Acylglycerol Determination in Biodiesel by RSLC with Charged Aerosol Detection

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The Application Notebook

The Application Notebook, The Application Notebook-06-01-2010, Volume 0, Issue 0

Many methods have been proposed to determine acylglycerols in biodiesel samples. This analysis is commonly performed by GC/FID (ASTM D6584/EN14105 methods).

Deanna C. Hurum and Jeffrey S. Rohrer, Dionex Corporation

Many methods have been proposed to determine acylglycerols in biodiesel samples. This analysis is commonly performed by GC/FID (ASTM D6584/EN14105 methods). However, GC methods typically require derivatization of the sample and high inlet temperatures can lead to analyte decomposition, causing underestimation of acylglycerols prone to thermal degradation. HPLC analysis with charged aerosol detection (CAD), is emerging as an alternative method for biofuel analysis. This technique is well-suited to determine components with varying degrees of response using other detection technologies. Previous work has shown that the ASTM GC method and HPLC methods can be statistically equivalent (1). By using HPLC, samples can be injected with no derivatization requiring only dilution. Additionally, by using a small particle size column, resolution can be improved while maintaining short run times. In this application brief, an RSLC biodiesel analysis method is shown that determines acylglycerols in fuels with a 10 min analysis time. This method avoids exceptionally toxic or environmentally damaging solvents, such as propionitrile and dichloromethane, which have previously been used for acylglycerol determination (2). Figure 1 shows the separation of a B99 biodiesel sample produced from used fryer oil that has been diluted in ethyl acetate. The monooleoyl glycerol and diacylglycerols are identified. Triacylglycerols elute later than the fuel methyl esters and are well separated and easily detected using the Acclaim® RSLC 120 C18 and the Corona® ultra™ Charged Aerosol Detector. With a broad detector dynamic range, oleoylglycerols can be determined between 5–100 μg/mL with peak area RSDs of <1.1 and logarithmic calibration correlation coefficients of 0.999. This range has the sensitivity to determine acylglycerols in biodiesel samples at and below the 0.24% and 0.25% total glycerol limits in ASTM D6751 and EN 14214, respectively. This leads to a rapid and sensitive method for determining acylglycerols in biofuels with minimal sample preparation.

Figure 1


UltiMate® RSLC system with a HPG-3400RS pump, Corona ultra detector, and WPS-3000TRS autosampler.

An Acclaim® RSLC 120 C18 2.2 μm, 2.1 × 150 mm column was used with an ethyl acetate – acetonitrile gradient at 0.5 mL/min.

Gradient: 0–15% ethyl acetate in 0.5 min, 15–65% ethyl acetate in 3.5 min, 65–80% ethyl acetate in 5 min, 80% A for 1 min. 2 min equilibration before injection at 0% ethyl acetate.

Column Temperature: 15 °C

Inj. Volume: 3 μL

Detector Settings: Nebulizer = 15 °C, Filter = none, Nitrogen Pressure = 35 psi

Biofuel samples were prepared by dilution in ethyl acetate to 5 mg/mL prior to analysis.


(1) T. A. Foglia, K. C. Jones, A. Nunez, J. G. Phillips, and M. Mittelbach, Comparison of Chromatographic Methods for the Determination of Bound Glycerol in Biodiesel, Chromatographia, 60, 305 (2004).

(2) M. Buchgraber, F. Ulberth, H. Emons, and E. Anklam, Triacylglycerol Profiling by Using Chromatographic Techniques, Eur. J. Lipid Sci. Technol., 106, 621 (2004).

Corona and UltiMate are registered trademarks, and ultra is a trademark of Dionex Corporation.

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