Photophysical Investigation of Green Vitamin C Adducts as Synthetic pH Indicators: Solvatochromism, Halochromism, and DFT Calculations Integrating Optical Band Gap and Electronic Transition Analysis

Srood Omer Rashid 1,2✉ Emailsrood.rashid@univsul.edu.iq

1 Department of Chemistry, College of Science University of Sulaimani Sulaymaniyah Kurdistan Region Iraq

2 Department of Chemistry, College of Science Charmo University, Sulaymaniyah - Kurdistan Region 46023 Chamchamal Iraq

Srood Omer Rashid ^a,b*

^*a Corresponding author: Department of Chemistry, College of Science, University of Sulaimani, Sulaymaniyah, Kurdistan Region, Iraq.

^*b Department of Chemistry, College of Science, Charmo University, 46023 Chamchamal, Sulaymaniyah - Kurdistan Region, Iraq.

E-mail address

srood.rashid@univsul.edu.iq (Srood Omer Rashid).

ORCID: https://orcid.org/0000-0002-0179-2981

Abstract

The chemistry of vitamin C adducts was revisited to investigate their pH sensitivity, photophysical properties, optical behavior, and assess their potential as eco-friendly pH indicators. In this study, a series of ascorbic acid adducts were synthesized through a straightforward, high-yield method. Their acid–base responsiveness is driven by a reversible protonation–deprotonation process between the hydrazineyl and azo groups, causing observable color changes. Analysis of substituent effects demonstrated that strong electron-withdrawing groups like nitro (–NO₂) significantly impact the electronic structure, particularly when located at the ortho or para positions of the aromatic ring. Solvatochromic studies revealed that the ortho-nitro adduct 3(b) and para-nitro adduct 3(d) exhibited positive solvatochromism, with their absorption wavelengths increasing in more polar solvents. Notably, 3(b) displayed a consistent yellow color with λₘₐₓ around 396 nm across solvents, indicating its structural stability and solvent-independent optical behavior, making it a robust and versatile pH indicator. Halochromic studies in aqueous media revealed pronounced bathochromic shifts for both 3(b) and 3(d) under alkaline conditions. The 3(b) exhibited a color change from yellow to dark purple (λₘₐₓ=534 nm), while the 3(d) transitioned from colorless (λₘₐₓ=361 nm) to dark rosewood (λₘₐₓ= 472 nm). Spectrographic analysis determined that 3(b) had a pKa of 8.99 and 3(d) a pKa of 10.68.

The band gap analyses conducted under both alkaline and acidic conditions provide detailed insights into the optical properties and electronic transitions of the most promising adducts, 3(b) and 3(d). The optical band gap energies obtained from UV–Vis spectroscopy, E_g(optical) and E_g(Tauc), showed strong agreement with the electrochemically measured band gap, E_{g(electronic)}. These findings were further corroborated by computational results from DFT (B3LYP/6-311 + G(d,p)), where the theoretically calculated band gaps, E_g(DFT), validated the experimentally derived values and provided additional information through frontier molecular orbital (FMO) analysis, indicating the presence of an efficient push–pull electronic system throughout the adduct structures. Correlation of DFT outputs with Tauc-plot analyses clarified the nature of the electronic transitions, including π→π*, n→π*, and intramolecular charge-transfer (ICT) processes, and determined whether these transitions were direct or indirect, and whether they were symmetry-allowed or forbidden. Biochemical assays further supported the proposed neutral proton-transfer mechanisms operating under both acidic and basic conditions for the promising adducts. Thermal analysis divulged thermal stability up to 210°C. Acid-based titration tests showed that 3(b) and 3(d) produced sharp and accurate endpoints comparable to the standard methyl red indicator, while 3(c) did not exhibit a reliable color transition. Given their green origin, tunable structures, and visual responsiveness, these ascorbic pH indicators hold promising candidates for sustainable applications in food coloration, cosmetics, and medical diagnostics.

