Introduction

Plant biomass is the most intensively studied and promising renewable feedstock for a sustainable global economy (Ragauskas et al. 2006). The transformation of plant biomass polysaccharides into high-demand chemicals has long been a major focus of biorefinery. The history of chemical processing of lignocellulosic biomass has shown that, among its main components, lignin is the most difficult to utilize (Abu-Omar et al. 2021; Tarabanko 2021).

Lignin is a complex heteropolymer, usually highly branched. Lignin monomers are phenylpropane units – syringyl, guaiacyl, and hydroxyphenyl. During the processing of lignocellulosic biomass, lignin significantly changes, condenses, loses its reactivity, and becomes a waste (lignosulfonates, Kraft lignin). Such lignins are very cheap, but unwanted, since there has been a lack of efficient techniques for their valorization (Kuznetsov et al. 2018). The advantages of native lignins over technical lignins have led to the modern concept expressed in two words: «Lignin first» (Renders et al. 2017; Tarabanko 2021). This means that the processing of lignocellulosic biomass should begin with the conversion of lignin into high-demand products.

A way of the efficient and environmentally friendly processing of native lignin is the use of organosolv methods, i.e., the removal of significant amounts of lignin from the initial plant biomass with organic solvents at elevated temperatures and pressures (Kuznetsov et al. 2018). One of the most attractive solvents is ethanol, a cheap and readily available aliphatic alcohol, which is produced from the carbohydrates. The properties of the isolated lignin differ from the native one and depend on organosolv fractionation time (Tao et al. 2016), temperature (Meyer et al. 2020), type of lignocellulosic biomass (Xu et al. 2020). The ethanol lignin production technique was developed specially for extraction from hardwood (Pan et al. 2006). Organosolv lignins as well as ethanol lignin can be used in high-value-added applications as extracted and isolated: adhesives, membranes, carbon fibers, among others (Rabelo et al. 2023).

The aromatic units of lignin are suitable for further functionalization (Eraghi Kazzaz et al. 2019) and, with properly chosen substituents, which ensure the occurrence of new properties, high value-added products can be obtained. One of the most interesting reactions for the modification of lignin is azo coupling. First, this reaction is well known in organic chemistry. In addition, for lignin itself, it became a starting point in determining its structure (Karlivan 1959, Karlivan 1960).

Secondly, the obtained azo derivatives of lignin are interesting for a wide application range. They exhibit a high efficiency in pyrocondensates processing as inhibitors of polymerization (Hai et al. 2011). Due to their ability to supramolecular assembly (Ago et al. 2017), the azo derivatives can serve as precursors for the production of anodes from nitrogen-doped carbon nanospheres (Zhao et al. 2016). Water solubility of lignin can be improved by adding hydrophilic substituents, for example, the 4-diazobenzenesulfonic acid (Borovkova et al. 2023). An azo-coupling-modified lignin is most commonly used in coloring and reflecting coatings (Frolova et al. 2020; Pandian et al. 2020).

Lignin is currently considered to be a promising ingredient of sunscreens for use in cosmetics industry (Qian et al. 2017; Gordobil et al. 2020). In addition, it exhibits the high antioxidant activity (Barapatre et al. 2016), as well as the antitumor, antiviral, and antimicrobial activities, which opens up new prospects for pharmacology and biomedicine (Spiridon et al. 2018). It must be taken into account, however, that there is evidence of the pronounced cytotoxic effect of lignin (Barapatre et al. 2016; Gordobil et al. 2020). At the same time, various azo dyes demonstrate bioaffinity and attract attention of researchers in the field of biomedicine (Alsantali et al. 2022). Although the azo derivatives of lignin have not yet evoked much interest of the biomedical community, they may prove to be effective drugs in therapy and components of personal care products. The combination of the optical and photosensitive properties of these derivatives and their stimuli-response properties can find high-tech applications, e.g., in biosensors, drug delivery systems (Wu et al. 2013), and nanomaterials (Urban 2009).

In this study, we discuss the use of ethanol lignin from hardwood raw materials in the production of azo and sulfated-azo modified materials. A source of native lignin was the aspen wood (Populus tremula), a forestry waste attracting attention as a raw material for deep chemical processing (Borovkova et al. 2022). The aim of this study was to develop methods for modifying aspen ethanol lignin by azo coupling with photosensitive and antioxidant properties.

