The fish Lepidocephalus guntea (Ham.) of three different sizes were collected from various canals and local ponds in the North-Bangal of Bangladesh during the period March, 2006 to November, 2006.
Extraction of lipid from Lepidocephalus guntea (Ham.) fish: The lipid was extracted from the body muscle (edible portion) of Lepidocephalus guntea (Ham.) fish by the method cited in the literature. About 20 g of wet fish was ground well in a homogenizer (model AM-5, Ogawa Seiki Co. Ltd., Japan) with about 200 mL distilled water to make pulp. The pulp was transferred to a volumetric flask and 600 mL of chloroform-methanol (2:1v/v) mixture was added and shook well for about 3 h. For complete extraction, it was kept overnight at room temperature, preferably in the dark. The resulting suspension was subjected to centrifugation (12x103 rpm) where three layers were found. Chloroform layer with lipid was separated. To ensure complete extraction, the process was repeated and the combined extract was dried with a flow of nitrogen gas. Lipid thus obtained was purified by removing non lipid substances.
Characterization of the lipid of Lepidocephalus guntea (Ham.): Physical properties of the lipid i.e., specific gravity, refractive index, were determined by the standard method (Molla et al., 1994). Chemical characteristics of the lipid i.e., iodine value, acid value, saponification value etc were also determined using the standard method cited in the literature.
Qualitative and quantitative analysis of lipid: The fish lipid was saponified and the fatty acid mixture of the lipid was converted to the corresponding methyl ester by BF3-methanol complex according to the standard method of AOAC (1990). The methyl ester mixtures thus obtained were subjected to Thin Layer Chromatography (TLC) and Gas-liquid Chromatography (GLC) for the identification and estimation of the individual fatty acid components.
Thin layer chromatographic examination of methyl esters: Thin layer chromatography is an excellent tool for micro-preparative separation of mixtures. The methyl ester mixtures were charged on thin layer (20x20 cmx0.25) of silica gel-GF254 and the plate were developed by ascending technique using the following solvent system.
| • |
Petroleum ether (40-60°C):ether (60:40) |
| • |
Petroleum ether (40-60°C):ether (80:20) |
| • |
Petroleum ether (40-60°C):ether:acetic acid (80:20:1) |
| • |
Petroleum ether (40-60°C):ether:acetic acid (85:15:1) |
| • |
Hexane:ether (80:20) |
After development, the plates were dried at room temperature and sprayed with 2,7-dichlorofluroescein (0.2 g 2,7-dichlorofluroescein in 100 mL ethanol). The fatty acid methyl ester gave yellow coloured spot with the reagent under UV-lamp. The coloured spots were marked and the Rf values of the spots were calculated.
Gas liquid chromatographic examination of methyl esters: The experiment was carried out with a PYE UNICAM 4500 U model capillary chromatograph (Philips, England) fitted with a flame-ionization detector containing SE-54 as a packing material. The temperature was programmed from 120-220°C at a rising rate of 4°C per minute and the temperature of the injection port, detector and oven were maintained at 220, 230 and 190°C, respectively. Hydrogen was used as the carrier gas at a flow rate of 5 mL min-1. The GLC graph (Fig. 1) represents some peaks of individual fatty acids. The identities of the individual fatty acids were achieved by co-chromatography with standard reference compounds. The peaks were tentatively identified by comparing their relative retention times with known values and by plotting the logarithm of the relative retention time curve against carbon number.
Determination of the average molar mass (M) of the lipid:
The average molar mass (M) of the lipid was calculated by the following:
|
M/g mol-1 = 3(σxiMi) + 38.05 g mol-1
|
| xi |
= |
The mole fraction of fatty acids |
| Mi |
= |
The molar mass of the group CHCCH |