Isolation and Identification of Phenolic Compounds

Maria Inês Rouxinol

doi:10.5772/intechopen.1005224

Abstract

Isolation and identification of phenolic compounds are crucial processes in the field of natural product chemistry and biochemistry. Phenolic compounds are secondary metabolites widely distributed in plants, exhibiting diverse biological activities with potential health benefits. The isolation involves extracting these compounds from plant sources using various techniques such as solvent extraction, steam distillation, or solid-phase extraction. Following isolation, identification is accomplished through sophisticated analytical methods like high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy. These methods allow researchers to characterize and quantify specific phenolic compounds, elucidating their structures and understanding their roles in plant physiology and human health. The isolation and identification of phenolic compounds contribute significantly to the exploration of natural resources for pharmaceutical, nutritional, and industrial applications.

Keywords

phenolic compounds
natural products
analytical methods
secondary metabolites
plant extracts

Author Information

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Maria Inês Rouxinol*
- MED—Mediterranean Institute for Agriculture, Environment and Development & CHANGE—Global Change and Sustainability Institute, Institute for Advanced Studies and Research, Universidade de Évora Pólo da Mitra, Évora, Portugal

*Address all correspondence to: mir@uevora.pt

1. Introduction

Polyphenols represent a broad group of compounds primarily occurring in plants as secondary metabolites. They are produced as a natural part of plant growth and in reaction to diverse biotic and abiotic stress. This class of compounds, which includes simple phenols, hydroxybenzoic acids, cinnamic acid derivatives, flavonoids, coumarins, stilbenes, tannins, and others, is derived from the amino acids phenylalanine and tyrosine [1]. The selection of appropriate methods for the extraction and quantification of phenolic compounds is extremely important to find the intricate chemical profiles present in complex matrices [2]. Beyond its influence on the precision and trustworthiness of analytical findings, it’s also essential for a method to effectively capture the unique characteristics of specific matrices. Phenolic compounds are widely distributed in nature. They exhibit structural diversity and reactivity, making the choice of methods used to extract and quantify them critical to the success of any analytical endeavor. In order to gain meaningful insight into the complex world of phenolic compounds across different scientific disciplines, it is essential to tailor methods to the nature of the matrices under investigation and to align them with the desired results. This chapter explores the central role of method selection in achieving precision and relevance in phenolic compound analysis.

Phenolics are commonly extracted using solvents, which could either be organic or inorganic. The yield of phenolics is influenced by various factors such as extraction duration, temperature, solvent-to-sample ratio, number of extractions, and solvent type. The achievement of optimal phenolic recovery varies across samples, relying on the plant type and its active compounds. Extraction solvents include water, acetone, ethyl acetate, and various alcohols (such as methanol, ethanol, and propanol), along with their combinations [2]. Due to all their properties, there is a growing demand for highly sensitive and selective analytical methods for the determination of polyphenols. The methodologies for phenolic compound quantification are based on the extraction and isolation of compounds. Extraction is a very important step, however there is no single and standard extraction method. In typical procedures, it’s essential to disrupt the samples, whether through grinding, drying, or lyophilizing, before conducting solvent extraction. In addition, with the realization of these methodologies, non-phenolic compounds such as sugars, organic acids, and proteins are also extracted, which may require requiring subsequent purification processes (for example, solid-phase extraction). At analytical level, the extraction method has a great influence on the phenolic amount and composition [3]. The selection of an appropriate extraction method is crucial for the recovery of phenolic compounds from plant samples, as single-step extraction and unsuitable methods can impact the overall recovery rate [4]. Determining and quantifying phenolic compounds can present challenges due to their intricate nature and structural diversity. Consequently, numerous methods are recognized and employed for quantifying phenolic compounds in plant extracts [5].

