Simultaneous Analysis of Glycerides and Free Fatty Acids in Palm Oil

Corona ® Charged Aerosol DetectorSimultaneous Analysis of Glycerides(mono, di, and triglycerides) and FreeFatty Acids in Palm OilApplication notePalm oil is the second largest edible oilproduced in the world. It is comprised ofmainly triglycerides with some mono anddiglycerides. Free fatty acids and some othersediments and components in the crude oilare considered impurities and may beremoved in the refining process. The level ofrefinement varies depending on source andfinal use.Analytical methods for oils, like palm oil,often use GC/FID methods with derivatization.An HPLC method was developed forthe analysis of palm oil using a binary pumpsystem with the Corona ® Charged AerosolDetector (CAD ® ) for detection. The methodrequires simple sample preparation and noderivatization. Comparisons were made tothe AOAC official method 993.24 which usesa differential refractometry detector. Therepeatability and selectivity of the systemwas checked as well as the limit of detection(LOD) for individual components.Figure 1. Replicate analysis of palm oil sample by HPLC-CAD(n=4; ~74µg on column).Method ParametersColumn:C18 MGIII,4.6mm I.D. x 250mm, 5µm at 30°CNebulizer Heater: 30°CFilter:Mobile Phase A:None75% Acetonitrile, 12% Methanol,8% HPLC grade water, 4% THF,and 0.3% Acetic AcidMobile Phase B: 90% Acetone and 10% AcetonitrileGradient Profile: Table 1Flow Rate: 1.5-1.9 mL/minRun Time: 70 minutesSample Preparation and AnalysisThe material analyzed was a commercially availablesample of Red Palm Oil from Jungle Products. The oil islisted as being from the fruit of the Dura plant and in itsnatural state without refinement. The sample was simplydissolved and diluted in a 1:1 solution of methanol andtetrahydrofuran (THF). The concentration used foranalysis was ~7.4mg/mL. The palm oil sample wasinjected multiple times on multiple days to showreproducibility and robustness.Standards of palmitic acid (Sigma P-0500), 1-oleoyl-racglycerol(Sigma M7765), 1,3 diolein (Sigma D3627), andtriolein (Sigma T7140) were also prepared in a 1:1solution of methanol and THF. The standards werediluted and run at concentrations from 250 µg/mL to3 µg/mL. 10µL injections were made through out theanalysis for all standards and samples. The data wereprocessed and the response versus the column load wasplotted.

Corona ® Charged Aerosol DetectorApplication noteTime %A %B Flow Rate0.00 100 0 1.55.00 100 0 1.511.00 96 4 1.524.00 37 63 1.535.00 32 68 1.545.00 18 82 1.958.00 15 85 1.960.00 5 95 1.563.00 100 0 1.565.00 100 0 1.5Table 1. Gradient Conditions.Results and DiscussionReproducibility of the method was tested by comparingfour different injections of the palm oil sample at 74µgcolumn load. The peak areas for an individual peak aswell as groups of peaks were observed and the resultsare shown in Table 2. The peak retention times exhibitedless than 1% variation over the gradient run for allsamples observed. Four overlays of the palm oil areshown in Figure 1. The chromatograms for the fourstandards run are illustrated in Figures 2A and 2B. Thecorrelation is done using a polynomial fit and was greaterthen >0.999 for all points (Figure 3). The LOD of the was