1 The Cinnamic Acid Skeleton and Principal Structural Types

1.1 The C6–C3 skeleton and terminal carboxyl group constitute the common structural basis of cinnamic acid derivatives

The basic structure of cinnamic acid is:

C₆H₅–CH=CH–COOH

The aromatic ring provides the C6 portion, while the —CH=CH–COOH side chain constitutes the C3 portion. The C=C double bond lies between the aromatic ring and the carboxyl group, giving the side chain a continuous Ar–C=C–C=O conjugated structure.

The carbonyl group within the carboxyl group is therefore not isolated; instead, it forms an extended π-electron system with the C=C bond and the aromatic ring. This structure not only gives cinnamic acid derivatives pronounced ultraviolet absorption but also distinguishes them from saturated 3-phenylpropionic acid derivatives in terms of electronic structure and spectroscopic behavior.[1–3]

In plant phenylpropanoid metabolism, L-phenylalanine is converted to trans-cinnamic acid through the action of phenylalanine ammonia-lyase. Corresponding intermediates in the pathway subsequently undergo aromatic-ring hydroxylation and hydroxy-group O-methylation, producing a series of hydroxy- and methoxy-substituted cinnamic acid derivatives.[1–2]

1.2 Hydroxylation and methoxylation form the principal representative compounds

Cinnamic acid itself contains no phenolic hydroxy group. Progressive replacement of hydrogen atoms on the aromatic ring with —OH and —OCH₃ produces structural series with different electronic and physicochemical properties.

p-Coumaric acid, caffeic acid, ferulic acid, and sinapic acid are among the most common representative hydroxycinnamic acids (HCAs).[3]

Hydroxy and methoxy groups have different hydrogen-bonding capacities and electronic effects:

① —OH can both donate and accept hydrogen bonds and can provide a phenolic hydrogen for participation in radical reactions;

② —OCH₃ cannot donate a phenolic hydrogen but can accept hydrogen bonds and alter the electron density of the aromatic ring through resonance effects;

③ Differences in the positions of —OH and —OCH₃ also alter intramolecular hydrogen bonding, steric effects, and electron delocalization after radical formation.

1.3 Double-bond configuration constitutes another level of structural variation

The cinnamic acid-type side chain, Ar–CH=CH–COOH, meets the structural requirements for E/Z geometric isomerism.

Naturally occurring hydroxycinnamic acids are generally more commonly found in the trans (E) form, although cis (Z) forms may also occur. Light exposure and sample-processing conditions can promote trans–cis isomerization of some trans-hydroxycinnamic acids. HPLC–DAD studies have separately detected the trans/cis isomers of caffeic acid, p-coumaric acid, and ferulic acid and have shown that the cis and trans structures exhibit different ultraviolet spectra and chromatographic retention behavior.[8]

2 Acidity, Ionization, and Solubility Behavior Determined by the Carboxyl Group

2.1 The carboxyl group is the principal source of the acid–base properties of phenylpropanoid acids

Cinnamic acid and hydroxycinnamic acids all contain:

—COOH ⇌ —COO⁻ + H⁺

The carboxyl group can dissociate a proton; these compounds are therefore weak organic acids.

p-Coumaric acid, caffeic acid, ferulic acid, and sinapic acid also contain phenolic hydroxy groups, but the carboxyl group generally dissociates before the phenolic hydroxy group. Experimental measurements show that the first acid-dissociation constants of various hydroxycinnamic acids lie at approximately pKₐ 4.2–4.6, with the specific values varying according to substituents, solvent, temperature, and ionic strength.[4,6]

Under acidic conditions substantially below the carboxyl-group pKₐ, the molecules exist mainly in the nonionic Ar–CH=CH–COOH form.

As the pH rises above the carboxyl-group pKₐ, the equilibrium gradually shifts toward Ar–CH=CH–COO⁻.

Phenolic hydroxy groups are substantially less acidic than the carboxyl group, and their appreciable deprotonation generally requires a higher pH.

2.2 Ionization gives the same compound different forms of existence

The nonionic carboxylic acid contains both a relatively large aromatic conjugated structure and a polar carboxyl group, giving the molecule both a hydrophobic region and a hydrophilic functional group.