Keywords:

pH sensor

Ascorbic acid

Synthetic dyes

Acid-base indicators

Computational chemistry

Optical properties

Band gap

1. Introduction:

The synthesis, advancement, and application of organic dyes as pH-responsive indicators are of significant scientific interest, as they facilitate precise and rapid pH detection through distinct color shifts. While these dyes have a broad spectrum of valuable applications, their potential continues to drive research toward the design and development of novel organic colorants and highly efficient pH indicators for diverse analytical and industrial applications.[1]–[5] The precise monitoring and assessment of pH, a fundamental chemical parameter that quantifies hydrogen ion concentration, are essential across a wide range of disciplines, including medical diagnostics, chemical process regulation, and environmental study.[6]–[8]

Recent efforts have focused on overcoming the drawbacks of commercial synthetic organic acid-base indicators, such as toxicity, high cost, limited thermal stability, and narrow pH response ranges.[9]–[13] In addition to the widespread use of natural organic dyes as indicators for various applications,[14]–[17] the development of new, sustainable synthetic organic dyes remains a promising approach,[18]–[21] particularly for achieving precise equivalence point determination in acid-base reactions during volumetric quantitative analysis.[22]–[26] The chromatic shifts observed in organic dye, arising from either ionic or nonionic protonation-deprotonation processes, are intrinsically linked to alterations in their molecular structure.[2], [10], [27]–[29] Therefore, a deeper understanding of the structure-function relationships is essential for broadening their practical applications and advancing the development of more robust sensing systems.[30]–[33] To this end, several studies have explored the influence of substituents on the acid-base and photophysical properties of organic dyes, including halochromic behavior, absorption intensity, and protonation-deprotonation mechanisms.[34]–[40] Research has also revealed that the position of electron-withdrawing substituents, such as the nitro group, within the aromatic ring significantly impacts π-electron delocalization across the conjugated system, crucially modulating the optical properties and pH sensitivity of organic acid-base indicators.[37], [41]–[46] Recent studies have predominantly concentrated on the preparation of vitamin C adducts as masked hydrazine intermediates[47],[48] for the synthesis of nitrogen-based heterocyclic organic molecules, such as pyrazoles and indoles.[49]–[51] Surprisingly, little attention has been paid to considering the vitamin C adducts as final products, nor to investigating their other significant physicochemical properties, particularly with regard to enhancing optical behaviors and photophysical applications. In our previous studies, the nitro derivative of the ascorbic acid adduct, employed as an organic dye, played a key role in developing a green nano-photoredox catalyst that operated efficiently under visible light for two distinct applications. The synthesized nanocatalyst (ZnONPs@VCA) significantly improved the photocatalytic performance of ZnO nanoparticles (ZnO NPs) in degrading an aqueous solution of Congo red.[52] Moreover, the same nitro adduct was successfully incorporated into the synthesis of a highly efficient hybrid nanocomposite (MgONPs@VCA), which significantly enhanced the thiocyanation process of anilines and phenols.[53] Furthermore, ascorbic acid adducts were successfully doped to improve the optical properties of biopolymers using the casting method.[54], [55] Fig. 1 illustrates the developments in the field of ascorbic acid adducts.

Fig. 1

Applications of ascorbic acid adducts in various fields.

Spectroscopic techniques serve as indispensable tools for the characterization of synthetic organic colorants, and they have been extensively employed to investigate various aspects of dye behavior and functionality, including solvatochromism, halochromism, and optical band gap properties.[56]–[63] Complementing these experimental methods, computational chemistry—particularly Density Functional Theory (DFT)—has become increasingly integrated into modern characterization workflows to strengthen result validation, reduce experimental workload, and provide atomic-level insights into electronic transitions and band structure. Among the commonly applied DFT approaches, the B3LYP hybrid functional combined with the 6-311 + G(d,p) basis set is frequently utilized to compute essential molecular parameters, such as HOMO–LUMO energy levels, band gap (E_g), dipole moment (µ), orbital symmetry, and frontier molecular orbital (FMO) distributions.[64]–[68] Further analytical depth is provided by non-spectroscopic techniques. Methods such as cyclic voltammetry (CV), biochemical assays, and thermogravimetric analysis (TGA) yield valuable complementary information regarding electrochemical activity, intermolecular interactions, and thermal stability, respectively.[8], [23], [69]–[73] The synergistic integration of spectroscopic, computational, and non-spectroscopic methodologies therefore enables a comprehensive and multidimensional characterization of synthetic dyes, offering critical insights into their structural features, functional behavior, and potential applications.

In light of these considerations, the present study revisits the chemistry of vitamin C adducts to evaluate their pH sensitivity as well as their electronic and optical properties by introducing a series of ascorbic acid–based derivatives as eco-friendly, neutral organic dyes. These hydrazineyl-based compounds were synthesized and comprehensively characterized using a suite of spectroscopic techniques, including ¹H NMR, ¹³C NMR, FT-IR, and UV–Vis spectroscopy. Their potential utility as organic pH indicators was systematically assessed through UV–Vis analysis, with particular attention given to substituent effects—especially those of nitro groups—on solvatochromic and halochromic behavior. A portion of this study focuses specifically on elucidating the nature of the electronic transitions, optical properties, and proton-transfer processes within the most promising nitro-substituted adducts.