2. Methods for phenolic compounds extraction

2.1 Conventional extraction

Maceration, a widely used traditional extraction method for phenolic compounds, relies on the principle of “like dissolves like.” Typically employing ethanol or methanol as solvents, this technique extracts organic phenolic compounds from plant samples [4]. Accelerating the process often involves using a shaking incubator for enhanced contact between samples and solvent, with temperature control. While higher temperatures increase solubility and diffusion, it’s crucial to avoid overheating to prevent solvent loss and phenolic compound decomposition [4, 6]. The effectiveness of conventional extraction is influenced by the duration within a specific time range. Higher extraction durations generally lead to increased efficiency until a solute equilibrium is reached between the inside and outside of the solid material. To achieve a higher extraction yield, a greater solvent-to-solid ratio is necessary. However, an excessively high ratio results in the use of excessive extraction solvent, demanding a longer extraction time. Balancing these factors is crucial for optimizing extraction efficiency [4].

2.2 Ultrasonic-assisted extraction (UAE)

Ultrasound is employed in extraction processes to reduce extraction time and enhance quality by inducing solvent-producing cavitation and high shear forces. Ultrasound-assisted extraction (UAE) is a modern method known for its simplicity, energy efficiency, and high reproducibility, offering a substantial yield of active compounds [7]. UAE is more efficient, requiring less solvent and a shorter extraction duration compared to conventional methods [4]. Mainly utilized in solid/liquid systems, UAE disrupts the cellular walls of plant materials, facilitating mass transfer across membranes and increasing solvent access to analytes. Extraction efficiency in UAE is influenced by factors like solvent composition, solvent-to-sample ratio, ultrasound amplitude and cycle, solvent pH, and temperature [8, 9]. While stronger ultrasonic applications can accelerate changes, cost considerations in food industries often lead to optimized applications for the best results with minimal energy usage [9]. To enhance extraction efficiency, factors such as amplifying ultrasound power, minimizing moisture content in food materials, and controlling temperature are considered. Proper selection of ultrasound frequency is essential, impacting the size of bubbles produced during resonance [4]. Solvent selection and temperature were identified as crucial factors impacting UAE efficiency [8, 9]. For highly polar phenolic compounds, extraction with pure organic solvents may exhibit low efficiency, making ethanol/methanol mixtures with water in various proportions commonly used as effective extraction solvents [9].

2.3 Reflux extraction

Reflux extraction, also known as solvent recycling reflux extraction, simultaneously extracts and concentrates the solvent [10]. The process involves two main components: the extraction tank and the concentration tank. The solvent is pumped from the extraction tank to the concentration tank during extraction, where it is heated, evaporated, condensed, and returned to the extraction tank. This cycle is repeated to accumulate extracts. Reflux extraction offers advantages such as shorter extraction time, reduced solvent cost, and lower fixed investment compared to conventional extraction. However, it has drawbacks, including potential contamination or decomposition of phenolic compounds during the concentration and heating stages. While reflux extraction has its advantages, it cannot fully replace conventional extraction [4].

2.4 Microwave-assisted extraction (MAE)

In recent years, there has been a growing emphasis on reducing the use of organic solvents in extractions, leading to the development and optimization of microwave-assisted extraction (MAE). Microwaves, characterized by electric and magnetic fields oscillating perpendicularly in a high-frequency range (0.3–300 GHz), induce localized heating. As a consequence, the plant matrix is destroyed, thereby facilitating the smoother diffusion of the desired compounds into the solvent [1, 11]. Usually, microwave powers ranging from 300 to 900 W and extraction temperatures spanning from 50 to 100°C are utilized in MAE [9]. In comparison with conventional extraction, MAE facilitates selective migration of target compounds within a shorter timeframe due to highly localized temperature and pressure. MAE achieves similar or higher recoveries than conventional extraction while requiring less space, time, and solvent [4, 12, 13]. In MAE, product recovery is boosted by the heating effect of microwaves, akin to conventional extraction methods. Although microwave heating is faster than conventional heating, it can lead to increased energy expenses. When comparing MAE to UAE and conventional methods, it’s crucial to consider the precise energy costs, particularly factoring in electricity expenses [8]. However, the potential for super boiling during MAE must be considered, especially when the penetration depth characteristic for the solvent is larger than the sample size. Scaling up MAE from laboratory to industrial levels requires careful consideration of solvent and sample sizes to avoid false results and safety issues [13]. The primary challenge associated with this extraction technique is achieving maximum extraction yield by effectively breaking down cellular tissue without compromising the chemical structure of the natural compounds. This balance is crucial for obtaining high-quality extracts through MAE [1, 9, 11, 14].

