Optical density measurements of the solutions before and after immobilization were carried out using EMC-30PC-UV-1800 and UV-5100 UV-VIS spectrophotometers, which enabled quantification of the immobilized reagent. Absorption and reflectance spectra were recorded with an X-Rite eye-one-pro mini-spectrophotometer (Russia).

UV spectra of complexes formed between the organic reagent EBT and metal ions were registered using a Bruker Invenio S-2021 FT-IR spectrometer (Germany). Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) were performed using a Jeol JSM-IT200LA scanning electron microscope (Japan).

X-ray fluorescence analysis was performed on a Rigaku NEX CG EDXRF Analyzer with Polarization (No. 9022190000, Japan). The pH values of the solutions were measured using a pH-12.mV TEMP Meter R 25 (Korea).

All chemical reagents, including chromium(III) chloride hexahydrate (CrCl₃•6H₂O), EBT, and buffer components, were obtained from “Khimreaktivinvest” LLC (Tashkent, Uzbekistan) and had a minimum purity of 99.8%. All solutions were prepared using double-distilled water.

A standard Cr(III) solution (1.0 × 10⁻³ mol/L) was prepared by dissolving 0.266 g of CrCl₃•6H₂O (molar mass 266.44 g/mol) in a small volume of distilled water, followed by dilution to 1.0 L in a volumetric flask. Working solutions with lower concentrations were prepared by serial dilution.

The EBT reagent solution (structural formula: C₂₀H₁₂N₃O₇SNa, Mr = 461.381 g/mol) was prepared by dissolving 0.004613 g of the substance in 100 mL of distilled water, yielding a concentration of 1.0 × 10⁻⁵ mol/L. The solution remains stable for up to one month when stored in a dark, tightly sealed container.

A universal buffer solution was prepared from phosphoric (H₃PO₄), acetic (CH₃COOH), and boric (H₃BO₃) acids. To prepare 1 L of a 0.04 M buffer, 2.2 mL of H₃PO₄, 3.5 mL of CH₃COOH, and 2.48 g of H₃BO₃ were added to a volumetric flask and diluted to the mark with distilled water. After 5–10 minutes, the pH of the solution was measured using a Mettler Toledo pH meter and found to be pH = 1.81. To obtain buffers with different pH values, calculated volumes of 0.2 M NaOH were added to this solution according to published recommendations [145].

An acetate buffer solution with a pH range of 3.8–6.3 was prepared by mixing 1 N acetic acid (from 421.5 to 59.3 mL) with the appropriate volume of 1 N NaOH solution, depending on the required pH.

At the initial stage, various fibrous sorbents were tested, and materials with optimal values of static and dynamic ion exchange capacities (SIEC and DIEC, mg-eq/g) were selected for further study as solid-phase carriers.

To evaluate the dynamic ion exchange capacity, a known amount of sorbent was placed in a column with a diameter of 10 mm. A 1.0 M hydrochloric acid solution was passed through the column at a constant flow rate. After saturation, the column was rinsed with distilled water until the pH reached approximately 7. Then, 25 mL of the eluate was titrated with a 0.1 M NaOH solution using methyl orange as an indicator.

Immobilization of the organic reagent EBT onto the selected fibrous carrier – silk fibroin – was performed under static conditions. Fiber samples with a diameter of 2 cm and a mass of 0.2 g were placed into a beaker containing 50 mL of 0.1 M HCl solution to convert them to the Cl⁻ form. After multiple rinses with distilled water, the fibers were transferred into 50 mL of an EBT solution with a concentration of 1.0 × 10⁻⁵ mol/L.

Immobilization was carried out at room temperature for 5–24 hours. Upon completion, the fibers were removed using a glass rod, rinsed twice with 50 mL portions of distilled water, and stored moist in a Petri dish.

The amount of unbound (free) reagent remaining in the solution was determined via UV transmittance spectroscopy, while the content of EBT immobilized on the fiber was measured using diffuse reflectance with a reflectance spectrophotometer.

