Determination of Free and Total Glycerol and Mono-, Di-, and Triglyceride Contents in Biodiesel According to EN 14105

Significance of the topic

The reliable quantification of free and total glycerol and mono-, di- and triglyceride contents in biodiesel is essential for quality control, regulatory compliance and performance assurance of biofuels. These parameters influence fuel stability, injector fouling, cold-weather performance and compliance with standards such as EN 14214. Robust analytical methods that meet EN 14105:2024 enable producers and testing laboratories to verify feedstock impacts and process control across diverse raw materials (rapeseed, soybean, palm, recycled oils).

Objectives and study overview

This application study evaluated the performance of the Nexis GC-2060 gas chromatograph equipped with a multi-mode injection unit (MMI) and FID for determination of free and total glycerol and mono-, di-, and triglycerides in biodiesel according to EN 14105:2024. Key aims were to: compare on-column direct injection (OCI) and splitless programmed temperature vaporization (PTV) using the MMI, verify method suitability across rapeseed-, soybean- and palm-derived biodiesel, demonstrate use of hydrogen as a carrier gas, and confirm compliance with the standard’s quantification limits and instrument performance criteria.

Methodology and sample preparation

Samples were prepared following EN 14105:2024. Free glycerol and glycerides (mono- and diglycerides) were derivatized with MSTFA immediately before analysis due to limited derivatization stability; fresh preparations were made for each injection mode. Analysis was performed by GC-FID with a 1 µL injection of the prepared solution. Quantification strategies used internal standard calibration:

• Free glycerol: internal standard 1,2,4-butanetriol (series of calibration standards).
• Mono-, di- and triglycerides: species-specific internal standards — 1-glyceryl monononadecanoate (Mono C19), 1,3-glyceryl dinonadecanoate (Di C38), glyceryl trinonadecanoate (Tri C57).

Total glycerol was calculated from measured species using the formula specified in EN 14105:

G_total = G_free + 0.255·M + 0.146·D + 0.103·T

Instrument performance was monitored by a relative response factor (RF) between Di C38 and Tri C57; the RF must remain below 1.8 per the protocol.

Used instrumentation

The configuration and analytical conditions reported in the study were:

• GC: Nexis GC-2060 with multi-mode injection unit (MMI) and autosampler AOC-30i.
• Detector: Flame ionization detector (FID), set at 380 °C with default gas flows.
• Column: SH-I-5HT, 15 m × 0.32 mm × 0.1 µm (high-temperature 5% phenyl-type phase).
• Carrier gas: Hydrogen at 80 kPa (cost-effective carrier; system includes built-in safety measures).
• MMI modes: Direct on-column injection (simple OCI) or splitless PTV (programmable temperature vaporization) selectable via liner and software mode.
• MMI temperature program: start 50 °C, ramp 50→370 °C (specified rates up to 250 °C/min).
• Column temperature program: 50 °C (0.5 min) → 15 °C/min → 180 °C → 7 °C/min → 230 °C → 10 °C/min → 370 °C (hold ~9.7 min).

Main results and discussion

Comparative analysis of OCI (direct mode) and splitless PTV using the MMI showed highly comparable chromatograms and quantitative results across feedstocks. Key findings:

• RF monitoring for Di C38/Tri C57 remained constant at ~1.2 for all injections in both MMI modes, well below the 1.8 threshold.
• PTV did not introduce additional triglyceride discrimination in the inlet and provided good recovery of high-boiling glycerides, supporting it as a practical alternative to direct OCI.
• Quantification limits specified in EN 14105 (0.10 % for glycerides and 0.001 % for free glycerol) were met. Calibration linearity for glycerol showed R² ≈ 0.9996 and %RSD < 1.8 for standards.
• Representative results (direct vs PTV) for three biodiesel types were consistent: rapeseed (Mono 0.42 %, Di 0.12 %, Tri 0.10 %, free glycerol ~0.008 %, total glycerol ~0.142 %), palm (Mono <0.10 %, Di 0.10 %, Tri 0.14–0.15 %, free glycerol 0.005 %, total glycerol ~0.05 %), soybean (Mono 0.64–0.65 %, Di 0.12–0.13 %, Tri 0.02 %, free glycerol 0.003 %, total glycerol ~0.186–0.188 %).
• All samples were below the maximum limits defined in EN 14214:2019 for biodiesel (free glycerol ≤ 0.02 %, total glycerol ≤ 0.25 %, monoglycerides ≤ 0.70 %, diglycerides and triglycerides ≤ 0.20 %).

Benefits and practical applications

The validated approach demonstrates several practical advantages for routine biodiesel testing laboratories:

• Flexibility: The Nexis GC-2060 MMI supports either OCI or PTV workflows, enabling labs to adopt the injection strategy that best fits their operational preferences without sacrificing accuracy.
• Cost and throughput: Hydrogen carrier gas offers cost-effectiveness and fast separations when safety systems are in place.
• Regulatory compliance: The method achieves the sensitivity and linearity required by EN 14105:2024 and produces results compatible with EN 14214 acceptance criteria.
• Robustness: Stable RF values and reproducible results across feedstocks indicate reliable instrument performance for diverse biodiesel matrices.

Future trends and possible applications

Anticipated developments and opportunities to enhance biodiesel glyceride/glycerol analysis include:

• Further automation of derivatization steps to reduce variability from limited MSTFA stability and to increase sample throughput.
• Broader adoption of hydrogen carrier gas coupled with enhanced safety and leak-detection systems to reduce operating costs and improve chromatographic speed.
• Application of high-temperature stationary phases and optimized inlet strategies (e.g., PTV programs) to improve recovery of very high-boiling components and to reduce discrimination.
• Integration with laboratory information management systems (LIMS) for streamlined regulatory reporting and QC trending.
• Exploration of complementary detectors or MS coupling for structural confirmation when complex feedstock contaminants are suspected.

Conclusion

The Nexis GC-2060 equipped with the MMI and FID provides a reliable, standards-compliant solution for determination of free and total glycerol and mono-, di- and triglycerides in biodiesel according to EN 14105:2024. Both direct on-column injection and splitless PTV modes deliver comparable performance, stable RF behavior and measured values within EN 14214:2019 limits across rapeseed, soybean and palm oil feedstocks. Fresh derivatization prior to analysis and proper instrument configuration are important for reproducible results. The approach offers laboratories flexibility, regulatory confidence and cost advantages when using hydrogen carrier gas under appropriate safety protocols.

Reference

1. EN 14105:2024 - Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride content.
2. EN 14214:2019 - Liquid petroleum products - Fatty acid methyl esters (FAME) for use in diesel engines and heating applications - Requirements and test methods.