Quality analysis of virgin olive oils – Part 3

Significance of the topic

Chlorophyll degradation in virgin olive oil influences oil color and quality. Analysis of pheophytins and pyropheophytins provides insight into storage conditions and refining treatments.

Objectives and overview of the study

The study evaluates the determination of chlorophyll a degradation products pheophytin a pheophytin a prime and pyropheophytin a using an Agilent 1260 Infinity LC system with fluorescence detection. Sample preparation follows ISO 29841 2009 E. Both a conventional 30 minute run and a rapid 8 minute run were developed and applied to various virgin and partly refined olive oils.

Methodology

• Sample preparation by solid phase extraction on silica cartridges following ISO 29841 2009 E
• Standards prepared from chlorophyll a by acidification to yield pheophytins and thermal treatment to produce pyropheophytin
• Chromatography on Agilent 1260 Infinity Quaternary LC with fluorescence detection
• Mobile phase water methanol acetone ratio 4 36 60 isocratic flow
• Long run ZORBAX SB C18 250 x 4.6 mm 3.5 micrometer column 1 mL per min 30 minutes
• Short run ZORBAX RRHT SB C18 50 x 4.6 mm 1.8 micrometer column 1.2 mL per min 8 minutes
• Fluorescence detection excitation 430 nm emission 670 nm temperature 20 degC injection volume 20 microliter

Used instrumentation

• Agilent 1260 Infinity Quaternary Pump G1311B
• Agilent 1260 Infinity Autosampler G1367E
• Agilent 1290 Thermostatted Column Compartment G1316A
• Agilent 1260 Infinity Fluorescence Detector G1321B
• Columns ZORBAX SB C18 and ZORBAX RRHT SB C18

Main results and discussion

• Chromatographic separation achieved for chlorophyll a pheophytin a pheophytin a prime and pyropheophytin a
• Fresh virgin olive oil three months old contained minor pyropheophytin levels around 1.5 percent
• Virgin oils from harvest 2011 showed pyropheophytin levels of 8 to 14 percent
• Partly refined olive oil exhibited pyropheophytin content around 18 percent indicating heat induced decarbomethoxylation
• Relative pyropheophytin content calculated by peak area ratio formula independent of absolute concentration
• Short 8 minute run provided sufficient resolution for routine screening

Benefits and practical applications of the method

• Rapid and sensitive assessment of olive oil quality and storage history
• Detection of oil aging and potential refining processes
• Supports quality control in industrial and research laboratories

Future trends and potential applications

• Integration of faster ultra high pressure liquid chromatography techniques
• Coupling with mass spectrometry for enhanced identification
• Application to a wider range of edible oils and color stability studies
• Automation of sample preparation for high throughput analysis

Conclusion

The Agilent 1260 Infinity LC with fluorescence detection offers an effective tool for quantifying chlorophyll degradation products in olive oil. Both long and short chromatographic runs deliver reliable separation and quantitation supporting quality monitoring of fresh aged and refined oils.

References

1. Agilent Application Note 5991-1894EN Quality Analysis of Virgin Olive Oils Part 1 2013
2. Agilent Application Note 5991-1895EN Quality Analysis of Virgin Olive Oils Part 2 2013
3. M I Mínguez Mosquera et al Pigments present in virgin olive oil Journal of the American Oil Chemists Society Volume 67 Issue 3 1990
4. A Giuliani et al Chlorophylls in Olive and in Olive Oil Chemistry and Occurrences Critical Reviews in Food Science and Nutrition Volume 51 2011
5. K Aitzetmueller Chlorophyll Abbauprodukte in pflanzlichen Oelen European Journal of Lipid Science and Technology Volume 91 Issue 3 1989
6. R Aparicio Ruiz Thermal degradation Kinetics of Chlorophyll Pigments in Virgin Olive Oils Series a Journal of Agricultural and Food Chemistry Volume 58 Issue 10 2010