Deprotonation of the carboxyl group generates a negative charge: —COOH → —COO⁻

The molecule's hydration capacity increases markedly, ion–dipole interactions with the aqueous phase become stronger, and its relative hydrophobicity decreases.

The actual solubility behavior of hydroxycinnamic acids therefore depends on whether the carboxyl group is ionized, the numbers of phenolic hydroxy and methoxy groups on the aromatic ring, temperature, solvent composition, and whether salts are formed.

The free acid under acidic conditions and the carboxylate under neutral or mildly alkaline conditions can exhibit pronounced differences in aqueous solubility, extraction partitioning, and chromatographic retention.

2.3 The carboxyl group also provides sites for salt formation and esterification

The carboxyl group can form the corresponding carboxylate salt with a base and can also undergo esterification.

Free hydroxycinnamic acid: Ar–CH=CH–COOH

can be converted to: Ar–CH=CH–COOR

This transformation masks the acidity of the carboxyl group and changes the molecule's polarity and lipophilicity.

In natural plants, hydroxycinnamic acids frequently occur in bound forms. For example, caffeic acid combines with quinic acid to form caffeoylquinic acids, while ferulic acid can also form ester bonds with structures such as cell-wall polysaccharides.[3]

When plant samples are analyzed, “free hydroxycinnamic acid content” and “total hydroxycinnamic acid content after hydrolysis” do not have the same meaning.

3 Modulation of Electronic Structure by Phenolic Hydroxy and Methoxy Groups

3.1 p-Coumaric acid: a single para phenolic hydroxy group

The structure of p-coumaric acid is:

4-HO–C₆H₄–CH=CH–COOH

The phenolic hydroxy group at the 4-position can donate electron density to the aromatic ring through resonance and can also provide a phenolic hydrogen.

After loss of a hydrogen atom, the unpaired electron of the resulting phenoxy radical can delocalize through the aromatic π system and is further influenced by the conjugated side chain. Accordingly, p-coumaric acid possesses characteristic phenolic redox properties that cinnamic acid itself lacks.[3,5]

3.2 Caffeic acid: the 3,4-catechol structure

Caffeic acid contains 3-OH and 4-OH.

The two adjacent phenolic hydroxy groups form a characteristic catechol structure.

This structure has two important effects.

① The two adjacent hydroxy groups can participate in favorable intramolecular and intermolecular hydrogen bonding.

② Oxidation of the phenolic hydroxy groups can form semiquinone and ortho-quinone structures. The two oxygen atoms and the aromatic π system jointly participate in electron delocalization, providing favorable stabilization of the corresponding oxidation intermediates.[3]

Electrochemical studies show that the oxidation behavior of caffeic acid is closely related to its catechol structure; methylation of one hydroxy group to form ferulic acid changes both the oxidation mechanism and the oxidation potential.[3]

3.3 Ferulic acid: coexistence of a phenolic hydroxy group and an ortho methoxy group

Ferulic acid contains 3-OCH₃ and 4-OH.

The methoxy group at the 3-position replaces one of the phenolic hydroxy groups in caffeic acid.

This change produces two effects simultaneously:

① The number of phenolic hydroxy groups decreases from two to one, eliminating one OH group capable of directly donating a hydrogen atom;

② The ortho methoxy group has a pronounced resonance electron-donating effect and can influence the electron delocalization and stability of the phenoxy radical formed from 4-OH.

Although the methoxy group cannot donate a phenolic hydrogen, it can still influence the entire aromatic conjugated system through its electronic effects.

The relative antioxidant activities of caffeic acid and ferulic acid vary with the assay system, solvent, pH, and evaluation mechanism. In electrochemical studies, the catechol structure of caffeic acid generally exhibits a lower oxidation potential, whereas methoxylation in ferulic acid changes its oxidation mechanism.[3,5]

3.4 Sinapic acid: two ortho methoxy groups jointly modulate the phenolic hydroxy group

Sinapic acid contains 3,5-di-OCH₃ and 4-OH.

Methoxy groups are present on both sides of the phenolic hydroxy group at the 4-position.

