Dear All,
I am working with E. coli and want to determine its fatty acid content using GC-MS. I have converted fatty acids into FAMEs by chloroform/methanol method and have my samples in n-Hexane. I have never operated GC-MS hence I have no idea about its program to be set in order to achieve good resolved peaks.
Can anyone please help me with a standard protocol to analyse FAMEs using GC-MS?
Thanks in advance!
Dear Neha Sawant,I do not have a workfllow for FAME, but I have developed quite a few methods for very different applications. FAMEs were also present in some samples.
I use a moderately polar separation column for GC-MS application, currently the Agilent Factor Four 5MS, 30 m x 0.25mm x 0.5 µm.
I would also recommend a comparable column for FAME analysis.
The temperature program then also depends on the injection mode.
Split injection:
Inlet: 320 °C, or with PTV 50 to 320 °C with maximum heating rate, 320°C for 3 min.
50° for 3 min, 10°/min after 180°C, 35°C/ min after 320°C, 320°C for 5 min.
Column flow: 0.8 ml /min or mean carrier gas velocity 32cm/sec
MS interface: 280°C
Splitless injection:
Inlet: 320°C, or at PTV 50 to 320°C with maximum heating rate, 320°C for 3 min
50°C 3min, 40°C/min to 180°C, 10°C/min to 320°C, 320°C for 5 min
Column flow: 0.8 ml /min or mean carrier gas velocity 32cm/sec
Solvent delay for the MS for 5 min, depending on solvent
MS interface: 280°C
Just try these methods and see if there is a need for optimization. You can adjust the heating rates and temperatures if needed.
Good luck with your work and best regards
Joachim Horst
You may follow the white paper below.
https://www.agilent.com/cs/library/applications/5989-3760EN.pdf
Dear All,
I thank everyone for their valuable support. I could standardize my protocol.
Regards
Neha Sawant
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