The experimentally determined electrochemical band gap energy, E_{g(electronic)}, obtained via cyclic voltammetry, was compared with the optical band gap energies derived from UV–Vis measurements, including absorption-edge band gap values E_g(optical) and Tauc-plot band gap estimates E_g(Tauc), and further correlated with DFT-calculated band gap energies E_g(DFT). Notably, all band gap values showed strong agreement. The integration of Tauc extrapolation analyses with other DFT data provided a coherent interpretation of the electronic transitions, allowing determination of whether they were direct or indirect and whether the transitions were symmetry-allowed or forbidden. Based on these correlations, an appropriate push–pull electronic system was suggested. Complementary non-spectroscopic techniques—including thermogravimetric analysis (TGA), cyclic voltammetry (CV), and bacterial Gram-staining protocols—offered further insights into the thermal stability, redox behavior, and reversible protonation–deprotonation characteristics of the hydrazineyl–azo framework. Finally, the practical applicability of the nitro-hydrazineyl dyes as acid–base indicators was demonstrated through their successful performance in standard titration experiments.

2. Experimental

2.1. Materials

All reagents and solvents were utilized in their original form without undergoing any additional purification. The aniline derivatives were purchased from BIOCHEM Chemopharma, while all solvents employed in this study were obtained from Merck. Other chemicals were procured from Aldrich.

2.2. Synthesis of vitamin C adducts (VCA)

Scheme 1

i) 1) H₂O (5 mL), HBF₄ (2 eq.), 2) NaNO₂ (1.1 eq., 0°C), stirred for 20 min. at RT ii) 1) 7 mL MeCN and 15 mL H₂O, 2) Ascorbic acid (2.5 eq.).

i) Diazotization: A suspension of anilines (1.0 equivalent) in 5 mL of water was treated with 2.0 equivalents of 48% HBF₄, and the mixture was stirred for 2 minutes. It was then cooled to 0°C, and a solution of NaNO₂ (1.1 equivalents) in 10 mL of water was added dropwise. After the addition, the reaction was stirred at 0°C for 15 minutes. The resulting solid was filtered and washed with 5 mL of ice-cold water and 10 mL of diethyl ether, yielding a slightly pale yellow to orange crude product. This product was purified by re-precipitation from an acetone solution using diethyl ether, resulting in diazonium salts as a white solid.

ii) Adduct Formation: In a 30 mL cylindrical vial equipped with a magnetic stirrer, diazonium salt 2 (1.0 equivalent) was dissolved in 7 mL of acetonitrile (MeCN), followed by the addition of 15 mL of H₂O. The resulting turbid yellow solution was treated with L-ascorbic acid (2.5 equivalents), causing the mixture to become a clear, intense yellow. After 20 minutes of stirring, a yellow precipitate started to appear, and stirring continued for another 10–20 minutes until the stirrer bar ceased movement. The mixture was then vacuum-filtered and washed several times with water to eliminate any excess ascorbic acid. The precipitate was dried in a desiccator, yielding the final compound as a light yellow to yellow solid.

(3R,4S)-4-hydroxy-2-oxotetrahydrofuran-3-yl 2-oxo-2-(2-phenylhydrazineyl)acetate 3(a)

White solid (5.01 g, 98%); FT-IR (υ): 3396, 3370, 3294, 2977, 2985, 1784, 1767, 1698, 1603, 1523, 1496, 1210, 1007, 758 cm^− 1. ¹H NMR (499 MHz, DMSO) δ 10.97 (s, 1H), 8.03 (s, 1H), 7.25–7.08 (m, 3H), 6.87–6.68 (m, 5H), 6.15 (s, 1H), 5.69 (d, J = 7.9 Hz, 1H), 4.70 (q, J = 7.8 Hz, 1H), 4.51 (t, J = 8.0 Hz, 1H), 4.06 (t, J = 8.4 Hz, 2H). ¹³C NMR (126 MHz, DMSO) δ 170.67, 162.34, 159.34, 156.41, 148.48, 129.28, 112.80, 76.36, 70.02, 69.81. Microanalysis for C₁₂H₁₂N₂O₆, require: C, 51.43, H, 4.32, N, 10.00%; Found: C, 51.72, H, 4.31, N, 9.98%.