Materials and methods

The initial sample for the synthesis and physicochemical study of the azo and sulfated derivatives of ethanol lignin (EL) was prepared from wood of Populus tremula aspen by the original technique (Kuznetsov et al. 2018). Aspen wood sawdust collected near Krasnoyarsk (Russia) was crushed on a BP-2 vibrating mill and a fraction of less than 0.5 mm was selected. Composition of materials: 46.3% – cellulose; 20.4% – lignin; 24.1% – hemicellulose; 5.2% - extractives; 0.5 – ash (Sharypov et al. 2016). Ethanol lignin was isolated by treatment of 60 wt% ethanol-water mixture at 190 °C during 3 h. The solution was separated by filtration and ethanol lignin was recovered with ice water at 4 °C.

The obtained ethanol lignin was modified using the well-known method for obtaining water-soluble sulfated ethanol lignin (SEL) proposed in Malyar et al. (2022). Per 1 g air-dry EL 25 mL of 1,4-dioxane, 5 g of sulfamic acid and 3 g of urea were taken and placed into three neck flask. The prepared mixture was heated to 90 °C under constant stirring for 3.0 h. After that, the solvent was decanted and the residue was dissolved in 25 mL of water, neutralized with aqueous ammonia and purified by dialysis.

Synthesis of the azo derivatives

Synthesis of azo lignin using p-nitroaniline

To obtain the diazonium salt based on p-nitroaniline (4-nitrobenzenediazonium): water − 1.125 ml, concentrated HCl − 1.125 ml and 0.5 g of p-nitroaniline were mixed and cooled to 0 °C in an ice bath and added with a solution of 0.35 g of NaNO2 in 1 mL of water cooled to 0 °C. In a separate beaker, 0.9 g of lignin and 2 ml of the 9% NaOH solution were mixed and cooled to 0°С.

The alkaline solution of lignin was gradually added with a diazonium salt solution under stirring at ~ 0 °C. The reaction mixture was incubated in an ice bath for 0.5 h. The EL and SEL samples modified with p-nitroaniline based diazonium salt are hereinafter referred to as EL-azo-NO2 and SEL-azo-NO2, respectively. The water-insoluble EL-azo-NO2 precipitate was then filtered with a Büchner funnel and dried in air. The water soluble SEL-azo-NO2 samples were subjected to dialysis in an MF-503-46 MFPI dialysis bag (US) with a pore size of 3.5 kDa against water for 10 h with changing water every hour. After dialysis, the solution was evaporated to dryness on a rotary evaporator under vacuum until a water-soluble solid residue was obtained.

Synthesis of azo lignin with sulfanilic acid

To obtain the diazonium salt based on sulfanilic acid (4-diazobenzenesulfonic acid): sulfanilic acid − 1 g, 2 М NaOH − 2.5 ml and 0.4 g of NaNO2 in 5 mL of water were placed in a glass beaker (100 ml). The solution was cooled to 0 °C in an ice bath and added with 10 ml of 2 M HCl cooled to 0°С. In a separate beaker, 0.9 g of EL (SEL) and 2 ml of the 9% NaOH solution were mixed and cooled to 0°С.

The alkaline solution of lignin was gradually added with the diazonium salt solution under stirring at a temperature of ~ 0 °C. The reaction mixture was incubated for 0.5 h in an ice bath. To purify the product it was subjected to dialysis and dried, same as described above. The EL and SEL samples modified with sulfanilic acid are hereinafter referred to as EL-azo-SO3H and SEL-azo-SO3H, respectively.

Fourier transform infrared (FTIR) spectroscopy

The Tensor 27 spectrometer was used to record FTIR spectra in the wavelength range of 4000–400 cm–1 with a resolution of 4 cm− 1, and the number of scans was 32. Specimens for the FTIR study were prepared in the form of tablets in a potassium bromide matrix. The substance concentration in the tablets was constant and amounted to 4 mg per 1000 mg of KBr.

Nuclear magnetic resonance (NMR) spectroscopy

NMR data were collected on a Bruker Avance III 600 spectrometer system at 295 K. Samples of 5–10 mg of lignin were placed into a 5 mm NMR tube and dissolved in 0.5 ml of DMSO-d6. The two-dimensional multiplicity edited 1-13 C heteronuclear single quantum correlation (HSQC) spectra were recorded with four scans of 2048 data points, 256 increments and relaxation delay of 2.5 s. All spectra were acquired and processed using Top Spin 2 software supplied with the spectrometer.

