Journal of the American Oil Chemists' Society

, Volume 94, Issue 2, pp 285–299 | Cite as

Bias and Imprecision in the Determination of Free Glycerin in Biodiesel: The Unexpected Role of Limitations in Solubility

  • Richard W. Heiden
  • Sigurd Schober
  • Martin Mittelbach
Original Paper


Residual free glycerin (FG) is a critical marker of fatty acid methyl ester (biodiesel) fuel quality. Yet, routine determinations by standard methods display excessive imprecision, and the uncertainties undermine confidence in the data. Various degrees of agitation are used here to evaluate the possibility of heterophase formation as a contributor to imprecision and potential bias. Statistical markers from the analysis of 13 biodiesel samples reveal that seven contain unexpected heterophases, which, due to settling, cause underestimates of 15–68%. Agitation alone re-suspends heterophases for brief periods, easing potential biases, but also increases imprecision. A promising new sample pretreatment is presented, which reduces the deleterious effects of heterophases. Solubility limitations are explored as possible contributing factors, and measurements made at 23 °C in purified soy FAME reveal an equilibrium solubility which is more than 80% below previously published estimates. Strong interactions of liquid FG with small amounts of moisture reduce solubility to below international quality limits, while interactions of initially dissolved forms of moisture and FG produce bias-inducing heterophases at 0.02% FG. The unexpected low solubility of FG, exacerbated by interactions with impurities, is seen as an important contributor to underestimates and imprecision, and a strong influencing factor on the fate of residual FG in commercial biodiesel fuels.


Biodiesel Glycerin Bias Solubility 



FAME saturated with moisture at 40 °C

20% D

0%D FAME diluted to 20% v/v DDMF (100%D)


0%D FAME diluted to 40% v/v DDMF (100%D)


0%D FAME diluted to 60% v/v to DDMF (100%D)


0%D FAME diluted with 80% v/v to DDMF (100%D)


100% DDMF, moisture <50 ppm


95% Confidence interval


95% Confidence interval, n = 1


95% Confidence interval, n = 3


Concentration of FG after agitation


Arbeitsgemeinschaft Qualitätsmanagement Biodiesel, e.V.


American Society for Testing and Materials


An approximately 100.0 mg subsample of B100


Time interval in seconds between ~100.0 mg subsamples


Commercial Grade of FAME biodiesel




US Center for Disease Control


Comité Européen de Normalisation-European Committee for Standardization


Coefficient of variation


Distilled, dry, methanol free


Electron ionization


European normalization


European Union


Fatty acid methyl esters: here refers to a research grade of biodiesel


Free glycerin


Gas chromatography


Gas chromatography/mass spectrometry


Gas chromatography-flame ionization detection


Interlaboratory study


Institute for Reference Materials and Measurements


An approximately 6.0000 g subsample for pretreatment by pyridine augmentation (PA)


Time interval between 6 g intermediate subsamples




Magnetic stirring with 2.5 cm stir bar, surface turbulence, no vortex


Magnetic stirring with 3.7 cm stir bar and 2 cm vortex




National Institute of Science and Technology


Treatment procedure


Pyridine augmentation at a level of 30.0000% of B100 ISUB weight


Reproducibility-between lab variations


Repeatability-within lab variations


Results of six consecutive injections of a single derivatized subsample


Portion of a B100 process stream or fuel lot submitted to a lab in a container for analysis


Standard deviation




A portion of an SMP for analysis by a test method




Unagitated-concentration of FG before agitation


US Department of Energy


Vigorous wrist agitation for 10 s


10 s interval between agitation and 100 mg subsample


EU rejection limit


Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


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Copyright information

© AOCS 2016

Authors and Affiliations

  • Richard W. Heiden
    • 1
  • Sigurd Schober
    • 2
  • Martin Mittelbach
    • 2
  1. 1.R. W. Heiden Associates LLC, Laboratory/Burle Business ParkLancasterUSA
  2. 2.Institute of ChemistryUniversity of Graz-NAWI GrazGrazAustria

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