2.4. Fatty acid methyl esters (FAME)
Lipid analysis was performed at the end of BGP and LIP by concentrating
the biomass (5000 rpm; 5 min at 30 C). Microalgae lipid
(100 mg) with methanol–sulphuric acid (2%) mixture (40:1)
were refluxed for 4 h by monitoring the reaction by thin layer chromatography
(TLC) using n-Hexane and ethyl acetate (EA) mixture
(90:10) as mobile phase (Venkata Mohan et al., 2011). Reaction
was continued till the oil spot disappeared on the TLC plate. After
the reaction time (4 h), the contents were washed with 25 ml of
water, the aqueous layer was extracted with EA (2 25 ml) and
pooled. The extract was dried over anhydrous Na2SO4 and concentrated
under vacuum. The dried FAME was analyzed using GC-FID
(Venkata Mohan and Devi, 2012).
After conversion of fatty acids to methyl esters, the concentrated
sample was used for the detection of FAME composition
by GC with FID (Nucon-5765) through capillary column [Valcobond
(VB) 30 mm (0.25 mm 0.25 lm)] using nitrogen as carrier
gas (1 ml/min). The temperature of the oven was initially maintained
at 140 C (for 5 min), later increased to 240 C at a ramp
of 4 C/min for 10 min. The injector and detector temperatures
were maintained at 280 and 300 C respectively with a split ratio
of 1:10. FAME composition was compared with the standard FAME
mix (C8–C22; LB66766, SUPELCO).