2.5 Soxhlet extraction

Soxhlet extraction stands out among conventional methods for phenolic compound extraction, requiring less solvent and time, resulting in low processing costs. The extraction device is user-friendly and suitable for initial and bulk extraction with a good recovery rate [15]. This methodology is an enhanced method derived from reflux extraction that integrates the advantages of percolation by enabling continuous extraction through reflux and siphon mechanisms. Although it offers automation and requires less solvent and time compared to conventional extraction methods, Soxhlet extraction’s reliance on thermal processes may lead to thermal degradation with prolonged heating [4]. This method has the potential to yield higher total phenolic and tannin content than conventional extraction. However, it’s crucial to note that with more stages, the high temperature involved in Soxhlet extraction may lead to the decomposition of phenolic compounds, as shown in an example by Ouahida et al. [16].

2.6 Pressurized liquid extraction (PLE)

Pressurized liquid extraction (PLE) (also known as accelerated solvent extraction—ASE) involves placing solid samples into a robust container saturated with extraction solvents, followed by a 5–15 minutes extraction period conducted at elevated temperatures and pressures [8]. Increased pressure enables solvents to stay in a liquid state above their boiling points, boosting the solubility and diffusion rate of lipid solutes and aiding solvent penetration into the matrix. PLE, when contrasted with conventional extraction methods, diminishes solvent and time demands while showcasing improved repeatability [4]. Widely employed by scientists for extracting natural products such as anthocyanin and saponins, PLE requires the same or even lower volumes of solvents compared to conventional techniques. It is a time-saving method that minimizes sample handling [6, 17, 18]. However, the mechanism of PLE, allowing solvents to remain in liquid form at high pressure and temperature, can lead to heat degradation. To enhance efficiency, Ju and Howard [17] recommend combining PLE with less efficient solvents at low temperatures, like water [17]. By adjusting process parameters, PLE enables faster extraction and enhanced selectivity for specific compound groups. The use of high pressure guarantees that solvents maintain their liquid form even when temperatures are raised, thereby enabling efficient extraction at elevated temperatures. Such conditions enhance the solubility of desired compounds and promote faster desorption from plant matrices. Furthermore, because PLE is carried out within a sealed system, the likelihood of oxidation reactions occurring is minimized [9]. Studies indicate that ASE efficiency is optimized when solvent mixtures, such as methanol or ethanol in water, are used instead of pure solvents, considering polarity compatibility [9]. Working pressures within the range of 4–20 MPa impact solvent diffusion into the matrix pores, promoting better contact between target compounds and the solvent [11, 19]. Temperature is a crucial parameter, and research suggests that an increase in phenolic extraction efficiency occurs within the temperature range of 40–120°C [11].

2.7 Supercritical fluid extraction (SFE)

Supercritical fluid extraction (SFE) is an eco-friendly technology utilizing supercritical fluids (SFs), characterized by critical values of pressure and temperature, to extract bioactive components from vegetal materials [11, 20]. It uses SF, such as supercritical carbon dioxide (S-CO₂), as a solvent for extraction. SF exhibits properties similar to both liquids and gases, making it ideal for dissolving a wide range of natural materials. S-CO₂, a commonly used SF, offers advantages such as low critical temperature, selectivity, inertness, and non-toxicity, making it suitable for extracting non-polar materials like lipids. However, for extracting phenolic compounds, co-solvents are often needed to enhance solubility [4]. SFE is recognized for producing clean extracts, avoiding oxidation, and degradation of phenolic compounds that can occur with conventional extraction methods. It is also acknowledged as safe by regulatory bodies like the European Food Safety Authority (EFSA) and the Food and Drug Administration (FDA) of the United States [11, 21]. However, a limitation of this approach is that SC-CO₂, being a non-polar solvent with an affinity for non-polar or low-polar compounds, exhibits low solubility for polyphenols, resulting in low extraction yields [20]. To overcome this constraint, research has investigated incorporating chemical modifiers or co-solvents, including water, methanol, ethanol, acetone, acetonitrile, or a mixture of acidified ethanol and water, to alter the non-polar characteristics of supercritical CO₂ [21]. Despite its efficacy, the high cost limits the widespread use of SFE, making it primarily applicable to high-value products [22, 23].