(3R,4S)-4-hydroxy-2-oxotetrahydrofuran-3-yl 2-(2-(2-nitrophenyl)hydrazineyl)-2-oxoacetate 3(b)

Yellow solid (4.02 g, 95%); FT-IR (υ): 3366, 3323, 3277, 2924, 1791, 1721, 1702, 1612, 1576, 1495, 1272, 1155, 751 cm^− 1. ¹H NMR (500 MHz, DMSO) δ 11.42 (s, 1H), 9.45 (s, 1H), 8.12 (dd, J = 8.5, 1.6 Hz, 2H), 7.62 (t, J = 7.7 Hz, 2H), 7.13 (d, J = 8.5 Hz, 1H), 6.92 (t, J = 7.7 Hz, 2H), 6.23 (s, 1H), 5.73 (d, J = 7.9 Hz, 2H), 4.72 (t, J = 7.9 Hz, 1H), 4.56–4.48 (m, 2H), 4.14–4.05 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 170.20, 158.41, 155.72, 144.01, 136.51, 132.06, 125.93, 118.40, 114.75, 76.10, 69.72, 69.39. Low-Resolution Mass Spectrometry (LRMS, ESI⁺): C₁₂H₁₁N₃O₈ requires 325; found (ES⁺) 326 [M + H]⁺, 348 [M + Na]⁺, (ES⁻) 324 [M-H]^͞ ; High-Resolution Mass Spectrometry (HRMS, ESI⁻): C₁₂H₁₀N₃O₈ [M-H]^͞ requires 324.0462; found 324.0463, (∆= 0.30 ppm).

(3R,4S)-4-hydroxy-2-oxotetrahydrofuran-3-yl 2-(2-(3-nitrophenyl)hydrazineyl)-2-oxoacetate 3(c)

Pale yellow solid (4.14 g, 98%); FT-IR (υ): 3394, 3362, 3273, 3094, 2920, 1760, 1672, 1621, 1531, 1354, 1214, 734 cm^− 1. ¹H NMR (499 MHz, DMSO) δ 11.24 (s, 1H), 8.75 (s, 1H), 7.60 (dd, J = 8.1, 2.3 Hz, 1H), 7.53 (s, 1H), 7.49 (t, J = 8.1 Hz, 1H), 7.20 (dd, J = 8.3, 2.2 Hz, 1H), 6.22 (s, 1H), 5.73 (d, J = 8.0 Hz, 1H), 4.73 (q, J = 7.9 Hz, 1H), 4.53 (t, J = 8.0 Hz, 1H), 4.09 (t, J = 8.3 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 169.43, 157.79, 155.13, 148.55, 147.81, 129.52, 117.78, 112.71, 105.14, 75.25, 68.78, 68.55. (LRMS, ESI⁺): Calculated for C₁₂H₁₁N₃O₈: 325; found 326 [M + H]⁺, 324 [M − H]⁻. (HRMS, ESI⁻): Calculated for C₁₂H₁₀N₃O₈ [M − H]⁻: 324.0462; found: 324.0467 (∆ = 1.54 ppm).

(3R,4S)-4-hydroxy-2-oxotetrahydrofuran-3-yl 2-(2-(4-nitrophenyl)hydrazineyl)-2-oxoacetate 3(d)

Pale yellow solid (4.14 g, 98%); FT-IR (υ): 3500, 3299, 3213, 3089, 2981, 2937, 2866, 1764, 1695, 1603, 1503, 1486, 1351, 1215, 1114, 1016, 845, 749 cm^− 1. ¹H NMR (DMSO, 400 MHz): δ11.31 (s, 1H), 9.31 (s,1H), 8.11 (d, J = 8.9 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.19 (s, 1H), 5.71 (d, J = 7.9 Hz, 1H), 4.71 (q, J = 8.0 Hz, 1H), 4.52 (dd, J = 8.18, 7.74 Hz, 1H), 4.08 (dd, J = 8.30, 8.33 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 170.64, 158.87, 156.20, 154.09, 139.08, 126.35, 111.42, 76.54, 70.11, 69.81. LRMS (ES⁺) C₁₂H₁₁N₃O₈ requires 325; found (ES⁺) 326 [M + H]⁺, 348 [M + Na]⁺, (ES⁻) 324 [M-H]^͞ ; HRMS (ES⁻) C₁₂H₁₀N₃O₈ [M-H]^͞ requires 324.0462; found 324.0465, (∆= 0.92 ppm).