Gel permeation chromatography (GPC)

The molecular weight characteristics of lignin samples were determined by the GPC technique using an Agilent 1260 Infinity II Multi-Detector GPC/SEC System with triple detection: refractometer, viscometer, and light scattering. The eluent flow rate was 1 mL/min and the injected sample volume was 100 µl. Before the analysis, the water-soluble samples were dissolved in water (1.5 mg/mL) and the remaining samples, due to their insolubility in water, were dissolved in THF (1.5 mg/mL) and filtered through a 0.45-µm Millipore PTFE membrane filter. For aqueous solutions separation was performed on two combined PL Aquagel-OH Mixed-M columns (7.5 × 300 mm) using the mixture 0.2 M NaNO3 + 0.01 M NaHPO4 as a mobile phase. For organic solutions PlGel Mixed-E column (7.5 × 300 mm) using tetrahydrofuran (THF) stabilized with 250 ppm butylated hydroxytoluene as a mobile phase was used. Calibration was carried out using polydisperse standards of polyethylene glycol and polystyrene.

Thermal analysis

The TGA study was carried out on a NETZSCH TG 209 F1 thermobalance. The thermal decomposition of the samples was analyzed in nitrogen in the temperature range from 25 to 700 °C in the dynamic temperature regime (10 °C/min) using cylindrical corundum crucibles. The protective and blow out gas flow rate was 20 mL/min.

Spectrophotometry analysis

The UV-vis spectra were measured on Shimadzu UV-Vis-NIR 3600 plus scanning spectrophotometer (Japan) at a 1-nm spectral gap in a 1-cm quartz cuvette. The samples were dissolved in dimethyl sulfoxide (DMSO). The cuvettes were irradiated by LED assemblies with wavelengths of 360 and 450 nm and a luminous flux specific power of 50 mW ∙ cm–2. Photoreactor walls were darkened to prevent additional irradiation of the sample caused by reflection.

Study of the antioxidant activity

Based on the data on the absorption capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH), which served as a reference free radical compound (Rumpf et al. 2023), the antioxidant activity of lignins was determined by the somewhat modified method from Lu et al. (2012) and Alzagameem et al. (2018). Before the UV measurements, a DPPH solution in ethanol (0.2 mmol/L) was prepared. The SEL-azo samples were dissolved in ethanol (Alzagameem et al. 2018) in a concentration series of 0.05, 0.1, 0.2, 0.5, 2, and 5 mg/mL. The SEL-azo solutions (1 mL) were thoroughly mixed with 2 mL of the freshly prepared DPPH solution and 2 mL of ethanol. The mixtures were well-stirred and incubated at room temperature in the dark for 30 min. After that, the absorbance was measured at 517 nm (Alzagameem et al. 2018) on a SPEKOL-1300 spectrophotometer (Analytik Jena AG, Germany) against a blank. In this study, vitamin C (Vc) was used as a positive control. The experiments were repeated for three times and the values obtained were averaged.

The DPPH radical scavenging ability was calculated as

$$\:\text{D}\text{P}\text{P}\text{H}\:\text{R}\text{a}\text{d}\text{i}\text{c}\text{a}\text{l}\:\text{S}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}\:\text{A}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\left(\text{\%}\right)=\left(1-\frac{{\text{A}}_{\text{S}}-{\text{A}}_{\text{B}}}{{\text{A}}_{\text{C}}}\right)\times\:100\text{\%},$$
(1)

where AC is the absorbance of the DPPH solution without a sample, AS is the absorbance of the test sample mixed with the DPPH solution, and AB is the absorbance of the sample without the DPPH solution.

Results and discussion

Aspen ethanol lignin was obtained with yield 12.5 mass %, i.e. half of native lignin was isolated. Further, the sulfated and azo-coupling derivatives were obtained. The initial ethanol lignin is insoluble in water, but soluble in organic solvents (THF, DMSO, ethanol, etc.). Sulfation introduces ionic groups, which make the SEL polymer water-soluble (Malyar et al. 2022), but reduce its solubility in organic solvents.

