Methylation of Oil sample :

1) Add 10 ml of 0.5 M methanolic sodium hydroxide at approximately 100mg of oil sample

2) Reflux the mixture for 20 minutes

3) Add 10ml BF3 in methanol solution to the reflux and continue boiling for 20 minutes

4) Add 100ml of HPLC grade heptane and continue boiling for 20 minutes

5) Cool the mixture and separate the organic upper layer

6) Add drying agent such as sodium sulfate to the separated organic layer

7) Inject 1 microliter of the above solvent to the GCMS system

GC–MS Condition:

The GC–MS techniques were used to identify % of Fatty Acid oil ME, which was examined by gas chromatograph with mass spectrometer. SLBTM-5ms fused-silica capillary column (30 m × 0.25 mm, film thickness 0.25 µm) was utilized to perform the separation. The temperature of the oven, including the column, was set to rise by 3°C every minute, Oven temp. beginning at 100°C and ending at 240°C 15 min. Helium 99.999% was used as a carrier gas at a constant flow rate of 1 mL/min during the whole chromatographic run. Injector temperature 290°C, 1 µL of the Methylated Oil injected in split mode with a split ratio of 1:50. Solvent Delay 0-8 min, Source temp.250 C , MS Scan Mass 50.0 to 500 EI+ form 8min to 65.67 min ,Standard F.A.M.E Mix From RM-1 To RM-6 (SIGMA-ALDRICH)

The National Institute of Standards and Technology’s MS Data Center reference spectra were compared to the mass spectra of the chemical components, and their Kovats retention indices were compared to values given in the literature.

The Kovats Retention Index for each compound was calculated using the retention time value from the Hydrocarbon Alkane Standard (C10-C40(alleven) (SIGMA-ALDRICH)

this method is good for separation C18:2 C18:1 C18:0 and conjugated Linolenic acid Isomers but the problem its difficult to Separate C18:1 as Oleic Acid ME and C18:3 α-Linolenic acid ME in Standard RM-2 FAME (sigma Aldrich) for this problem i used another method for GC

which was examined by gas chromatograph with mass spectrometer. SLBTM-5ms fused-silica capillary column (30 m × 0.25 mm, film thickness 0.25 µm) was utilized to perform the separation. The temperature of the oven,50 °C for 7 min including the column, was set to rise by 1°C every minute, Oven temp. beginning at 50°C 7min with rate 1 °C/min to 177°C ,from 177°C to 186°C Rate 0.5 °C/min then from 186°C with Rate 2 °C/min and ending at 283°C . (run time 200.50min) Helium 99.999% was used as a carrier gas at a constant flow rate of 1 mL/min during the whole chromatographic run. Injector temperature 290°C, 1 µL of the Methylated Oil injected in split mode with a split ratio of 1:50. Solvent Delay 0-8 min, Source temp.250 C , MS Scan Mass 50.0 to 500 EI+ form 8min to 200.5 min ,Standard F.A.M.E Mix From RM-1 To RM-6 (SIGMA-ALDRICH)