Procedure
Extraction of lipid by Folch’s method
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Homogenise 10 g of fish sample in a pestle and mortor and transfer to a beaker
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Add 30 ml of chloroform : methanol (1 : 1) and keep for 10 min.
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Filter using a strainer first and then with Whatman No. 1 filter paper.
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To the precipitate, add again 30 ml of chloroform : methanol (2 : 1)
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Extract in a blender and filter again in Whatman No. 1 filter paper.
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Combine the filtrate and transfer to a separating funnel.
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Add 0.74% KCl, shake well and remove the lower chloroform layer.
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Extract again with 10 ml of chloroform : methanol : 0.74% KCl (3 : 48 : 47).
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Collect the lower chloroform layer.
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Add few grams of sodium sulfite to remove the moisture and filter again through Whatman No. 1 filter paper.
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Concentrate the chloroform layer by distillation.
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Dissolve the fat in diethyl ether and transfer to the pre-weighed flask.
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Evaporate the ether and note down the weight of the fat.
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Compute the fat content for 100 g of fish sample.
Direct esterification of lipid by BF3 - methanol method
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Take lipid fraction (250mg) in a round bottom flask.
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To which, add 4ml of 0.5M alcoholic NaOH and reflux for 5 – 10 min until droplets of fat disappears.
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Then, add 5ml of BF3 methanol and reflux for another 1 min
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Cool the contents and add 15 ml of saturated sodium chloride (25%).
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Over to it, add 5 ml of hexane, shake well and remove the upper hexane layer.
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Repeat the extraction with hexane once again.
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Combine the hexane layers and evaporate to dryness in a rotary flask evaporator set at 55 - 60°C.
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Again reconstitute in 1ml of hexane for injection in GC.
GC analysis of methyl esters
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Switch “ON” the instrument
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Open the carrier gas, Nitrogen
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Create a method with the following conditions:
Injector temperature – 250oC
Detector temperature – 300oC
Carrier N2 pressure – 20 psi
Split ratio - 1:50
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Set the gradient temperature programming as follows:
Rate
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Temperature
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Hold
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-
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70oC
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1 min
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8oC/min
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180oC
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1 min
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3oC/min
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210oC
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30 min
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- Ignite the “FLAME” by opening the zero air and hydrogen gases
- Allow to condition for 30 min until the autozero value becomes stable
- Then, inject 0.5μl of standard FAME mixture on to the Gas chromatograph.
- Once the run is over, reset the programme again and inject 0.5μl sample methyl esters.
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Last modified: Saturday, 12 November 2011, 7:25 AM