Via azo coupling, new functional groups are introduced into the polymer. The general scheme of the reaction is shown in Fig. 1. Diazonium salts react with lignin in an alkaline medium to form azo derivatives. The substitution of aromatic H proceeds in the ortho position to phenolic hydroxyl. Thus, guaiacyl and hydroxyphenyl lignin units can be modified, syringyl units cannot. Therefore, the use of softwood lignin, which contains 90–95% guaiacyl units (Li et al. 2015), for modification by azo coupling looks more promising than hardwood and grass lignin, which contains 50–75% and 25–50% syringyl units (Li et al. 2015). Also, phenylpropane units having esterified phenolic hydroxyl groups do not react (Gogotov and Luzhanskaya 2005). The latter fact is interesting because with the sulfation method used, phenolic hydroxyls are usually not sulfated (Levdansky et al. 2022; Malyar et al. 2022). Obviously, different methods of lignin extraction affect the modification by azo coupling. First of all, methods leading to a strong rupture of alkyl-aryl ether bonds (e.g., α-O-4 and β-O-4) and minimal condensation processes (e.g., 5–5’, α-5, α-6) should be considered favorable. Kraft wood pulping and other alkaline pulping processes destroy most parts of alkyl-aryl ether bonds in native lignin, but lead to the condensation reactions (Evstigneyev et al. 2017). Milled wood lignin is considered most similar to the unaltered lignin; alkyl-aryl ether bonds are disrupted in the least way, and there are almost no condensation processes (Li et al. 2015). Organosolv lignins seem to be among the best for modifying via azo coupling. During high-temperature processing, the destruction of alkyl-aryl ether bonds, depolymerization and a low degree of condensation in phenolic unit are observed (Patil et al. 2020). All 3 factors open up possibilities for the reaction to proceed along the described route (Fig. 1).

Fig. 1
figure 1

General scheme of the azo coupling reaction phenylpropane units, the building blocks of lignin

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The 4-nitrobenzenediazonium-modified EL-azo-NO2 and SEL-azo-NO2 compounds contain nitro groups; the EL-azo-NO2 derivative is water-insoluble. The coupling lignin units with 4-diazobenzenesulfonic acid (EL-azo-SO3H and SEL-azo-SO3H) improves the solubility of the product in the water, which is maintained in the pH range of 2‒12.

FTIR study of the polymers

The chemical changes of ethanol lignin were studied by FTIR spectroscopy (see the spectra in Fig. 2). The spectra of all the samples contain a set of characteristic absorption bands near 1594, 1423, 1328, 1214 and 1122 cm‒1 (L in Fig. 2). These wavenumbers are typical of the guaiacylsyringyl-type lignin, i.e., lignin from hardwood (Levdansky et al. 2019). However, the intensity of these absorption bands strongly changes from one sample to another. Their intensity is maximum for initial ethanol lignin and significantly decreases for the SEL and azo derivative samples. Another characteristic difference of the modified samples from the initial one is a drastic change in the shape of the absorption band between 3100 and 3700 cm–1, which corresponds to OH groups.

A decrease in the intensity of the signal of C‒H vibrations in methyl and methylene at 2930 and 2840 cm–1 characteristic of organosolv lignins (Yáñez-S et al. 2014; Michelin et al. 2018) is related to a decrease in the content of the aliphatic part at the sulfation and azo coupling modification of ethanol lignin.

Fig. 2
figure 2

FTIR spectra of ethanol lignin and its derivatives

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The p-nitroaniline-modified EL-azo-NO2 and SEL-azo-NO2 samples are characterized by the intense absorption bands with maxima at 1518 and 1345 cm‒1, which belong to the NO2 in nitrobenzene group (Sundaraganesan et al. 2007). The medium-intensity absorption bands at 700, 750, and 855 cm–1 are also attributed to the NO2 vibrations (Khaikin et al. 2015).

In the spectra of the EL-azo-SO3H and SEL-azo-SO3H samples with the grafted azobenzensulfonic acid groups, there are bands at 1030, 1006 and 835 cm‒1 characteristic of sulfonic groups (Wang et al. 2002). There is an absorption band with the maximum at ~ 1190 cm‒1 corresponding to C = S stretching vibrations. In addition, absorption bands associated with the presence of sulfate and sulfonic groups are located in the region of 660 –500 cm− 1.

Nuclear magnetic resonance study of azo lignins

The structural features of the modification of aspen ethanol lignin by the azo coupling reaction were studied by the example of EL and EL-azo-NO2 using 2D HSQC NMR. The comparison of these two samples is most expedient, since there are no changes, except for the azo coupling. For example, the water-soluble samples are likely to be fractionated during the dialysis purification.

