On-Line TOC Analyzer for Pure Water TOC-1000e

Significance of the topic

The valorization of agricultural pruning residues from Olea europaea represents a practical opportunity to recover high-value bioactive compounds (phenolics, carotenoids, tocopherols) while supporting circular-economy strategies. Developing a single-step, scalable, and environmentally compatible extraction workflow reduces solvent hazards and operator handling compared with conventional multi-step liquid extractions and enables more efficient industrial processing of olive leaf biomass.

Objectives and study overview

The study aimed to optimize a one-step supercritical fluid extraction (SFE) protocol that simultaneously isolates polar phenolics (exemplified by oleuropein), medium-polarity carotenoids (lutein, β-carotene), and non-polar tocopherols (α-tocopherol) from dried olive leaves. Ultrasound-assisted extraction (UAE) procedures were used as validated references to evaluate SFE recovery across compounds spanning wide log P values.

Methodology

Sample preparation and matrix: Olive leaves collected in Messina, Italy (Feb 2025) were washed, air-dried to constant weight, milled and sieved to <0.3 mm particle size. For SFE, 100 mg of dried powder was loaded into 0.2 mL stainless-steel extraction vessels.

Optimization strategy: The protocol explored static and dynamic extraction stages, CO2-only steps and CO2 modified with bio-ethanol (bio-EtOH), varying modifier percentage (10–40 %), extraction pressure (15–35 MPa), temperature (40–60 °C), dynamic times, and flow rates (1–2 mL·min−1). Recoveries were benchmarked against compound-specific UAE procedures used as references.

Optimized SFE conditions

The best compromise method (MET.6) consisted of:

• 2 min static extraction with 100 % CO2 to purge and fill the vessel
• 5 min dynamic extraction with 100 % CO2 (non-polar fraction)
• 5 min dynamic extraction with 10 % bio-ethanol modifier
• 25 min dynamic extraction with 30 % bio-ethanol modifier
• Operating parameters: flow rate 1 mL·min−1, pressure 35 MPa, temperature 40 °C

Used instrumentation

The SFE work used a Shimadzu Nexera UC system equipped with CBM-40 controller, SFE-30A module, LC-30ADSF CO2 pump, LC-40DXR modifier pump, LC-40D make-up pump, SFC-30A back pressure regulator, degasser (DGU-405) and FRC-40 fraction collector.

Analytical characterization of extracts employed HPLC with different detectors and interfaces:

• Phenolics: Reversed-phase C18 HPLC, PDA (280 nm) and ESI-MS (LCMS-2020)
• Carotenoids: C30 column, PDA (450 nm) and APCI-MS
• Tocopherols: Normal-phase (silica) HPLC, fluorescence detection (Ex 290 nm / Em 333 nm)

Main results and discussion

Recovery performance (optimized MET.6) relative to UAE references:

• Oleuropein (phenolic marker): 76 % recovery
• α‑Tocopherol: 84 % recovery
• Lutein: 88 % recovery
• β‑Carotene: 90 % recovery

Key observations from the optimization:

• Pure CO2 preferentially extracts non-polar components; a polar modifier (bio‑EtOH) is necessary to co-extract phenolics and polar pigments.
• Stepwise dynamic extraction (CO2 then low % modifier then higher % modifier) balances extraction of broad polarity ranges within a single run.
• Higher pressure (35 MPa) markedly improved overall recovery compared with 25 MPa; raising temperature above 40 °C gave mixed effects and reduced oleuropein yield, supporting 40 °C as optimal.
• Excessively high modifier fractions (>30–40 %) degrade supercritical CO2 solvent properties and give limited benefit; higher flow rates shorten contact time and can reduce recovery for polar targets.

Quantitative composition highlights of the optimized SFE extract (representative values, mg·kg−1): oleuropein ~64,519; hydroxyoleuropein ~3,675; verbascoside ~2,298; lutein ~865; trans‑β‑carotene ~217; α‑tocopherol ~171. These values indicate the extract is strongly enriched in oleuropein and contains appreciable amounts of carotenoids and tocopherols.

Benefits and practical applications

Advantages of the single-step SFE protocol demonstrated in this work:

• Shorter processing time (~40 min per sample) compared with ~3 hours required by three separate UAE procedures.
• Reduced reliance on hazardous organic solvents by using CO2 and bio-ethanol, improving operator safety and environmental profile.
• Automation and high-throughput potential via SFE instrumentation and fraction collection.
• Feasibility for scale-up and integration into circular-economy workflows to produce natural antioxidants, nutraceuticals, or food additives from orchard residues.

Future trends and applications

Potential developments and uses stemming from this approach include:

• Process scale-up and techno-economic assessment for pilot- and industrial-scale extraction of olive leaf bioactives.
• Refinement of fractionation strategies (in-line modulators or gradient modifiers) to selectively enrich specific compound classes.
• Integration with downstream purification (chromatography) or formulation platforms to produce standardized ingredients for nutraceutical, cosmetic, or food applications.
• Life-cycle and sustainability assessments comparing SFE with conventional solvent extraction to quantify environmental and economic benefits.

Conclusion

A single-step SFE workflow using CO2 and bio-ethanol was successfully optimized to co-extract phenolics, carotenoids and tocopherols from olive pruning residues with good recoveries (76–90 % for representative markers). Although recoveries were slightly lower than the most selective UAE methods, SFE delivered faster processing, improved safety, and better environmental compatibility, making it attractive for scalable valorization of agricultural residues into high-value bioactive extracts.

References

1. Olmo-García L., et al., Molecules, 2018, 23, e2419.
2. Dugo L., et al., Food Analytical Methods, 2020, 13, 1027–1041.
3. Silva Coelho T. L., et al., Ultrasonics Sonochemistry, 2022, 84, e105980.
4. Shimadzu Corporation, Supercritical fluid extraction of bioactive compounds from Olea europaea pruning wastes, Technical Report C190‑E349, First Edition July 2026.