####19/05/2026

##homework peotocol

#my protocol- extraction of fatty acid in GC

##step 1:

weighte 50ul fish tissue

##step 2:

make 1M sodium methylate+MeOH (use the big 5ml pipetorand the 250 spescial red or blue pipetor), for 4ml- 0.155ml sodium (in the fridge), 3.85ml methanolput it in a glass tube and add 1ml to each sample

##step 3:

boil for 30 minutes, vortex every 10 minutes

##step 4:

add 100 ul internal standart FAME, for 10 ml stock- 1000 ug/ml + actiton

##step 5:

PH control-add 1-2 drops of phenolphthalein (in the frieg), add slowly 500 ul 1M HCL (for 20000ul- 16000ul 1.25 HCL, 4000ul methanol) use normal pipetor

##step 6:
vortex, and boil for 5 minutes

##step 7:

add 2ml NaCL (in the fridge), vortex (for 50 ml-0.35g/ml)

##step 8:

add 0.5ml MTBE (in the fridge)use the blue secial pipetor, invert

##step 9:

centrifuge for 5 minutes, 3000rpm

##step 10:

put the samples in small narrow tubes, and take the upper layer in GC vial

##step 11:

put in the GC-MS and start the progrem

##Figure 1: inpute of the fatty acid in the GC-MS | Fish ID | Season | Temperature (C) | Length (cm) | Weight (g) | FA % | |—|—|—|—|—|—| | 1 | Winter | 18 | 12.4 | 35 | 22 | | 2 | Winter | 17 | 13.1 | 38 | 25 | | 3 | Summer | 28 | 11.8 | 30 | 18 | | 4 | Summer | 29 | 12.0 | 31 | 17 | | 5 | Winter | 16 | 13.5 | 40 | 26 | | 6 | Summer | 30 | 11.5 | 29 | 16 |

##scientific references##

Optimised preparation of fish tissue samples for fatty acid extraction

##online protocol of analysis of fatty acid composition by gas chromatography

Analysis of fatty acid composition by gas chromatography(GC)