The HSQC spectra of initial ethanol lignin include characteristic correlation peaks of phenylpropane units, β-aryl ethers, and pinoresinol and phenylcoumarane lignin fragments (Figs. 3a and 4a) (Li and Gellerstedt 2008; Kuznetsov et al. 2018; Levdansky et al. 2019).

Fig. 3
figure 3

Aliphatic region in the 2D HSQC NMR spectra of (а) the EL and (b) EL-azo-NO2 samples

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As compared with the case of the EL sample, in the aliphatic region of the EL-azo-NO2 spectra (Fig. 3b), one can see an abrupt drop in the intensity of the cross peaks corresponding to the structures of β-aryl ethers, phenylcoumarane fragments, and even methoxyl groups. This is obviously due to a change in their concentration in the sample, rather than the chemical transformations and fractionation. During the reaction, 4-nitrobenzenediazonium species are added to guaiacyl and hydroxyphenyl units, while the contents of other ethanol lignin structural elements decrease.

Fig. 4
figure 4

Aromatic region in the 2D HSQC NMR spectra of (а) the EL and (b) EL-azo-NO2 samples

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The most intense peaks in the aromatic structure region (δ1Н/δ13С 6.2–7.3/103–123 ppm) correspond to guaiacyl (G) and syringyl (S) structural units characteristic of hardwood lignins (Li and Gellerstedt 2008; Kuznetsov et al. 2018; Levdansky et al. 2019). After the modification the signal of the S structures is preserved in the spectrum, while the G structures completely disappear.

In the aromatic region, the most characteristic is the appearance of cross peaks of nitrobenzene structures (δ1Н/ δ13С 8.0-8.5/122–127) in the EL-azo-NO2 spectrum. The C atom signal positions (2, 3, 5, and 6) correspond to the simulation data using nmrdb.org (Banfi and Patiny 2008). At the same time, C atoms in positions 3 and 5 of the guaiacile structure of lignin, which has an N = N bond in position 2, are responsible for the peaks in the region of δ1Н/ δ13С 7.6–7.9/127–137.

Gel permeation chromatography

The chemical modification of ethanol lignin affects the distribution of molecular weights of polymer molecules, which is reflected in its properties and potential applications. The characteristics of the molecular weights and their differential distributions were studied by GPC in an aqueous medium for the water-soluble samples and in THF for the water-insoluble samples. According to the data given in Table 1, the azo coupling modification increases the number average (Mn) and weight average (Mw) molecular weights.

Table 1 Molecular weight characteristics of lignin samples
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Fig. 5
figure 5

Molecular weight distribution curves of lignin samples

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The comparison of the EL and EL-azo-NO2 samples analyzed in the THF medium revealed a shift in the molecular weight distribution towards higher molecular weights due to the azo coupling. The other modifications (SEL, SEL-azo-NO2, EL-azo-SO3H, and SEL-azo-SO3H) are water-soluble and their molecular weight distributions are strongly shifted towards larger values (Fig. 5). Their Mn and Mw parameters exceed the values for the initial EL sample by a factor of 2‒4.

One can estimate the molecular weight growth by comparing the molar weights of the model lignin structure ‒ coniferyl alcohol (180 g/mol) ‒ and its conjugate with diazosulfobenzene (366 g/mol). An increase in the molecular weight after the reaction should not exceed a twofold growth even in the simplest lignin model, when each monomer phenylpropane unit is modified. The low-molecular-weight part of the water-soluble samples was removed by dialysis (Kuznetsov et al. 2020), which strongly shifted the distribution towards higher molecular weights.

An unexpected decrease in the observed molecular weight, or rather the size of the molecules, is observed when SEL is modified via azo coupling with p-nitroaniline. Obviously, this is not due to depolymerization, but due to the compaction of the molecule due to the introduction of a more hydrophobic fragment and a decrease in the hydrate shell size.

Thermal analysis

The thermogravimetry (TG) and differential thermogravimetry (DTG) curves of the samples of ethanol lignin and its azo derivatives are shown in Fig. 6. The weight loss in the initial sample at 700 °C attained 74.1%. The DTG curve of the initial lignin sample contains a broad peak between 200 and 500 °C, which is typical of the thermal decomposition of aspen ethanol lignin (Fetisova et al. 2019). Further weight loss is due to graphitization (Ma et al. 2016).