The two ortho methoxy groups can donate electron density to the aromatic system and influence the resonance stabilization of the phenoxy radical. Electrochemical and radical-scavenging studies indicate that sinapic acid frequently exhibits strong electron- and hydrogen-donating capacity, although its specific antioxidant effect still depends on the experimental system.[3,5]

4 Structural Changes and Principal Physicochemical Properties

4.1 Aromatic ring–alkene–carboxyl conjugation produces characteristic ultraviolet absorption

The cinnamic acid-type structure contains a continuous Ar–CH=CH–COOH conjugated system.

Introduction of phenolic hydroxy and methoxy groups further changes the electron density of the aromatic ring and the energy of π→π* transitions; different hydroxycinnamic acids therefore have different ultraviolet absorption spectra.

In practical high-performance liquid chromatography–diode array detection, hydroxycinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid are often detected with high sensitivity at approximately 320 nm, although their actual absorption maxima vary according to the specific compound and analytical medium.[7,9]

For example, the reported absorption maxima of caffeic acid and ferulic acid in specific analytical systems can lie at approximately 323–324 nm.[6]

4.2 OH and OCH₃ jointly alter hydrogen bonding, polarity, and intermolecular interactions

Hydroxy and methoxy groups simultaneously alter hydrogen-bonding patterns, hydrophobic surface area, and the electron distribution of the aromatic ring. Increasing the number of phenolic hydroxy groups generally increases the numbers of hydrogen-bond donor and acceptor sites. Methylation of a phenolic hydroxy group to form a methoxy group removes one hydrogen-bond donor, retains the hydrogen-bond-accepting capacity of the oxygen atom, and increases the hydrophobic surface area contributed by the methyl group. The polarity, solubility, and chromatographic retention of the complete molecule remain jointly determined by the ionization state of the carboxyl group, the positions of substituents on the aromatic ring, and solvent conditions.

4.3 Phenolic hydroxy groups determine the principal sites of radical reactions

Hydroxycinnamic acids can participate in radical scavenging through different pathways, commonly involving:

① Hydrogen atom transfer (HAT)

② Single-electron transfer followed by proton transfer (SET-PT)

③ Sequential proton-loss electron transfer (SPLET)

The contribution of each mechanism is influenced by solvent polarity, pH, and the type of radical.[5]

The phenolic hydroxy group is an important hydrogen or electron donor. After a phenoxy radical is formed, the aromatic ring and unsaturated side chain can participate in electron delocalization.

4.4 Oxidation and photoisomerization affect sample stability

Hydroxycinnamic acids containing phenolic hydroxy groups can undergo oxidation. In particular, the catechol structure of caffeic acid can form semiquinone and quinone oxidation products; ferulic acid and sinapic acid can likewise form corresponding phenoxy radicals and undergo further coupling or oxidation.[3]

The C=C double bond also makes hydroxycinnamic acids susceptible to photoinduced E/Z isomerization. Experimental studies have found that cis isomers of caffeic acid, p-coumaric acid, and ferulic acid may be detected even when sample preparation is performed with efforts to minimize light exposure.[8]

Accordingly, the following factors generally require attention for standards and analytical samples: light exposure; contact with air; solution pH; temperature; and storage time.

When trans/cis isomers must be distinguished or accurate quantification is required, isomerization caused by sample handling cannot be ignored.

For ortho-hydroxycinnamic acids, the E/Z configuration can also influence the possibility of ring closure. The cis configuration can place the ortho hydroxy and carboxyl groups in a spatial relationship favorable for lactonization and formation of the coumarin skeleton.[10]

5 Detection, Identification, and Structural Confirmation of Phenylpropanoid Acids

5.1 Functional-group reactions provide only supporting information

There is no single color reaction that produces a highly specific result for all of cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid.

Acid–base reactions of the carboxyl group can demonstrate the presence of an acidic functional group. p-Coumaric acid, caffeic acid, ferulic acid, and sinapic acid contain phenolic hydroxy groups and can additionally undergo phenol-related color or oxidation reactions.

5.2 TLC is suitable for reference-standard-assisted qualitative analysis

Thin-layer chromatography (TLC) can be used for rapid preliminary screening of hydroxycinnamic acids.

The test sample and reference standard can be compared in terms of:

① Retardation factor (Rf);

② Appearance under ultraviolet light;

③ Visualization characteristics.

The ionization state of the carboxyl group affects interactions between the sample, stationary phase, and developing solvent; the acid–base conditions of the developing system therefore generally require control.

