Abstract
A system for separation and quantitation of free fatty acids by HPLC is described. This system provides for separation of short, medium, and long chain, saturated from unsaturated, branched from unbranched free fatty acids, and iso- from anteiso-branched acids of equal carbon number, and for their quantitation. An efficient method and inexpensive apparatus for performing the Schmidt degradation on separated acids is also presented. Standard and plant-derived branched and straight chain fatty acids were investigated using these methods. The latter were radiolabeled in vivo and released from trichome-exudated sugar esters by saponification. Free fatty acids were separated by reversed-phase C-18 HPLC using gradients of acetonitrile/H2O, containing 0.01 M H3PO4. Separated acids were converted to their potassium salts and transferred to easily and inexpensively fashioned, triple-well septum vials containing a center well for trapping CO2. Sulfuric acid was placed in a tip well atop the center well and the vial was closed. Tipping the vial allowed the acid to spill to the sample without the need to puncture the septum or transfer acid via a syringe. The system allowed for the introduction of an additional CO2-trapping reagent, midreaction. This arrangement improved CO2 recovery to 99-100% and its reproducibility to about ±3%.
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