The modified samples exhibit a significantly higher thermal stability. The EL-azo-NO2 and SEL-azo-NO2 weight losses at 700°С were 43.8 and 38.4%, respectively. The thermograms of the EL-azo-SO3H and SEL-azo-SO3H samples also show that their stability exceeds that of the EL, EL-azo-NO2, and SEL-azo-NO2 samples. Their weight losses were 37.0 and 31.7%. The azo coupling modification leads to the formation of thermostable condensed structures during pyrolysis.

Fig. 6
figure 6

Thermogravimetry (TG) and differential thermogravimetry (DTG) curves of ethanol lignin and its azo derivatives

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The weight loss in the EL-azo-NO2 and SEL-azo-NO2 samples at ~ 260°С is related to the decomposition of a nitro compounds (Simeonov et al. 1990); as the temperature further increases, the azo compounds and aromatic matrix of lignin decompose, but, in general, the modified ethanol lignin weight loss at 350 °C noticeably weakens.

The thermal event at 345 °C can be attributed to the decomposition of sulfate group in the SEL-azo-NO2 and SEL-azo-SO3H samples (Malyar et al. 2022). The decomposition of sulfonic groups in 4-diazobenzenesulfonic acid occurs at temperatures of 440–470 °C.

The results of the thermal analysis show that the azo derivatives of ethanol lignin are promising for use in the production of nitrogen-doped carbon materials. They exhibit a fairly high thermal stability; the weight loss at 700 °C is reduced by a factor of more than 2 as compared with the value for the initial ethanol lignin.

Photoisomerization study

The cis-trans photoisomerization reaction represents a change in the configuration of a molecule during the transition from the stable ground state to the excited state after absorption of a photon with a certain wavelength (Fig. 7). Azobenzene is a well-studied chromophore which demonstrates photoisomerization activity; its derivatives attract attention as photofunctional materials for use in biochemistry and materials science (Bandara and Burdette 2012).

Fig. 7
figure 7

Photosensitive behavior of the azo derivatives of ethanol lignin

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Since the synthesized azo lignins contain the N = N conjugated double bonds, they can form two isomers upon photoisomerization. The spectra, as well as the optical changes in the investigated samples at different excitation wavelengths are shown in Fig. 8. Although the initial spectra are rather poorly distinguishable from each other, an analysis of their spectral differences (ΔA-λ plots in Fig. 8) gives an idea about the different optical changes under the light excitation.

Fig. 8
figure 8

UV-vis absorption spectra (in DMSO) and ΔA-λ curves. The black curve corresponds to the mixture of isomers; the red curve – to the excitation at 360 nm; and the blue curve – the excitation at 450 nm

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Fig. 9
figure 9

ΔA-wavelength curves for the EL-azo-SO3H sample excited at (A) 360 and (B) 450 nm for exposure times of 1, 3, 7, and 10 min, (C) and photoisomerization kinetics

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Figure 9 presents the dependence between the absorbance (ΔA) and exposure time by the example of EL-azo-SO3H. It can be seen that the absorption maximum is an invariant for both excitation wavelengths and independent of the irradiation time, while the ΔA value depends only on the excitation time. The maximum ΔA is located at wavelengths of 355 and 378 nm for irradiation of 360 and 450 nm, respectively. These peaks can be attributed to the cis and trans forms. Slopes of the linear fits of photoisomerization kinetic curves are 0.021 ± 0.003 and 0.014 ± 0.0006 (Fig. 9C). These values can be interpreted as photoisomerization rate constants (k = ΔC = ΔA/ε) under the assumption of a zero-order reaction. Unfortunately, without reliable data on the molar absorption coefficient (ε) of various forms of azo lignins, it is impossible to estimate the concentrations of cis and trans isomers from spectroscopic data.

The characteristic variation region at 300–360 nm is typical of the π‒π* transitions in the azo compounds (Mirković et al. 2017; Lađarević et al. 2019). First, for all the investigated samples, the spectral maxima for the cis and trans forms are noticeably different. This indicates a fundamental redistribution of shapes of the molecular orbitals for the cis- and trans-forms of azo derivates ethanol lignins and, consequently, their different geometric structures. The second point to note is the nonuniformity of the spectral changes: for the two investigated samples (SEL-azo-SO3H and SEL-azo-NO2), the differences in the absorbance under appropriate irradiation are no more than hundredths. Obviously, this is due to the fact that the sulfate groups initially incorporated into the lignin structure reduce the content of inherent phenylpropane units, reducing the number of available reaction sites.

