Biochemical Techniques and Instrumentation

Procedure

Extraction of lipid by Folch’s method

1. Homogenise 10 g of fish sample in a pestle and mortor and transfer to a beaker
2. Add 30 ml of chloroform : methanol (1 : 1) and keep for 10 min.
3. Filter using a strainer first and then with Whatman No. 1 filter paper.
4. To the precipitate, add again 30 ml of chloroform : methanol (2 : 1)
5. Extract in a blender and filter again in Whatman No. 1 filter paper.
6. Combine the filtrate and transfer to a separating funnel.
7. Add 0.74% KCl, shake well and remove the lower chloroform layer.
8. Extract again with 10 ml of chloroform : methanol : 0.74% KCl (3 : 48 : 47).
9. Collect the lower chloroform layer.
10. Add few grams of sodium sulfite to remove the moisture and filter again through Whatman No. 1 filter paper.
11. Concentrate the chloroform layer by distillation.
12. Dissolve the fat in diethyl ether and transfer to the pre-weighed flask.
13. Evaporate the ether and note down the weight of the fat.
14. Compute the fat content for 100 g of fish sample.

Direct esterification of lipid by BF₃ - methanol method

1. Take lipid fraction (250mg) in a round bottom flask.
2. To which, add 4ml of 0.5M alcoholic NaOH and reflux for 5 – 10 min until droplets of fat disappears.
3. Then, add 5ml of BF₃ methanol and reflux for another 1 min
4. Cool the contents and add 15 ml of saturated sodium chloride (25%).
5. Over to it, add 5 ml of hexane, shake well and remove the upper hexane layer.
6. Repeat the extraction with hexane once again.
7. Combine the hexane layers and evaporate to dryness in a rotary flask evaporator set at 55 - 60°C.
8. Again reconstitute in 1ml of hexane for injection in GC.

GC analysis of methyl esters

1. Switch “ON” the instrument
2. Open the carrier gas, Nitrogen
3. Create a method with the following conditions:
   Injector temperature – 250^oC
   Detector temperature – 300^oC
   Carrier N₂ pressure – 20 psi
   Split ratio - 1:50
4. Set the gradient temperature programming as follows:

Rate	Temperature	Hold
-	70oC	1 min
8oC/min	180oC	1 min
3oC/min	210oC	30 min

5. Ignite the “FLAME” by opening the zero air and hydrogen gases
6. Allow to condition for 30 min until the autozero value becomes stable
7. Then, inject 0.5μl of standard FAME mixture on to the Gas chromatograph.
8. Once the run is over, reset the programme again and inject 0.5μl sample methyl esters.