2.8 Pulsed electric field extraction (PEF)

Pulsed electric field (PEF) is a non-thermal extraction method that employs short, high-voltage pulses to disrupt membrane structures, enhancing extraction yield by releasing cellular content. Efficiency in PEF is influenced by factors such as field strength, specific energy input, pulse number, and temperature. The intact cytomembrane in plant cells acts as a semipermeable barrier, controlling substance movement in and out. PEF treatment disintegrates the cell membrane, increasing cell wall permeability, and allowing more bioactive compounds to be released into solvents. This structural disintegration destroys selective permeability, facilitating the extraction of more substances. PEF does not require heating, minimizing heat generation, and preventing the degradation of thermolabile compounds [4, 24].

2.9 Enzyme-assisted extraction (EAE)

Enzyme-assisted extraction (EAE) involves using enzymes to hydrolyze cell membrane components, disrupting their selective permeability and enhancing the extraction rate by releasing compounds [8]. Enzymatic hydrolysis, a commonly employed and safe extraction method in numerous food applications, utilizes the enzymatic action of cellulases, pectinases, and hemicellulases to break down cell walls, thereby improving the extraction of valuable compounds from plants [25]. Additionally, the enzymatic activity of lyases and hydrolases on glycosidic fractions of natural polyphenols improves their biological properties, enhancing bioactivity and bioavailability [26]. The cell membrane structure, primarily composed of macromolecules like polysaccharides and proteins, is susceptible to denaturation under high temperatures, affecting extraction efficiency. Enzymes, such as cellulase, are applied in EAE as a nonthermal and nontoxic treatment to boost efficiency [4]. Mixtures of enzymes, including pectinases, endo- and exo-glucanases, β-glucosidases, β-galactosidase, and cellobiases, are employed to achieve an overall synergistic effect [27, 28]. Cellulose and peptinase in enzymatic hydrolysis are utilized to release polymeric polyphenols, theoretically considered “non-extractable,” from the plant matrix [28]. However, EAE is more complex compared to chemical- and physical-assisted extraction methods. To achieve high-quality extracts, a detailed understanding of sample composition and the suitable enzyme for extraction is necessary. Additionally, enzyme activity is influenced by factors like pH, temperature, and substrate concentration, adding to the intricacy of EAE [4]. While an increase in temperature enhances mass transfer rates and solubility, temperatures below 60°C are typically used to avoid enzymatic denaturation. An environment with pH values ranging between 4.0 and 6.5 is optimal for enzymatic system activity [25, 27].

2.10 Extraction with ionic liquids

Recent research has delved into extracting bioactive constituents from herbal medicines or other plant-based sources using ionic liquids (ILs) and deep eutectic solvents (DESs) as alternative solvents. These studies showcase their potential to supplant traditional organic solvents [29]. ILs, a category of organic salts, exist in a liquid state below 100°C and comprise an organic cation and an inorganic or organic anion [8]. The choice of solvent polarity, hydrophobicity, viscosity, and miscibility can be customized by selecting either the cationic or anionic component. Various combinations of cations and anions lead to a broad spectrum of physicochemical properties, influenced notably by the nature and size of the cation, especially the anion. Depending on their composition, resulting ILs may display hydrophobic or hydrophilic traits, varying viscosity, compatibility with water or other organic phases, and distinct electrochemical characteristics [6].