On the other hand, sulfate groups create steric obstacles for the attachment of the N = N chromophore group. In the other two azo lignins (EL-azo-NO2 and EL-azo-SO3H), on the contrary, the optical changes are several tenths. The difference between the extinctions of the cis- and trans-forms is most likely due to the kinetic difficulties (the transition kinetics are much faster for the last two samples) and the fact that the EL-azo-NO2 and EL-azo-SO3H samples undergo the most dramatic transformation of the electronic structure of molecular orbitals.

All the synthesized azo derivatives can photoisomerize, but, depending on the properties of lignin and the nature of the azo component, the activity and depth of the cis‒trans‒cis transitions change.

Study of the antioxidant activity

The free radical scavenging assay of 1,1-diphenyl-2-picrylhydrazyl (DPPH) is based on the redox reaction of DPPH with an antioxidant, which results in a decrease in the color intensity in proportion to the antioxidant concentration (Alzagameem et al. 2018).

According to the literature data (Alzagameem et al. 2018; Du et al. 2022), lignins can exhibit the antioxidant properties, and, as a rule, their inhibitory effect on DPPH increases with the lignin concentration. The complex structure of lignin, which includes aromatic rings with hydroxyl and methoxyl functional groups, is responsible for the antioxidant potential. This depends, first of all, on the termination of oxidation reactions due to hydrogen donation and single electron transfer reactions. The ability of the samples and vitamin C solutions of different concentrations to absorb the DPPH free radicals is illustrated in Fig. 10.

Fig. 10
figure 10

DPPH radical scavenging activity: SEL and SEL-azo obtained using p-nitroaniline and sulfanilic acid and Vc as a positive control at different concentrations

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According to the data obtained, the SEL-azo-NO2 sample exhibits a higher ability to inhibit free radicals than the SEL-azo-SO3H sample. The maximum antioxidant activity of SEL-azo-NO2 was achieved at 5 mg/mL and amounted to 64.6%, while the maximum value for SEL and SEL-azo-SO3H at the same concentration was merely 6.2 and 3.3%, respectively. Such a strong difference is explained by several factors.

Previously, it has been repeatedly reported (Alzagameem et al. 2018; Du et al. 2022) on the inverse relationship between the molecular weight characteristics of lignins and their antioxidant activity. In particular, the SEL and sample SEL-azo-NO2 has a lower weight average molecular weight (4.0-4.2 kDa) than SEL-azo-SO3H (9.2 kDa). In addition, according to the molecular weight distributions in these samples (Fig. 5), the SEL-azo-NO2 sample is a mixture of fractions with different molecular weights, in which phenolic hydroxyls may be present in greater quantities, causing increased antioxidant activity.

In addition, the increase in the antioxidant activity of the phenolic part of lignin in the SEL-azo-NO2 sample may be caused by the influence of the nitro group, which has a negative mesomeric effect. It is likely that the shift in electron density along the p-bond system increases the mobility of hydrogen in the phenolic hydroxyl, which causes inhibition of DPPH. In the case of the sulfonic group, which also has a negative mesomeric effect, a similar effect is not observed due to the replacement of hydrogen in the sulfonic group with sodium, which greatly reduces the acceptor properties of the substituent.

Conclusion

A method for modifying Populus tremula aspen ethanol lignin via azo coupling with diazonium salts based on p-nitroaniline and sulfanilic acid was developed. These reactions were studied also for sulfated ethanol lignin. The novel synthesized polymers were examined by FTIR and NMR spectroscopy, which confirmed their successful functionalization. The p-nitroaniline modification does not make ethanol lignin water-soluble. The introduction of azobenzenegroups with a sulfonic acid group improves the water solubility of the polymer, as does sulfation. It was established that the modification via the azo coupling reaction increases the lignin molecular weights. The study of the photosensitive properties showed that the synthesized azo derivatives exhibit the photoisomerization ability, but it depends on the properties of lignin and the nature of the azo component. The modified lignins, especially p-nitroaniline based, are proven to be antioxidants.