For the sorption preconcentration of Cr(III) ions, sample solutions with volumes ranging from 10 to 200 mL were passed through a microcolumn containing 0.2 g of immobilized EBT reagent on a silk fibroin matrix. Sorption was carried out at a constant flow rate of 1–10 mL/min at room temperature.

The optimal pH values were determined experimentally to ensure maximum extraction efficiency of Cr(III) from the aqueous phase. Throughout the procedure, the flow rate was maintained constant to ensure the reproducibility of results.

For the analysis, an aliquot of solution containing 5–50 µg/mL of Cr(III) was transferred into a 25 mL volumetric flask. To the sample, 5 mL of a 0.005 M aqueous solution of the sodium salt of EBT and 10 mL of a universal buffer solution (pH = 8.0) were added to ensure optimal complexation conditions.

The volume was adjusted to the mark with distilled water. Five minutes after preparation, the absorbance of the samples was measured using EMC-30PC-UV-1800 and UVS-755 B spectrophotometers, with standard quartz or glass cuvettes of 1 cm optical path length.

Seven polymeric materials were investigated as solid-phase supports for the immobilization of EBT, including three anion-exchange fibers of the SMA series, a granular sorbent (PPM-1), two polyacrylonitrile-based fibers (PPA and PPF), and modified silk fibroin. The amino groups introduced via ethylenediamine, hydroxylamine, or hexamethylenediamine (SMA-1/-2/-3), as well as polyethylene polyamine (PPA) and phosphinyl groups (PPF), enable ion-exchange or hydrogen-bond interactions with EBT. Fibroin, composed mainly of Gly, Ala, Tyr, and Ser residues, contains sufficient –NH₂ and –OH functionalities for dye sorption. Silk fibroin showed the best performance (Table 3) (A ≈ 0.32; fixation efficiency ≈ 88%), as confirmed by reflectance spectra in Kubelka–Munk coordinates (Fig. 2). The high sorption capacity is attributed to the combination of hydrophilic (–OH, –NH₂) and hydrophobic regions in the protein matrix.

Silk fibroin was selected as the optimal support for subsequent stages, including complex formation with Cr(III), optimization of analytical conditions, and evaluation of the method’s analytical performance.

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To confirm the formation of a complex between the reagent EBT, immobilized on silk fibroin fibers, and chromium(III) ions, Fourier-transform infrared (FTIR) spectroscopy was employed.

Silk fibroin fiber obtained from the production waste of the “Urganch TEX” company was used as the solid-phase carrier. IR spectra were recorded for the native silk fiber, the fiber with immobilized EBT reagent, and the resulting complex with Cr(III) ions. Comparative IR spectra of all three samples are presented in Fig. 11.

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Figure 11. IR spectra of the complex formed by silk fibroin fiber, EBT reagent, and Cr(III) ion.

Spectral analysis revealed the characteristic vibrational bands: stretching vibrations of –OH groups in the range of 3276–3279 cm⁻¹; C–H bands at 2975–2976 cm⁻¹; –CH = N– vibrations at 1616–1618 cm⁻¹; N = N group bands between 1375 and 1437 cm⁻¹; stretching vibrations of the –SO₃⁻ group in the range of 1066–1164 cm⁻¹; and a new band around 607 cm⁻¹ corresponding to Cr–O coordination bonds, which confirms the formation of a chelate complex between the reagent and the Cr(III) ion. The observed frequencies are summarized in Table 9.

The presence of a distinct band at 607 cm⁻¹ in the complex spectrum confirms the formation of a Cr–O coordination bond. Shifts in the –CH = N–, –N = N–, and –NO₂ bands, as well as changes in the intensity of other characteristic vibrations, indicate the involvement of these functional groups in the chelation with Cr³⁺. Meanwhile, the minimal changes observed in the –SO₃⁻ bands suggest their primary role is in reagent immobilization on the fibroin fiber rather than in metal coordination.

To confirm the composition of the resulting complex and to identify the functional groups involved in immobilization and complexation, X-ray fluorescence (XRF) analysis was carried out on three samples: raw silk fibroin fiber, fiber with immobilized EBT reagent, and the EBT–Cr(III) complex.

