Principles of Biochemistry (2+1)

6.6.1 Quantitative and qualitative fatty acid analysis

For fatty acids, gas - liquid chromatography has proved particularly useful.

Lipids present in the sample muscle are extracted using chloroform. The extracted lipids are then esterified using sulfuric acid – methanol and converted to methyl esters. The fatty acid methyl esters are separated individually in the gas chromatograph based on their number of carbon chains.

The individual fatty acids in the sample were identified by comparing the retention time of the individual fatty acids in the standard mixture and the concentration of the individual fatty acid was calculated from the area intensities with the help of authentic fatty acid standards.

Extraction of lipid by Folch’s method:

10 g of sample was homogenized in a beaker and 30 ml of chloroform: methanol (1:1) was added and kept for 10 min. Filtered using a strainer and then with filter paper. Added 30 ml of chloroform: methanol (2:1), to the precipitate and extracted in a blender and filtered. The filtrate was mixed with 0.74% KCl in a separating funnel. The lower chloroform layer removed. The sample was extracted again with 10 ml of chloroform: methanol: 0.74% KCl (3:48:47). The lower layer was collected. Sodium sulfite was added and left as such for 10- 20 min to remove the moisture. The chloroform layer was concentrated by distillation. The fat was dissolved in ether and transferred to the pre-weighed flask. The ether. Was evaporated and the weight of the fat was noted. The fat content was calculated for 100 g of fish sample.

Formation of Methyl esters

200 mg lipid sample was taken a screw capped tube. 4 ml of 0.5 N methanolic NaOH was added, the tube was closed and heated on a steam bath for 5 min. Then, 5 ml of BF3 – Methanol was added and boiled for 5 min. The contents were then transferred to the separating funnel and 10 ml of saturated sodium chloride was added until methyl esters float. The upper layer was removed and concentrated to dryness under vacuum on a rotary evaporator.

GC analysis of methyl esters  

For the analysis of fatty acid methyl esters Perkin Elmer Autosystem XL Gas Chromatograph equipped with flame ionization detector (FID) was used with a computing integrator attached with Turbochrom work station. GC was fitted with PF-225 (methyl cyanopropyl phenyl silicate) 30 m x 0.25 mm (ID) capillary column for the fatty acid analysis. Nitrogen was used as the carrier gas.

For the analysis, 0.5 µl of fatty acid methyl esters mixture prepared from sample was injected to the Gas chromatograph. The instrument was set under the following conditions.

 Injector temperature - 250^oC

 Detector temperature - 300^oC

 Carrier pressure - 20 psi

 Split ratio - ON

The initial column temperature maintained at 70^oC and then programme the temperature was set from 70 to 180^oC at the rate of 8^oC/min; then from 180 – 21-^oC at the rate of 3^oC / min. The flow rates of oxygen and hydrogen were 450 and 50 ml / min, respectively. Simultaneously, 0.5 µl of the fatty acid standard mixture was injected.