Key Points
- Polyphenols, which are plant secondary metabolites, are typically extracted using reversed-phase liquid chromatography (LC), though poor retention and resolution are observed with this process.
- Supercritical fluid chromatography (SFC) has been proposed as an alternative approach, being capable of coupling with mass spectrometry (MS) and handling polar compunds.
- SFC has potential to become a more commonly used technique due to how it differs from LC.
Scientists from the Sapienza University of Rome (Rome, Italy) explored how supercritical fluid chromatography (SFC) can be used to analyze polyphenols as an alternative to reversed-phase liquid chromatography (LC). Their findings were published in the Journal of Chromatography A (1).
Night view of the Basilica St Peter in Rome, Italy | Image Credit: © Mapics – stock.adobe.com
The plant kingdom is said to produce between 100,000 to 1 million metabolites, with their main functions being to regulate growth and development, protect against biotic stresses (parasites, herbivores, and other living organisms), and improve tolerance to abiotic stresses (drought, frost). However, each plant species only synthesizes limited numbers of these metabolites, likely between 5000 and tens of thousands, with significant variations within a species. The ability to characterize natural compounds is an important tool for understanding plant adaptation mechanisms to their environment, which can also have implications in the quantitative and qualitative improvement of agricultural production. There is also significant research and commercial interest in natural compounds that exhibit bioactivity including therapeutic or anti-inflammatory properties, or organoleptic characteristics.
Polyphenols are a type of plant secondary metabolite, typically synthesized through the phenylpropanoid pathway. This group is comprised of flavonoids, stilbenes, lignans, and simple phenolic acids. Characterizing these natural compounds can be challenging due to the complex nature of plant extracts, the variety of polyphenols formed in plant organs, and the limited availability of authentic standards. Polyphenol analysis is mostly achieved through reversed-phase liquid chromatography (LC), though for polar polyphenols, poor retention and resolution are often observed.
Supercritical fluid chromatography (SFC), which involves using a supercritical fluid as a mobile phase, is viewed as a versatile chromatographic technique and a greener alternative to traditional LC (2). Further, the technique has been deemed suitable for a heterogeneous class of compounds as polyphenols, where very polar or chiral compound separation is requested. Advancements in instrumental technology allowed coupling with mass spectrometry (MS) and the introduction of sub-2 µm stationary phases, allowing the technique to be used for the analysis of compounds.
In this review, the scientists explored the latest supercritical fluid chromatography (SFC) methods for characterizing polyphenols from 2019 to 2024. The authors discussed the coupling of MS and applications in plants, along with other selected applications in polyphenol characterization. According to the scientists, SFC is relatively underutilized in this field, especially in combination with MS. This largely stems from the need for extensive optimization of several critical parameters.
Much previous research are quantitative targeted studies, with only select reports using metabolomics approaches for large-scale polyphenol analysis. SFC applications have been limited to a small range of polyphenols rather than complex mixtures, making the technique uncompetitive with standard LC.
Regardless, SFC has potential for further development, especially in areas where LC falters. According to the researchers, LC typically poses challenges with retaining highly polar analytes and performing chiral separations, even with dedicated stationary phases. SFC has been effective for chiral separations, showing promise for highly polar compounds when proper co-solvents and additives are used. SFC–MS must be optimized for a given group of analytes. The interest in chiral separations achievable by SFC is more related to pharmacologically active molecules than polyphenols. Further, high-resolution MS (HRMS) detection in SFC is mostly limited to time-of-flight (TOF) instruments, rarely exploiting the full potential of metabolomics approaches for comprehensive characterization. With time, SFC–MS may continue to gain attention for bio-oil characterization, lipid profiling, and metabolomics.
References
(1) Taglioni, E.; Capriotti, A. L.; Cerrato, A.; et al. Recent Applications of Supercritical Fluid Chromatography in the Analysis of Polyphenols. J. Chromatogr. A 2025, 1758, 466198. DOI: 10.1016/j.chroma.2025.466198
(2) Supercritical Fluid Chromatography. ScienceDirect 2000. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/supercritical-fluid-chromatography (accessed 2025-7-18)