For structurally similar hydroxycinnamic acids and E/Z isomers, Rf values and visualization colors alone have limited specificity. Confirmation still requires liquid chromatography or spectroscopic analysis.

5.3 HPLC–DAD is a commonly used qualitative and quantitative method for hydroxycinnamic acids

High-performance liquid chromatography (HPLC) is particularly suitable for free hydroxycinnamic acids.

These compounds:

① Have low volatility;

② Exhibit strong ultraviolet absorption;

③ Contain an ionizable carboxyl group;

④ Exhibit different hydrophobicity and hydrogen-bonding behavior as a result of aromatic-ring substitution.

Reversed-phase HPLC coupled with a diode array detector (DAD) is therefore well suited to their analysis.[7,9]

Mobile-phase pH is an important parameter governing separation behavior.

The carboxyl-group pKₐ values of hydroxycinnamic acids lie at approximately 4.2–4.6. When the mobile phase in reversed-phase chromatography is maintained at approximately pH 3, ionization of the carboxyl group is suppressed, the proportion of the nonionic form increases, and retention on hydrophobic stationary phases such as C18 generally becomes stronger; the peak shape of some weakly acidic compounds may also improve.

When the mobile-phase pH rises substantially above the pKₐ: —COOH → —COO⁻

The proportion of carboxylate increases, affinity for the aqueous phase rises, and retention on conventional reversed-phase stationary phases can decrease markedly. A UHPLC study of hydroxybenzoic and hydroxycinnamic acids found that these weak acids were largely dissociated and exhibited poor reversed-phase retention at pH > 6, whereas adjustment to approximately pH 3 with formic acid or acetic acid suppressed dissociation and increased retention.[6]

This relationship can be expressed as:

Low pH → increased proportion of COOH → relatively greater hydrophobicity → increased RP-HPLC retention

High pH → increased proportion of COO⁻ → greater affinity for the aqueous phase → decreased RP-HPLC retention

The actual method is also influenced by the organic-phase proportion, column stationary phase, temperature, and buffer system; the complete elution order cannot be predicted from pH alone.

5.4 DAD can utilize the conjugated absorption of hydroxycinnamic acids

Multiwavelength DAD detection can simultaneously utilize retention time and ultraviolet spectral characteristics.

In a UHPLC–DAD method developed for plant-derived phenolics, approximately 320 nm was used to increase the detection selectivity for hydroxycinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid.[7]

Compared with detection at a single fixed wavelength, DAD can acquire the complete ultraviolet absorption spectrum and can be used to assess:

① Whether the ultraviolet absorption characteristics are consistent with a cinnamic acid-type conjugated system;

② Whether the spectrum of the target peak matches that of the reference standard;

③ Whether co-eluting impurities may be present.

Ultraviolet absorption and retention time may also differ between trans/cis isomers. Studies have observed that the absorption maxima of cis-hydroxycinnamic acid isomers undergo a degree of hypsochromic shift relative to those of the trans isomers.[8]

5.5 LC–MS preferentially uses negative-ion mode to obtain information on carboxylic acids

Liquid chromatography–mass spectrometry (LC–MS) is suitable for analyzing multiple phenylpropanoid acids and their bound derivatives in complex plant samples.

Free carboxylic acids readily lose a proton: M → [M−H]⁻ + H⁺

Negative-ion mode electrospray ionization (ESI) is commonly used for the analysis of hydroxycinnamic acids. Typical nominal deprotonated ions of the free acids include:

When tandem mass-spectrometric (MS/MS) fragments are also examined, information such as loss of the carboxyl group and methoxy-related fragmentation can be observed. In complex samples, confirmation generally also requires the retention time of a reference standard, accurate mass, and fragment spectra; a structure cannot be identified from a single [M−H]⁻ ion alone.[6–7]

LC–MS mobile phases involve an analytical balance: acidic conditions favor reversed-phase chromatographic peak shape and retention, whereas negative-ion ESI requires the analyte to form an anion. Method development generally uses an appropriate concentration of formic acid, acetic acid, or a volatile buffer to balance chromatographic separation and ionization efficiency.

5.6 IR and NMR are used for final structural confirmation

Infrared (IR) spectroscopy can provide information on the broad O—H absorption of a carboxylic acid, carboxyl C=O absorption, aromatic-ring and C=C vibrations, phenolic hydroxy groups, and C—O-related features.

