Fatty acid methyl ester

Significance of the Topic

Fatty acid methyl esters (FAMEs) are key markers for profiling lipid composition in food, biological and clinical samples. Accurate separation and quantification of FAMEs is essential for quality control in food and feed industries, nutritional studies and metabolic research. Using highly inert GC stationary phases minimizes adsorption and peak tailing, improving reliability when analyzing unsaturated and trans isomers.

Objectives and Study Overview

The primary goal of this application note was to demonstrate the separation efficiency and inertness of an InertCap® 225 column in GC–FID analysis of a comprehensive 41-component FAME mixture. The study provides an overview of chromatographic conditions, retention behavior of saturated, cis- and trans-unsaturated FAMEs up to C24, and highlights the ability to resolve positional and geometric isomers.

Methodology and Instrumentation

System and Conditions:

• Instrument: Gas Chromatograph with Flame Ionization Detector (GC–FID)
• Column: InertCap 225 custom for MS, 0.25 mm × 60 m, df = 0.25 µm
• Temperature Program: 80 °C hold 1 min, ramp 20 °C/min to 180 °C, ramp 3 °C/min to 235 °C
• Carrier Gas: Hydrogen at 50 cm/s linear velocity
• Injection: Split mode, 23.9 mL/min, injector at 250 °C
• Detector: FID at 250 °C
• Sample: 1.0 µL of FAME mixture in hexane

Main Results and Discussion

The InertCap 225 column achieved baseline separation of 41 FAME components, ranging from C6:0 to C24:1 and polyunsaturated species such as C20:4, C20:5 and C22:6. Key observations:

• Saturated FAMEs (C6–C24) eluted in increasing retention order with sharp, symmetric peaks.
• Monounsaturated cis- and trans-isomers (e.g., C16:1(9c/t), C18:1(9c/t), C20:1) were fully resolved, demonstrating high phase inertness against double-bond interactions.
• Positional and geometric isomers of di- and tri-unsaturated FAMEs (e.g., C18:2, C18:3) showed distinct peak separation, supporting detailed fatty acid profiling.
• Long-chain polyunsaturated FAMEs (C20:4, C20:5, C22:6) were eluted without significant peak broadening, indicating stable phase performance at high temperatures.

Practical Benefits and Applications of the Method

By combining a hydrogen carrier gas and a highly inert stationary phase, the method delivers:

• Improved sensitivity and reproducibility for unsaturated FAME analyses.
• Enhanced resolution of cis/trans and positional isomers crucial for nutritional labeling and trans fat regulations.
• High throughput profiling suitable for food authenticity, clinical lipidomics and bioprocess monitoring.

Future Trends and Possibilities

Advancements may include coupling the InertCap 225 with mass spectrometry for structural confirmation, implementation of faster temperature programs for high-throughput screening, and integration of AI-driven deconvolution algorithms to automate isomer identification. Expanding column chemistries to target highly polar or conjugated fatty acid derivatives can further broaden application scope.

Conclusion

The InertCap 225 GC column demonstrates excellent inertness and resolving power for complex FAME mixtures, enabling precise separation of saturated, cis/trans unsaturated and polyunsaturated isomers up to C24. This robust GC–FID method supports diverse analytical needs in food, clinical and research laboratories.

Reference

Chromatogram data provided by Dr. Kouhei Yamamoto (Osaka Prefecture University, Japan).