Chromatography of FAMEs Using Cyanopropyl Capillary Columns

Importance of the Topic

Analysis of fatty acid methyl ester (FAME) isomer composition in hydrogenated vegetable fats such as corn margarine is critical for quality control and nutritional assessment. Trans fatty acids formed during partial hydrogenation pose health concerns, so reliable methods to separate and identify cis- and trans-isomers are essential in food analysis laboratories.

Objectives and Study Overview

This study compared the performance of two strongly polar cyanopropyl capillary columns (BPX-90 and Rtx-2560) in the gas chromatographic separation of C18 FAME isomers extracted from corn margarine. Key goals were:

• To evaluate chromatographic resolution of cis- and trans-C18:1, C18:2 and C18:3 isomers.
• To establish equivalent chain length (ECL) temperature dependencies for isomer differentiation without specialized standards.
• To apply the method to real margarine samples after methyl esterification.

Methodology and Instrumentation

Sample Preparation:

• Corn margarine was saponified and converted to fatty acid methyl esters via acidic methylation.

Chromatographic Conditions:

• Instrument: Shimadzu GC-2010 with FID detector.
• Columns:
  
  • BPX-90, 100 m or 160 m × 0.25 mm I.D., film 0.25 µm.
  • Rtx-2560, 100 m × 0.25 mm I.D., film 0.20 µm.
• Carrier gas: Helium at 20 cm/s.
• Column temperatures investigated from 160 °C to 190 °C (isothermal runs).
• Injection: Split (1:30), injector 280 °C; detector 280 °C.

Main Results and Discussion

Separation Performance:

• Both columns achieved baseline separation of single- and di-double bond C18:1 and C18:2 isomers, with enhanced resolution at higher temperatures.
• Minor unknown peaks (e.g., adjacent to C18:1 trans-13) were observed on BPX-90 at ≥ 180 °C, indicating potential unidentified trans isomers.

ECL Temperature Dependencies:

• C18:1 isomers: On Rtx-2560 the slope of ECL vs. temperature was ~0.0046–0.0054 for cis and 0.0025–0.0032 for trans; on BPX-90 it was ~0.0055–0.0063 for cis and 0.0029–0.0038 for trans.
• C18:2 isomers: Cis, cis isomers showed higher slopes (~0.0108–0.0136) than mixed cis/trans (~0.0083–0.0105) and trans, trans (~0.0056–0.0067), on both columns.
• C18:3 isomers: Slopes decreased consistently with increasing number of trans double bonds, confirming ECL dependence on isomer geometry.

Analysis of Actual Margarine:

• Comparison of ECL slopes between standard and sample peaks allowed unambiguous assignment of cis and trans isomers in the margarine extract without requiring pure trans standards.

Benefits and Practical Applications

• The method provides robust differentiation of cis/trans FAME isomers using temperature-dependent ECL criteria.
• High-resolution columns allow detection of minor trans isomers in food matrices.
• Analytical simplicity: no need for expensive trans-fatty acid standards, leveraging temperature dependency for peak identification.

Future Trends and Potential Applications

• Integration with automated temperature programming and data processing for rapid isomer profiling in quality control.
• Extension to other edible oils and fats to monitor industrial hydrogenation processes.
• Coupling with mass spectrometry for structural confirmation of unknown trans isomers.

Conclusion

This investigation demonstrates that strongly polar cyanopropyl GC columns (BPX-90 and Rtx-2560) combined with equivalent chain length temperature profiling enable reliable separation and identification of cis and trans C18 FAME isomers in margarine. The approach enhances food analysis capabilities and supports health-related quality assessments without reliance on specialized standards.

Reference

No external literature was cited in the source document.