Biodiesel Quality Control according to DIN EN 14105 Determination of free and total glycerol and mono-, di, triglyceride contents (reference method)

Importance of the Topic

Biodiesel is a renewable alternative to fossil diesel, derived from plant oils and animal fats. Due to its lower viscosity and good low-temperature properties, fatty acid methyl esters (FAME) produced via transesterification can be used in existing diesel engines without modification. Quality control of biodiesel is essential to ensure compliance with regulations such as DIN EN 14105, which sets limits on residual glycerol and glyceride contents. Reliable quantification of these components ensures fuel performance, engine safety, and market acceptance.

Study Objectives and Overview

This application note describes the reference method according to DIN EN 14105 for determining free and total glycerol, mono-, di-, and triglyceride levels in biodiesel. The goals are to:

• Develop calibration protocols for representative glyceride compounds.
• Optimize sample derivatization to improve chromatographic performance.
• Demonstrate chromatographic separation and detection of target analytes in biodiesel samples.

Methodology and Instrumentation

Sample Preparation and Derivatization:

• Dissolve 100 mg of biodiesel in internal standard solution and add 100 µL of MSTFA.
• Shake for at least 15 minutes at room temperature to derivatize hydroxyl groups.
• Add 8 mL of n-heptane and mix to extract derivatized compounds.

Calibration Strategy:

• Use relative quantification with internal standards for glycerin and tricaprin.
• Prepare four calibration levels covering 0.005 % to 0.05 % glycerin and corresponding mono-, di-, and triglyceride concentrations.

Instrumentation Used:

• Gas Chromatograph: Shimadzu GC-2010AF equipped with AOC-20i autosampler.
• Injection Technique: Cool on column (OCI) or Simple On Column injection to avoid discrimination effects.
• Column Options: Non-polar HT5 (10–25 m length, 0.25–0.32 mm ID, 0.1 µm film) with or without retention gap for optimal resolution.
• Carrier Gas: Helium at constant linear velocity (approx. 50 cm/s).

Main Results and Discussion

Chromatographic performance demonstrated baseline separation of free glycerol, monoolein, monostearin, monopalmitin, diglycerides, and triglycerides in standard and sample runs. Typical retention times and peak shapes improved after MSTFA derivatization. Calibration curves showed linear responses across the concentration range, enabling precise quantification. Sample chromatograms confirmed efficient removal of excess methanol and clear detection of residual glycerol and glycerides within method limits.

Benefits and Practical Applications

The described method offers:

• Regulatory compliance with DIN EN 14105 and ASTM D6584.
• High reproducibility and sensitivity for trace glycerol and glycerides.
• Compatibility with routine quality control workflows in biodiesel production facilities.
• Flexibility to adapt column length and ID for faster or higher-resolution analyses.

Future Trends and Potential Applications

Advancements may include:

• Integration of two-dimensional GC or GC-MS detection for enhanced selectivity.
• Automation of derivatization and sample injection for higher throughput.
• Development of novel derivatization reagents to further improve sensitivity.
• Application of fast GC columns and advanced detectors to reduce analysis time.

Conclusion

The DIN EN 14105 reference method, combined with MSTFA derivatization and optimized GC conditions, provides a robust analytical approach for assessing glycerol and glyceride levels in biodiesel. Its precision and regulatory alignment make it suitable for routine quality assurance in biodiesel production and blending operations.