3. Chromatographic techniques for separation, identification, and quantification

The quantification, purification, separation, and identification of specific phenolic compounds (including anthocyanins) are dependent on expensive equipment with lengthy sample preparation. To identify and quantify anthocyanins, methods such as paper chromatography (PC), thin-layer chromatography (TLC), column chromatography, solid-phase extraction, counter-current chromatography, and high-performance liquid chromatography (HPLC) are used [30, 31, 32, 33]. Both gas chromatography (GC) and HPLC coupled with mass spectrometry (MS) have proven utility in detecting phenolic compounds in various samples [34, 35]. These methods are accurate and efficient, providing reliable results within short analysis times. As HPLC techniques, simultaneous identification of phenolics with a wide range of polarities becomes increasingly possible [34, 35]. Despite HPLC and GC being the most used techniques, they are expensive and require specific equipment, other chromatographic techniques are still used to determine phenolic compounds.

3.1 Paper chromatography (PC) and thin-layer chromatography (TLC)

Paper chromatography (PC) and thin-layer chromatography (TLC) are partitioning techniques utilized for the separation of phenolics in foods [36]. While PC is a simpler and less commonly used method compared to HPLC and GC, it has proven effective for separating and identifying phenolic compounds in tea leaves and green leafy vegetables [37, 38].

TLC emerges as an easy and reliable technique, especially for analyzing phenolics in crude plant extracts. Various TLC techniques are cost-effective and allow for multiple detections on the same TLC plate within a short analysis time. Silica gel TLC-based video imaging has been identified as a valuable complementary fingerprint technique for identifying phenolic acids and flavonoid fractions from different sage species [39, 40].

3.2 High-speed counter-current chromatography (HSCCC)

High-speed counter-current chromatography (HSCCC) is a biphasic liquid-liquid partitioning method widely employed for the isolation and separation of various natural compounds [36, 41, 42, 43]. Operating without a solid support, HSCCC facilitates the permanent adsorption of sample compounds, allowing for the isolation and purification of natural compounds from crude extracts without preparation [44].

3.3 Capillary electrophoresis (CE)

Capillary electrophoresis (CE) is a high-resolution technique utilizing a narrow capillary column with a solution of ions. CE is suitable for rapidly and efficiently identifying charged low- and medium-molecular-weight compounds, with low sample and reagent volume requirements [45]. Although there is a scarcity of research on utilizing CE for the separation and identification of phenolics in plant materials [46, 47, 48, 49], micellar electrokinetic chromatography, capillary electrochromatography, and capillary zone electrophoresis coupled with various detection methods are widely employed in CE separation [6, 50].

3.4 Supercritical fluid chromatography (SFC)

Supercritical fluid chromatography (SFC) stands out as a versatile technique for analyzing and identifying phenolics, offering high separation efficiency, resolution, short analysis time, environmental friendliness, and compatibility with different detectors [51, 52].

3.5 Gas chromatography (GC)

Gas chromatography stands as another exceptionally efficient method for isolating, identifying, and quantifying various phenolic species. The primary limitation in GC analysis is the low volatility of phenolic compounds, necessitating their derivatization [53, 54]. Numerous analytical techniques for identifying and quantifying phenolic compounds rely on gas chromatography-mass spectrometry (GC-MS) [55]. Nevertheless, the majority of methods concentrate on a limited number of specific compounds. There is generally a lack of an efficient approach for identifying and quantifying a broad spectrum of trace phenolic compounds in wastewater. The recent development of techniques such as retention time locking (RTL) and deconvolution report software (DRS) addresses this issue by enabling multi-residue analysis. These methods facilitate the simultaneous identification of numerous target compounds, even in cases where they may be obscured by co-eluting matrix compounds. Hence, it is recommended to develop a screening method for identifying target phenolic compounds from a considerable pool of candidates utilizing DRS and RTL. Despite DRS’s ability to qualify and quantify compounds listed in libraries, there is presently no dedicated library specifically designed for phenolic compounds [56].

