Sensitive analysis of polar lipids using HPLC with low temperature evaporative light scattering detection

Importance of the Topic

Polar lipids are essential nutritional and structural components in foods, pharmaceuticals and biological systems. Their analysis is demanding because many lipids lack strong UV chromophores and can degrade under high temperatures, necessitating robust, sensitive detection methods compatible with complex matrices.

Objectives and Overview of the Study

This application note evaluates the performance of high-performance liquid chromatography coupled with the Agilent 1290 Infinity evaporative light scattering detector (ELSD) for the sensitive separation and quantification of polar lipids such as cholesterol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine.

Methodology and Instrumentation

The chromatographic separation was performed on a silica column (250 × 4.6 mm, 3 µm) at a flow rate of 0.3 mL/min. Mobile phases comprised mixtures of isopropanol, hexane, water and ammonium hydroxide (eluent A: 57.8/40/2/0.2; eluent B: 51.8/40/8/0.2). A gradient from 0 to 100% B in 7 minutes (hold 8 minutes), followed by a return to 0% B in 5 minutes (hold 10 minutes), was used. Injection volume was 10 µL. The Agilent 1290 Infinity ELSD was operated with a nebulizer temperature of 40 °C, an evaporation temperature of 90 °C and a gas flow of 0.8 SLM, enabling low-temperature analysis of labile species.

Main Results and Discussion

The method achieved baseline resolution of five polar lipids under ambient conditions. Signal-to-noise ratios at a loading of 100 µg/mL ranged from 33 (sphingomyelin) to 158 (cholesterol), demonstrating excellent sensitivity. Calibration curves for each lipid exhibited high linearity (R2 ≥ 0.99), confirming the detector’s capability for accurate quantitation without derivatization.

Benefits and Practical Applications

The universal response of ELSD, combined with compatibility with gradient elution and ambient-temperature operation, eliminates the need for time-consuming derivatization and reduces thermal degradation. This approach is well suited for rapid lipid profiling in food quality control, pharmaceutical formulation analysis and academic lipidomics research.

Future Trends and Applications

• Integration with high-resolution mass spectrometry for structural elucidation and lipidomics workflows
• Development of lower-flow micro- and nano-HPLC ELSD systems to improve sensitivity and reduce solvent use
• Automation and high-throughput platforms for routine lipid screening in industry
• Advanced data processing and chemometric methods to handle complex lipid mixtures

Conclusion

The Agilent 1290 Infinity ELSD represents a next-generation detector for polar lipid analysis, offering universal detection, high sensitivity, linear quantification and ambient-temperature operation. It provides a robust alternative to UV and refractive index detectors, streamlining workflows for complex lipid mixtures.

References

• Agilent Technologies. Sensitive analysis of polar lipids using HPLC with low temperature evaporative light scattering detection. Application Note, 2015.