The positions of OH and OCH₃ substituents generally cannot be fully determined by IR alone.

Nuclear magnetic resonance (NMR) spectroscopy can further determine the aromatic-proton substitution pattern, the C7=C8 double bond, E/Z configuration, the number and positions of methoxy groups, the carboxyl carbon, and the overall connectivity of a hydroxycinnamic acid.

The two vinylic hydrogens of a trans-cinnamic acid-type double bond generally exhibit a large coupling constant, providing important evidence for the E configuration. ¹³C NMR can confirm the chemical environments of the carboxyl carbon, vinylic carbons, aromatic carbons, and methoxy carbons.

For an unknown purified compound, HPLC/LC–MS provides information on separation, molecular mass, and fragmentation, whereas NMR establishes connectivity and configuration; the two types of methods provide structural evidence at different levels.

6 Relationships Among Structure, Properties, and Analysis Jointly Determined by COOH, OH, and OCH₃

7 Structural Classification, Physicochemical Research Features, and Scientific Applications of Representative Phenylpropanoid Acid Products

Table 1. Cinnamic Acid Scaffold and Hydroxycinnamic Acids

Table 2. Methoxycinnamic Acids

Table 3. Hydroxy–Methoxy Mixed-Substituted Cinnamic Acids

Note: The products listed above are representative Aladdin products related to scientific research. Their specific uses should be determined in conjunction with the product specifications, batch COA, and the intended reaction or evaluation system. Additional information on product specifications, grades, and COAs can be retrieved from the Aladdin website using the product name, CAS number, or catalog number.

References

[1] Zhu Z, Chen R, Zhang L. Simple phenylpropanoids: recent advances in biological activities, biosynthetic pathways, and microbial production. Natural Product Reports, 2024, 41: 6–24. doi:10.1039/D3NP00012E.

[2] Vogt T. Phenylpropanoid biosynthesis. Molecular Plant, 2010, 3(1): 2–20. doi:10.1093/mp/ssp106.

[3] Teixeira J, Gaspar A, Garrido EM, Garrido J, Borges F. Hydroxycinnamic acid antioxidants: an electrochemical overview. BioMed Research International, 2013, 2013: 251754. doi:10.1155/2013/251754.

[4] Ozkorucuklu SP, Beltrán JL, Fonrodona G, Barrón D, Alsancak G, Barbosa J. Determination of dissociation constants of some hydroxylated benzoic and cinnamic acids in water from mobility and spectroscopic data obtained by CE-DAD. Journal of Chemical & Engineering Data, 2009, 54(3): 807–811. doi:10.1021/je800595x.

[5] Chen J, Yang J, Ma L, Li J, Shahzad N, Kim CK. Structure-antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids. Scientific Reports, 2020, 10: 2611. doi:10.1038/s41598-020-59451-z.

[6] Tais L, Schulz H, Böttcher C. Comprehensive profiling of semi-polar phytochemicals in whole wheat grains (Triticum aestivum) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. Metabolomics, 2021, 17: 18. doi:10.1007/s11306-020-01761-4.

[7] González-Domínguez R, Sayago A, Santos-Martín M, Fernández-Recamales Á. High-throughput method for wide-coverage and quantitative phenolic fingerprinting in plant-origin foods and urine samples. Journal of Agricultural and Food Chemistry, 2022, 70(25): 7796–7804. doi:10.1021/acs.jafc.2c01453.

[8] Istasse T, Jacquet N, Berchem T, Haubruge E, Nguyen BK, Richel A. Extraction of honey polyphenols: method development and evidence of cis isomerization. Analytical Chemistry Insights, 2016, 11: 49–57. doi:10.4137/ACI.S39739.

[9] Harnly JM, Bhagwat S, Lin LZ. Profiling methods for the determination of phenolic compounds in foods and dietary supplements. Analytical and Bioanalytical Chemistry, 2007, 389(1): 47–61. doi:10.1007/s00216-007-1424-7.

[10] Vanholme R, Sundin L, Seetso KC, et al. COSY catalyses trans–cis isomerization and lactonization in the biosynthesis of coumarins. Nature Plants, 2019, 5: 1066–1075. doi:10.1038/s41477-019-0510-0.

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