3.6 High-performance liquid chromatography (HPLC)

The quantification and identification of phenolic compounds have been widely studied using high-performance liquid chromatography (HPLC) coupled with a diode array detector (DAD) [57, 58, 59, 60, 61, 62]. In the analysis of complex matrixes, chromatographic techniques, specifically HPLC, are deemed more suitable due to their sensitivity, allowing the separation and identification of various anthocyanins in complex matrixes, and providing specific information. Although they are recommended, the multitude of protocols available can pose challenges in selecting the optimal method for determining phenolic compounds or anthocyanins. However, the use of HPLC for flavonoid determination enables the identification of different compounds in the samples, offering insights into the individual flavonoid profiles of various grape varieties. Nonetheless, the identification of each compound is challenging due to the limited availability of commercially accessible standards [58, 63, 64].

Currently, the most used methods for analyzing phenolic compounds involve HPLC coupled with ultraviolet detection, electrochemical detection, MS, or particle beam/electron ionization mass spectrometry. Moreover, GC coupled with MS, HSCCC, chiral CE, or Fourier transform near-infrared reflectance spectroscopy are frequently employed techniques. Hyphenated approaches, such as HPLC-MS and HPLC-MS/MS, which are built on HPLC separation, offer insights into the molecular mass and structural characteristics of compounds. These methods are deemed more advantageous than alternatives in terms of their efficiency, suitability for routine analysis, and effectiveness in the separation, identification, and quantification of phenolic content [65]. Reversed-phase HPLC is frequently employed for the analysis of various phenolic groups. Ultra-performance liquid chromatography has been used to enhance the analysis of phenolic compounds in diverse matrices [66].

3.6.1 Selective columns and stationary phases

Choosing appropriate columns and stationary phases is essential to ensure precise and selective determination of phenolic compounds in chromatographic techniques. It’s crucial to take into account the physicochemical properties of the compounds, the intended separation mechanism, and the specific analysis requirements when making these selections. Experimentation and method optimization may be required to achieve the best results for a particular set of phenolic compounds. A summary of the different columns and stationary phases that can be used to determine phenolic compounds can be found in Table 1.

Chromatography	Stationary phase	Selectivity	Applications	Refs.
Reverse-phase chromatography	C18 (Octadecylsilane)	Separation based on the hydrophobicity	Wide range of phenolic compounds	[60, 67]
Normal-phase chromatography	Silica gel or other polar materials	Separation based on their polarity	Useful for less polar phenolic compounds	[68, 69]
Ion-exchange chromatography	Positively or negatively charged resins	Separates based on ionic interactions	Effective for charged phenolic compounds	[70, 71]
Size-exclusion chromatography	Porous gels with different pore sizes	Separates based on molecular size	Useful for polymers and large phenolic compounds	[72, 73]
Hydrophilic interaction chromatography	Polar materials, such as silica with bonded polar functional groups	Separates based on both hydrophilic interactions	Suitable for polar phenolic compounds	[74, 75]
Chiral chromatography	Chiral selectors	Resolves enantiomers of chiral phenolic compounds	Necessary when stereoisomer separation is critical	[76, 77]
Mixed-mode chromatography	Combines different separation mechanisms	Offers versatility in separation	Useful for complex samples containing various phenolic compounds	[78, 79]

Table 1.

Different stationary phases are used to separate and identify phenolic compounds.

4. Spectroscopy in phenolic compounds quantification

Colorimetric techniques are extensively employed in UV/Vis spectrophotometry due to their simplicity, speed, suitability for routine laboratory use, and cost-effectiveness. Despite the numerous advantages of UV/Vis-based colorimetric methods, they require the use of reference substances (such as gallic acid) to ensure accurate quantification of the phenolic hydroxyl groups within samples [5]. When in need of determining the total phenolic content or anthocyanins, spectrophotometric methods like Folin-Denis, Folin Ciocalteu, or differential pH are widely used. However, these methods are widely used but since they are not specific for matrixes like grape juice, they tend to overestimate the content due to the lack of selectivity. For matrices such as grape juices or extracts, quantification and identification should be conducted using more specific methods, such as chromatographic techniques. Although these methods are more recommendable, the diversity of protocols found can difficult the choice to select the best to determine phenolic compounds [80].

