INTRODUCTION

Fatty acids or derivatives of fatty acids are used in a wide variety of application. Fatty acids are the major components of lipids. Its physical, chemical, and physiological properties of a lipid class depend on fatty acid composition itself. Saponification followed by methylation is the classical method for preparation of fatty acid methyl ester (FAME) from sterol ester and glycerolipids. FAME can be prepared by acid or base-catalyzed esterification. Base-catalyzed methanolysis proceeds much rapidly under mild temperature compared to acid-catalyzed reactions. Potassium hydroxide-catalyzed methanolysis can complete within 2 minutes from glycerolipids and about within 1 hour for sterol ester. BF3 is commonly used acid catalyst for methanolysis and methylation but it is harmful. On the other hand, H2SO4 is also an effective acid catalyst for FAME synthesis but it is very corrosive viscous liquid and must be handle with care. However, results are best in the presence of both reagents. Fatty acids are not sufficiently volatile for gas chromatography (GC) analysis. The acids need to be derivatized. Derivatization is the process to chemically modify a compound to produce a new compound which has the properties that are suitable for GC analysis. The compound need to be derivatize to permit analysis of compounds that is not directly amenable to analysis due to for example inadequate volatility and to improve the chromatographic behavior. This experiment introduces a procedure that is used routinely for fat analysis in which Non-volatile fatty acids are chemically converted to the corresponding volatile methyl esters. The resulting volatile mixture can be analyzed by gas chromatography. Gas chromatograph equipped with flame ionization detector(FID) is used in this experiment.

ANALYTICAL PROCEDURE

a) Preparation of fatty acid methyl ester samples from fat samples i. ii. iii. iv. v. Approximate 2g of oil or fat is weighed out and the exact weight is recorded. The sample is transferred into a 50 ml flask equipped with air condenser 5 ml of 0.5 M methanolic solution is added and refluxed for 3-4 minutes. 15 ml of esterification reagent is added and refluxed for 3 minutes. The mixture is transferred into a separatory flask. 50 ml of saturated NaCl and 25ml of diethyl ether is added. The mixture is shaken vigorously for 2 minutes and the aqueous layer is discarded. vi. Step v is repeated with another 25ml of saturated NaCl ant the aqueous layer is discarded once again. vii. The organic layer is transferred into a screw cap vial. Make sure that only the organic layer is injected into the GC as water can ruin the GC column. Note: Calibration of standards can be run while the esterification reaction is in progress. b) Instrument set-up (may vary depending on instrument): Injection port: split (40:1) Injection port temperature: 250C Column temperature: 100C to 290C at 40C /min Carrier gas flow rate: 30 ml/s Detector temperature: 250C

c) Quantitative analysis of FAME i. 0.4l of standard esters is injected to the column. The injection is repeated to get reproducible peak areas. ii. The amount of each fatty acid in the sample is calculated by using the data from the standard esters.

RESULTS
FAME sample Average amount of sample in FAME for 1st and 2nd injection FAME 1 (ppm) FAME 2 (ppm) FAME 3 (ppm) Average amount of sample in FAME (ppm) Laurate Myristate Palmitate linoleate 55.88 16.84 59.14 219.95 37.30 2.43 38.04 40.32 292.31 23.45 550.00 2076.2 128.50 14.24 215.73 778.82

Table 1: The average amount of each compound in FAME Samples Laurate Myristate Palmitate Linoleate Average tR (min) 1.124 1.547 2.407 3.841

Table 2: Average retention time of sample in standard mixture

Samples Laurate Myristate Palmitate Linoleate

Average tR in 3 replicates of FAME (min) 1.120 1.411 2.500 3.890

Table 3: Average retention time of sample in FAME analysis

CALCULATION
Response factor (RF) = Standard mixture ester compound Peak area of compound in standard mixture Amount of sample in FAME = RF x peak area of compound in FAME analysis I. 1st injection RF = 100 ppm 1140.163 = 0.09

Amount of methyl laurate in FAME sample = 0.09 x 618.400 = 55.656ppm II. Amount of methyl laurate in FAME sample = 0.09 x 623.282 (2nd injection) = 56.095ppm

DISCUSSIONS
The main objective of this experiment is to determine the compounds (methyl esters) that exist in standard mixture 5. Standard mixture 5 was injected two (2) times to ensure five (5) peaks appear on the chromatogram. The 5 peaks represent the methyl laurate, methyl myristate, methyl palmitate, methyl stearate and methyl linoleate. From table 1, average retention time (tR) for methyl laurate is 1.124 minute. While, the average tR for methyl myristate, methyl palmitate and methyl linoleate were 1.547 minute, 2.407 minute and 3.841 minute respectively. Methyl stearate was excluded because there is no peak appears at the chromatogram. For FAME sample, the average tR for laurate, myristate, palmitate and linoleate were 1.112 minute, 1.411 minute, 2.500 minute and 3.890 minute respectively. In this experiment, the response factor (RF) and amount of sample in FAME sample need to be calculated. Based on the result, RF for laurate, myristate, palmitate and linoleate were 0.09, 0.02, 0.04 and 0.14 respectively. From these values, the amount of sample can be determined. For methyl laurate, the average amount of sample in FAME is 128.50 ppm. As for methyl myristate, methyl palmitate and methyl linoleate, the average amount of sample were 14.24 ppm, 215.73 ppm and 778.82 ppm respectively. Standard mixture 5 was injected at temperature programming set up and the result was used to identify the compound present. First peak after the solvent peak appear to be methyl laurate. Next, followed by methyl myristate, methyl palmitate, methyl linoleate and methyl stearate. The carrier gas flow rate used is 30 mL/sec.

CONCLUSION
In conclusion, there are 5 compounds exist in standard mixture 5 which are laurate, myristate, palmitate, linoleate and stearate. This experiment was successfully done.

REFERENCES
1. H. Claus. (2007). Rapid identification of fatty acid methyl esters using a multidimensional gas chromatographymass spectrometry database. Journal of Chromatography A, 1177 (2008) 159169. Retrieved from

http://www.sciencedirect.com.ezaccess.library.uitm.edu.my/science/article/pii/S0021967 307018705 2. Esterification of fatty acids Fatty Acid Esters. Retrieved December 24th, 2012. Retrieved from website http://www.amberlyst.com/fatty_acids.htm 3. GC derivatization. (2006). Retrieved December 24th, 2012. Retrieved from website http://www.google.com.my/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CDsQ FjAB&url=http%3A%2F%2Fwww.registech.com%2FLibrary%2Fgcderrev.pdf&ei=u2bbU N7OGYurAf_xIGADg&usg=AFQjCNENTRA4DvIQKLrGkOfuyTCtO26LiA&sig2=yeYruOjCVnroLKE-KPZYw&bvm=bv.1355534169,d.bmk

APPENDICES A: SAMPLE CALCULATIONS

Response Factor of: 1. Methyl myristate = 100ppm 5941.77 = 0.02 2. Methyl palmitate = 150ppm 3483.671 = 0.04 2. Methyl linoleate = 350ppm 2534. 605 = 0.14