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Calculating fatty acids in seaweeds

MvH

I am trying to calculate the fatty acid content in g/100g seaweed on FID but I am unsure which formula to use. 

We use C17:0 FFA as internal standard.

Other information we have is: 

Volume of the internal standard = 50 uL of 1 mg/ml solution

Sample mass: 100 mg seaweed

Extracting with 4 ml of a 2% H2SO4 methanol solution and 2 ml chloroform (direct transesterification)

After transesterification we add 1 ml 0.9% saline and further extract FAMES 3 times with 2 ml hexane each time. 

Thereafter the hexane is evaporated. 

Then the FAMES are reconstitute with 200 ul isooctane, 1 uL injected with a 1:10 split. 

I hope this information suffices.  

  • 0

    Hi MvH, 

    I have some ideas, but I don't think you are going to be able to do this with one formula Slight smile  I should mention that this is more of a thought exercise.  I have not done this experiment before, and I encourage knowledgeable members of the community to chime in!

    Do you have any reference to what an expected range of FA should be present?  From the instrumental side, you will need a calibration curve to quantify the FAMES content of your final solution.  The way I would approach is to build a multipoint RRF calibration curve that brackets your expected range, using your internal standard as part of the calibration.  This assumes that you have adequate separation on the GC column and you can identify your peaks of interest.  If you calibrate with the 10x split ratio, you won't need to factor that into your math.  

    Once you have the quantitative result of FAMES in i-C8, you will work backwards through your extraction steps to determine a factor that can be applied to the final result.  

    The GC results will likely be in some ppm/ppb or mass/volume unit, so you will need to convert.  

    For example, lets say your GC found amount is 100ug/mL.  You would need to multiply that value by 0.2mL to get the mass of FAMES present before the reconstitution step.  This would give you 20ug in the vial at that point.   You would consider each step and the volumes used, the effectiveness of your transesterification process and the initial extraction to get to your compositional analysis, which would be a percentage of the original weight of the seaweed.     

    A second approach might be to find a null-FFA seaweed simulant (maybe 100mg of iceberg lettuce?) where you could spike a known amount of FFA (including blanks), then execute your sample prep steps to get the GC result (you would still need a GC calibration curve).  Doing this multiple times at a range of levels could provide a simplified yet supported correlation sans multi-step algebraic quests for your experiment.  

    Do either of these suggestions help?  

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