Based on the obtained XRF spectra, the following was established:

In the spectrum of the raw silk fibroin fiber, signals corresponding to carbon (C), oxygen (O), and nitrogen (N) were observed, which are typical for the proteinaceous nature of the material (see Figure. 12A). After immobilization of EBT, a characteristic sulfur (S) peak appeared, confirming the presence of the –SO₃H functional group in the reagent, which participates in binding to the carrier (see Fig. 12B). In the spectrum of the complex formed after the introduction of Cr(III) ions, a distinct peak corresponding to chromium (Cr) was detected, clearly indicating the formation of a chelate complex with the immobilized reagent (see Fig. 12C).

In addition, a decrease in the intensity of the chlorine (Cl) signal was noted in the modified fiber after immobilization, which may be associated with the displacement of Cl⁻ ions from salt forms and their substitution during complexation via the –SO₃H group.

The obtained results are fully consistent with the IR spectroscopy and quantum chemical modeling data, confirming the formation of a stable complex based on EBT and Cr³⁺ ions on the silk fibroin surface.

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A)

To investigate the morphological and elemental characteristics of the complex formed between the immobilized EBT reagent and Cr(III) ions on silk fibroin fibers, scanning electron microscopy combined with energy-dispersive spectroscopy (SEM-EDS) was conducted.

Figure 13A shows the SEM image of the fiber surface with immobilized EBT reagent prior to interaction with metal ions, while Fig. 13B presents the morphological changes observed after the formation of the Cr(III) complex. Visible alterations in surface texture indicate sorption and chelation processes occurring at the phase interface.

The elemental composition of the analyzed samples is summarized in Table 9. Before modification, the fiber contains carbon (C), nitrogen (N), oxygen (O), phosphorus (P), and sulfur (S). After acid activation, the appearance of chlorine (Cl) is noted. Following complexation, the chromium content reaches up to 20 wt%, confirming the high efficiency and selectivity of the interaction between the reagent and Cr³⁺ ions.

In addition, the EDS spectrum presented in Fig. 14 confirms the presence of key elements involved in complex formation. Characteristic peaks for carbon (C), oxygen (O), nitrogen (N), sulfur (S), chlorine (Cl), and chromium (Cr) are observed. The distinct Cr–Kα and Cr–Kβ peaks in the range of 5.4–5.9 keV further verify successful coordination of Cr(III) with the immobilized reagent. The intensity of these peaks is consistent with the high chromium content determined by SEM-EDS mapping (Table 10).

To evaluate the practical applicability of the developed sorption–spectrophotometric method for determining Cr(III) ions, samples of wastewater were collected from several industrial facilities: the Navoi Electrochemical Plant (Navoi Region), the Mubarek Gas Processing Plant, and the Almalyk Mining and Metallurgical Complex.

The analysis was performed using the standard addition method as follows: 5 mL of 0.5 M nitric acid (HNO₃) and 5 mL of universal buffer solution (pH = 8) were added to each water sample (sample volume: 2.0 mL). The solution was then passed through a carrier with the immobilized EBT reagent, after which the absorbance or reflectance spectra were recorded using a spectrophotometer.

Cr(III) ions were successfully detected in all tested samples. The results are summarized in Table 12.

As shown in the table, the measured concentrations closely match the added values of Cr(III) ions, confirming the accuracy and reproducibility of the proposed method for analyzing real environmental samples.

To evaluate the analytical performance of the developed sorption–spectrophotometric method for the determination of Cr(III) ions based on the immobilized reagent EBT, a validation study was conducted by comparing its parameters with those reported for other analytical methods. The validation included a comparative assessment of the limit of detection (LOD), analysis time, buffer systems used, and correlation coefficients of calibration curves. The results are presented in Table 13.

As shown in Table 12, the proposed method demonstrates superior sensitivity (LOD = 1.2 µg/L), shorter analysis time (10 minutes), and excellent linearity (R² = 0.9991) compared to conventional photometric and polarographic methods. These advantages make it a reliable and efficient tool for the determination of Cr(III) in environmental and industrial samples.