and protective clothes

at all times when you are in the lab! The combined separation and analytical technique, Gas Chromatography-Mass Spectrometry (GC-MS) can be used to characterize fatty acids in fats and oils (triacylglycerols) from plant and animal sources as their fatty acid methyl ester derivatives (FAMEs). The GCMS profile of FAMEs can also be obtained from biological membranes. (It is used, for example, as fingerprints for identifying bacterial species or for following their ability to alter the composition of their biological membranes in response to changing environmental conditions). Gas chromatography (GC) is employed to separate the fatty acid methyl esters. Mass Spectrometry (MS) is used to identify the individual components separated. The one requirement for GC-MS is that the compounds to be identified should be capable of being easily vaporized into the gas phase. Unfortunately, fatty acids are not very volatile and become even less volatile as the chain length increases. In addition, the polar H-bonding character of the carboxyl group causes what is called peak broadening and therefore mixtures of fatty acids are typically difficult to resolve into the pure components required for identification. This problem is easily overcome by converting the fatty acids to more volatile methyl esters before analysis. Aim In this experiment you will analyze the fatty acid content of commercial vegetable oils and animal fats using GC-MS. Materials Vegetable oil (corn oil, olive oil) Animal fat (lard, butter) Methanol:toluene (4:1 v/v %) Acetyl chloride Toluene 6% Na2CO3 solution Preparation of the samples week 1. You will work in groups of eight students during this experiment. Each group will have four samples: two vegetable oils and two animal fats. Each pair of students in the group has to prepare one sample only, but must analyze the data from all four samples. 1. Label a round bottom screw-cap Pyrex tube. Line the inside of its screw-cap with a piece of Teflon tape. Dissolve 3 drops of vegetable oil or a small piece of lard (about a drop size) in 2.5 mL methanol-toluene (4:1) in the labeled tube. 2. Add a small magnetic spin bar into each tube and stir for 1 minute. Then, using a glass syringe, very slowly add 200 L of acetyl chloride with continuous stirring. This step needs to be done in a chemical hood. NOTE: the acetyl chloride is a catalyst for the transesterification reaction that will produce fatty acid esters directly from triacylglycerols/membrane lipids. The methanol is providing the methyl group for the formation of the methyl esters. 3. Tightly close the tubes with Teflon lined caps. Place them in a 100C boiling water bath and incubate with continuous stirring for 1 hour. (Do not put the tubes directly on

the bottom of your water bath; the temperature might be higher there than 100 C and the solvents in your sample will evaporate.) Be careful while doing this step. You are heating a solvent in a tightly closed tube. Work behind a protective shield and monitor the boiling water level. 4. Cool the tubes to room temperature in water. Remove the small magnetic spin bar. (This can be easily done by moving a bigger magnet on the outer surface of the tube). Add 4 mL of 6% Na2CO3 solution slowly. Also add 1 mL of toluene. 5. Close the tube, gently shake/flip it a few times and allow the layers to separate. 6. Carefully remove a small portion of the upper toluene phase with a Pasteur pipette and transfer to a small sample vial. This will contain the derivatized fatty acids. Close the vial tightly with a screw-cap, and also cover it with parafilm. Label the vial and keep it in the refrigerator until the GC-MS analysis. GC-MS analysis and evaluation of the spectra week 2. Take your samples to the GC-MS and inject 1 L of each sample. The operation of this instrument will be explained to you. Your results will be printed automatically. The series of peaks that you will see on the GC chromatogram corresponds to the specific compounds that have emerged from the column. The size of the peak indicates the amount of the compound. The distance of the peak from the beginning of the trace (at the left) is indicated in minutes and is called the retention time. You will also get a library search report. It is the result of comparing the mass spectral fragmentation pattern obtained for each peak to that of 120,000 chemical substances in the computers library. Finally, the computer will indicate the ratio of the compounds the specific peaks represent, given as Area %. Questions 1. Write the reaction carried out for the transesterification of triacylglycerols. 2. Explain the elution order of the fatty acids in your chromatograms. 3. Compare your results obtained for the vegetable oils and animal fats. Make a table of the relative amounts for C12, C14, C15, C16, C17, and C18 saturated and unsaturated fatty acids. (It may happen that you can not see every one of these.) Explain the relationship between fatty acid chain length and degree of saturation and the physical property of melting point. Reference Schultz, E., and Pugh, M. E. Determination of the Fatty Acid Content of Biological Membranes: A Highly Versatile GCMS Experiment. Journal of Chemical Education 2001, 78, 944-946.