Polyphenols within plant extracts interact with redox reagents such as the Folin-Ciocalteu reagent, forming a blue complex that can be measured using visible-light spectrophotometry. The Folin-Ciocalteu reaction relies on creating a blue chromophore comprised of a phosphotungstic-phosphomolybdenum complex, with the maximum absorption of chromophores contingent upon the alkaline solution and the concentration of phenolic compounds within the plant extract. Due to the rapid degradation of the reaction in alkaline environments, lithium salts are incorporated into the Folin-Ciocalteu reagent to prevent turbidity and facilitate analysis [5].

There are also assays that are used for specific groups of flavonoids. Examples include aluminum complexation assays and the spectrophotometric method using 4-dimethylaminocinnamaldehyde. For instance, flavonols and flavones can produce yellow complexes with Al(III) under neutral pH conditions, with the solution’s absorbance measured in the 400–430 nm range. Furthermore, Al(III) can generate red complexes with specific flavonoids (such as rutin, luteolin, and catechins) when sodium nitrite is present in an alkaline environment. The absorbance of the resulting solution is subsequently measured at 510 nm [81, 82].

Conventional techniques like the Folin-Ciocalteu method and aluminum chloride complexation are employed for the assessment of total phenolic and flavonoid contents post-extraction [83]. However, the Folin-Ciocalteu reagent has a tendency to react with other non-phenolic reducing substances, leading to an overestimation of the total phenolic content [66].

5. Nuclear magnetic resonance in phenolic compounds identification

High-resolution 1H-NMR spectroscopy has recently demonstrated utility in analyzing complex mixtures without the need for prior separation of individual components within the mixture [84]. Specifically, 1H-NMR spectroscopy was employed for the quality control and authentication of olive oil [85]. The utility of 1H-NMR spectroscopy is increasingly acknowledged for its non-invasive nature, rapidity, and sensitivity to a broad array of compounds in a single measurement, obviating the necessity for sample pre-treatment [86]. At room temperature, the 1H-NMR resonances of phenolic ∙OH groups exhibit broad signals primarily because of intermolecular exchange between the ∙OH protons and protons from protic solvents or residual H₂O in aprotic solvents. Additionally, further exchange broadening may occur due to proton exchange among different ∙OH groups and between ∙OH and ∙COOH groups, particularly in low-polarity and low dielectric constant organic solvents, as a result of intermolecular association of solute molecules. The linewidths of phenol OH signals (the spectral width or breadth of the signals corresponding to the hydroxyl (OH) group) are crucial for the assignment and interpretation of 1H-NMR spectra [87].

6. Conclusions

In conclusion, the extraction and identification of phenolic compounds from plant sources involve a diverse array of methodologies, each offering unique advantages and applications. Traditional methods, such as maceration and reflux extraction, provide valuable insights into the extraction efficiency influenced by factors like time, solvent ratio, and temperature. The incorporation of UAE and MAE accelerates the process while maintaining extraction quality. Chromatographic techniques offer robust options for separating and identifying phenolics. In the realm of chromatography, GC and HPLC coupled with MS remain indispensable, providing accuracy and efficiency in phenolic compound detection across diverse samples. This comprehensive overview underscores the importance of a tailored approach in selecting extraction and identification methods based on the specific characteristics of the target phenolic compounds and the nature of the plant matrix. The synergistic application of these diverse techniques contributes to a deeper understanding of phenolic composition in plant materials and supports advancements in the fields of food science, pharmacology, and natural product research. The inclusion of more objective composite analyses as part of routine procedures will result in the classification of more reliable and consistent data and ensure the quality.

Acknowledgments

The author would like to thank the funding to FCT - Foundation for Science and Technology on MED (Mediterranean Institute for Agriculture, Environment and Development) under the project UIDB/05183/2020 (https://doi.org/10.54499/UIDB/05183/2020; https://doi.org/10.54499/UIDP/05183/2020) & CHANGE (Global Change and Sustainability Institute) under the project LA/P/0121/2020 (https://doi.org/10.54499/LA/P/0121/2020).

Conflict of interest

The author declares no conflict of interest.

Funding

This work is funded by National Funds through FCT - Foundation for Science and Technology under the Project UIDB/05183/2020.

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