Determination of Phenolic Compounds in Virgin Olive Oil Using Comprehensive 2D-LC - - Chromatography Online
Determination of Phenolic Compounds in Virgin Olive Oil Using Comprehensive 2D-LC

The Application Notebook
pp. 19

This application note demonstrates how comprehensive 2D-LC can be used to resolve the complex mixture of hydrophilic phenols found in virgin olive oil and investigates differences in the phenol composition of several olive oils.

Virgin olive oil is associated with the health and nutritional benefits of the Mediterranean diet. In this respect, the presence of antioxidants, which are represented by hydrophilic phenols among others, plays an important role. Hydrophilic phenols contained in virgin olive oil include phenolic acids and alcohols, flavonoids, secoiridoids, and lignans (1,2).

One-dimensional liquid chromatography is not able to resolve completely the complex mixture of hydrophilic phenols present in virgin olive oil (3). Due to its high separation capability, comprehensive 2D-LC can be deployed to improve the separation.

Experimental Conditions

Comprehensive 2D-LC analysis was achieved with the Agilent 1290 Infinity 2D-LC solution. The first dimension separation used an Agilent ZORBAX RRHD Eclipse Plus Phenyl-Hexyl column (2.1 150 mm, 1.8 m) with a gradient of water and methanol, each with 0.1% formic acid, at a flow rate of 0.05 mL/min. In the second dimension, an Agilent ZORBAX RRHD Eclipse Plus C18 column (3.0 50 mm, 1.8 m) was used with shifted gradients of water and acetonitrile, each with 0.1% formic acid, at a flow rate of 3.0 mL/min. Modulation was realized using the Agilent 2-position/4-port duo-valve, equipped with two 60 L loops and with a modulation time of 30 s. Detection was performed at 260 nm and by ESI-TOF-MS in negative ionization mode. Preparation of olive oil samples was carried out according to the protocol from the International Olive Council (4).


Four different olive oil samples with high phenol content (3) purchased from Italian olive oil farms were analyzed by comprehensive 2D-LC. Figure 1 (top) exemplarily shows the 2D-LC chromatogram of one olive oil.

Figure 1: Two-dimensional-LC chromatogram of an olive oil at 260 nm (top) and visualization of differences between the olive oils analyzed (bottom); blue circles indicate higher percent responses and white circles lower percent responses of substances detected, areas indicate differences.
MS detection showed that the main hydrophilic phenols present in the analyzed olive oils are aglycons of oleuropein, ligstroside, and their derivatives. Further, elenolic acid, luteolin, apigenin, hydroxytyrosol, and hydroxytyrosol acetate were identified in all olive oils analyzed. Compared to the olive oils A–C (from the same farm), olive oil D (from another farm) showed higher percent responses of the flavonoids apigenin and luteolin (Figure 1).


The Agilent 1290 Infinity 2D-LC solution can be used to improve significantly the separation of hydrophilic phenols contained in virgin olive oil. This enables the investigation of differences between the compositions of hydrophilic phenols in olive oils.


(1) M. El Riachy et al., Eur. J. Lipid Sci. Technol. 113, 678–691 (2011).

(2) Y. Ouni et al., Food Chemistry, 127, 1263–1267 (2011).

(3) S. Schneider, "Quality Analysis of Virgin Olive Oils – Part 6," Agilent Application Note, publication number 5991-3801EN, (2014).

(4) "Determination of biophenols in olive oils by HPLC," International Olive Council: COI/T.20/DOC. 29, (2009).

Agilent Technologies Inc.
5301 Stevens Creek Blvd., Santa Clara, CA 95051


blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:



Column Watch: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments. LATEST: When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: HPLC for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

MS — The Practical Art: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis

LC Troubleshooting: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: How Much Can I Inject? Part I: Injecting in Mobile Phase

More LCGC Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>

Source: The Application Notebook,
Click here