Authors: Wei Zou
Fatty acids are major energy provider for the human body. Over intake of fatty acids causes obesity.
I. External Standard Curve Setting Up
2.Make standard FAME solutions for the calibration curves (FAME standards are from Nu-Chek-Prep, Inc.):
3.Preparation and injection of different concentrations of standard FAMEs: Concentrate or dilute the standard FAMEs according to the values of the calibration points. At least 3 injections are needed at each concentration level for each fatty acid. Average the peak area for the injections. Type the theoretic amount and average peak area into the calibration table. Save the method.
4.Make standard FAME solutions for the determination of the retention time (RT): Weigh the 10 ml beaker, write down the value. Add about 100 mg 68D in the 10 ml beaker, weigh again and write down the value. Wash the 10 ml beaker with petroleum ether more than 5 times. Collect the wash in a 10 ml volumetric flask. Tip to the marker with petroleum ether. Calculate the theoretic amount (ng / inj. vol.). 68D is better for the determination of the RT because it has a unique fatty acids profiles.
ii. Extraction of fatty acids:
For tissue lipid extract:
Typical applications for different tissues:
iii. Sample preparation:
5.Add 2 mL 0.5 N methanolic NaOH into the tube. Vortex vigorously 9 seconds. Cap tightly.
6.Heat the tube in a boiling water bath for 15 minutes.
7.While waiting, prepare another set of 12×100 tubes. Add 0.15 g anhydrous Na2SO4. Prepare pasteur pipettes.
8.After cooling the tube for 5 minutes, add 2 mL 14% BF3. Vortex vigorously 9 seconds. Cap tightly. After using the BF3, blow some N2 into the BF3 bottle and seal it.
9.Heat another 15 minutes. Cool down the tube in ice for 5 minutes.
10.Use a dispenser to add 2 mL petroleum ether and 2mL automatic pipette to add 2 mL saturated aqueous NaCl into the tube.
11.Vortex the tube vigorously for 2 minute with the “vortex rack”. Centrifuge the tube for 5 minutes.
12.Use a pasteur pipette to transfer carefully the upper petroleum ether layer into the tube with anhydrous Na2SO4. Cap the tube and vortex 9 seconds. Let the tube stand for 20 minutes. Petroleum ether is very easy to drop from the pipette. Be careful! Don’t take all of the upper layer. It is better not to disturb the lower layer.
13.While waiting, choose a set of brown color glass vials and check the mouth of the tube. Wash the tubes with CM mixture and Pet ether. The cap of the vial should be Teflon.
14.Transfer the solution with pasteur pipette into the vials. Wash the tubes once with 2 mL petroleum ether. Add the washing solution into the vials. Be careful not to transfer the aqueous layer, otherwise the evaporation will take much longer time and there will be NaSO4 residue on the wall of the vial. The residue might affect the GC separation of fatty acids.
15.Evaporate the vials at 30-40 C under a very gentle stream of N2. Stop evaporating immediately after the vials are dry because FAMEs are very easy to evaporate.
16.Take out the vial and put it into the ice box to cool down for 15 seconds. Put the bottle of petroleum ether with BHT in ice also to avoid evaporating.
17.Use 250 µL syringe to add exactly 100 µL petroleum ether with BHT into the vial. Vortex gently. Cap tightly. Wrap the cap with Teflon tape.
18.Wait 3-4 hours for GC injection, otherwise the recovery rate will be low. From my experience, overnight standing is OK.
iv. GC injection:
For the GC 6890 system, check handbooks for the installation and hardware information. Check the online tutorial and printout help files for the software information.
19.Check if the gas tanks are full. The tanks should be changed to avoid GC column contamination if the pressure is below 400 psi.
20.Before GC warming on, check if the septa need to be changed. To change the new septa, wash it with HPLC graded methanol and put it on the inlet. Set GC temperature to 250 °C for 30 min. to clean the residues from the new septa.
21.GC warming up. At least half an hour before the sample injection, click the icon “instrument 1 online” on the desktop. In the popup windows, type in the name of the operator. In the ChemStation desktop, click the menu “File” – “Load Method” to choose the right method. After the warming up, run the “Instrument” – “StartColumnComRun” to set blank injection.
22.Click the “RunControl” – “Sample Info” to change the name of the operator, file prefix, and subdirectory for the file storing, etc.
23.Before injection, wash the 10 µL GC syringe with D.I. water, then acetone, then petroleum ether.
24.Put the vials in ice. Apply “Hot Needle Solvent Flushing Method” to inject sample into the GC. Wash 10 µL GC syringe with petroleum ether, take 2 µL pet ether, pull the needle a lot bit more in the air to make a small air plug in the syringe, then take exactly 3 µL sample final solution into the syringe (try to avoid contacting the vial with bare hands), measure the amount exactly, wipe the syringe needle with tissue paper, pull again to take some air into the syringe to avoid evaporation of the solution. Push the syringe needle into the GC inlet. Count 9 (3 seconds) to make the needle hot. Inject the sample as fast as you can. Pull the syringe out quickly. Push the “Start” button on the GC panel.
25.For the purpose of quality control, inject external STD first, then inject samples, finally inject external STD again to check if the GC is stable. Inject one ESTD for every 10 samples.
26.After injection, wash the 10 µL GC syringe with D.I. water, then acetone.
Detailed information regarding the Gas Chromatography Method can be found in the following Word Document.
Thanks to Koo, S.I. for mentoring and financial supporting efforts.
Wei Zou, Zou's lab, DTSC, California State Government
Correspondence to: Wei Zou ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.247. Originally published online 29 July 2011.