Analysis of Free and Total Glycerol and Triglyceride Content in B-100 Biodiesel Methyl Esters Using Agilent Select Biodiesel for Glycerides

Significance of the Topic

Ensuring the quality and performance of biodiesel requires accurate quantification of free glycerol, mono-, di-, and triglycerides. Residual glycerides can compromise engine function, contribute to deposit formation, and affect compliance with international standards such as EN-14105 and EN-14214. A robust analytical protocol is therefore vital for manufacturers and quality control laboratories to verify that biodiesel meets regulatory limits and delivers reliable performance.

Aims and Overview of the Study

This application note evaluates a gas chromatography method using the Agilent Select Biodiesel for Glycerides UltiMetal column and an on-column injector for the simultaneous determination of free glycerol and esterified glycerides in B-100 biodiesel. The study follows the European standard EN-14105 procedure and explores method linearity, repeatability, and chromatographic robustness for typical FAME matrices derived from rapeseed, sunflower, and soybean oils.

Methodology and Instrumentation

The procedure involves:

• Sample Preparation: 100 µL of homogenized biodiesel is weighed and mixed with two internal standards (butanetriol and tricaprin) and MSTFA in pyridine for derivatization. After 15–20 minutes, 8 mL of heptane is added.
• Derivatization: Conversion of glycerol and glycerides into trimethylsilyl derivatives using MSTFA.
• Instrumentation:
  • Gas chromatograph with on-column injector under full electronic flow control.
  • Agilent Select Biodiesel for Glycerides UltiMetal column (0.32 mm × 10 m, 0.1 µm, pre-installed retention gap).
  • Helium carrier gas at 4 mL/min; FID detector at 380 °C.
  • Oven program from 50 °C to 370 °C in multiple ramps.
• Calibration: External calibration for glycerol, monoolein, diolein, and triolein with regression coefficients (r2 ≥ 0.9992).

Main Results and Discussion

Calibration curves for all analytes demonstrated excellent linearity (r2 > 0.999). The method covers concentration ranges of 0.005–0.05% for free glycerol, 0.25–1.25% for monoglycerides, 0.05–0.5% for diglycerides, and 0.05–0.4% for triglycerides. Repeatability tests on both native and spiked biodiesel samples yielded RSD values below 1.6%. The UltiMetal column provided clear separation from the FAME matrix and exhibited low bleed at 370 °C, ensuring sustained performance over extended runs.

Benefits and Practical Applications

• Compliance with EN-14105 and ASTM D6584 requirements.
• High robustness and thermal stability of the UltiMetal column for high-temperature analyses.
• Minimal matrix interference and reliable quantification of trace glycerides.
• Ease of adaptation to routine quality control workflows in biodiesel production facilities.

Future Trends and Potential Applications

• Extension to biodiesel derived from non-conventional feedstocks (e.g., algae, waste oils) with tailored column chemistries.
• Integration with mass spectrometry detectors for enhanced selectivity and identification of minor by-products.
• Automation of derivatization and injection for high-throughput screening.
• Real-time monitoring of biodiesel production via in-line GC sampling.

Conclusion

The presented GC-FID method employing an on-column injector and the Agilent UltiMetal column reliably quantifies free and total glycerol, mono-, di-, and triglycerides in B-100 biodiesel, meeting EN-14105 standards. The system delivers high linearity, repeatability, and chromatographic robustness, making it well suited for routine quality control in biodiesel production.

Reference

• EN-14105. Fat and oil derivatives – Fatty Acid Methyl Esters (FAME) – Determination of free and total glycerol and mono-, di-, triglyceride content.
• Knothe G. (2006) Analyzing Biodiesel: Standards and other Methods. Journal of the American Oil Chemists' Society, 83, 823–833.
• EN-14214:2003. Automotive fuels – Fatty Acid Methyl Esters (FAME) for diesel engines – requirements and test methods.