(3R,4S)-4-hydroxy-2-oxotetrahydrofuran-3-yl 2-(2-(4-chlorophenyl)hydrazineyl)-2-oxoacetate 3(e)

White solid (4.31 g, 97%); FT-IR (υ): 3464, 3359, 3227, 2960, 2910, 1788, 1765, 1695, 1600, 1490, 1318, 1200, 1015, 810 cm^− 1. ¹H NMR (DMSO, 400 MHz): δ 11.03 (s, 1H), 8.22 (s, 1H), 7.21 (d, J = 8.7 Hz, 2H), 6.74 (d, ³J = 8.8 Hz, 2H), 6.17 (s, 1H), 5.68 (d, J = 7.9 Hz, 1H), 4.70 (td, J = 7.9 Hz, 1H), 4.51 (dd, J = 8.2 Hz and 7.8 Hz, 1H), 4.06 (dd, J = 8.3 Hz and 8.4 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 170.06, 158.60, 155.77, 146.87, 128.29, 122.40, 113.78, 75.81, 69.46, 69.22.

4-(2-(2-(((3R,4S)-4-hydroxy-2-oxotetrahydrofuran-3-yl)oxy)-2-oxoacetyl)hydrazineyl)benzoic acid 3(f)

White solid (4.49 g, 95%); FT-IR (υ): 3677 − 2065 (broad), 3359, 3269, 3078, 2983, 2811, 1714, 1651, 1601, 1510, 1420, 1380, 1214, 1173, 1122, 855, 774 cm^− 1. ¹H NMR (499 MHz, DMSO) δ 12.35 (s, 1H), 11.11 (s, 1H), 8.67 (s, 1H), 7.77 (d, J = 8.7 Hz, 2H), 6.77–6.73 (m, 1H), 6.18 (s, 1H), 5.69 (d, J = 7.8 Hz, 1H), 4.70 (q, J = 7.8 Hz, 1H), 4.51 (t, J = 8.0 Hz, 1H), 4.06 (t, J = 8.3 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 170.65, 167.68, 159.10, 156.34, 152.23, 131.39, 121.15, 111.60, 76.46, 70.04, 69.80. Elemental analysis for C₁₃H₁₂N₂O₈: Calculated: C, 48.16%; H, 3.73%; N, 8.64%. Found: C, 47.99%; H, 3.72%; N, 8.60%.

2.3. Instrumentals

2.3.1. FT-IR Spectrometer

IR sample disks were prepared by mixing the adduct powders with potassium bromide at a 1:10 ratio, then pressing the mixture hydraulically under 10 tons of pressure. The FT-IR spectra of the synthesized adduct powders were recorded using a Perkin-Elmer spectrophotometer (Waltham, MA, USA) operating within the 400–4000 cm⁻¹ range.

2.3.2. NMR Spectrometer

The proton and ¹³C NMR spectra of the synthesized ascorbic acid adducts were recorded using Bruker DRX-400 and DRX-500 MHz instruments (Billerica, MA, USA), with trimethylsilane (TMS) as the internal standard and deuterated dimethyl sulfoxide (DMSO-d₆) as the solvent. Chemical shifts (δ) are reported in ppm units, referenced downfield from TMS.

2.3.3. UV–Visible Spectrometer

Approximately 1–3 mg of each ascorbic acid adduct was dissolved in 30 ml of the selected solvents. UV–Vis absorption spectra of all vitamin C adducts were measured using an Agilent Technologies, Cary 60 UV–Vis spectrophotometer over a range of 200–800 nm. The influence of various solvents, pH levels, and substituents on the behavior of the adducts was also investigated.

2.3.4. Thermal gravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was performed using SDT Q600 V20.9 Build 20 TGA Analyzer to measure the thermal stability of the prepared vitamin C adducts. The analysis was conducted under an inert nitrogen atmosphere, with the temperature ranging from 25 to 955°C at a heating rate of 10°C/min.

2.3.5. PH meter

The pH values of the prepared adduct solutions were adjusted and measured using a Jenway 3540 pH & Conductivity Meter.

2.3.6. Cyclic voltammetry (CV)

Voltammetric experiments were conducted at room temperature using a 797 VA Computrace voltammetric analyzer (Metrohm), connected to a PC for data acquisition. Adduct solutions were prepared by dissolving 5 mg of each adduct in 10 mL of either distilled water (DW) or 0.1 M aqueous KOH. These solutions were then added to a 0.2 M supporting electrolyte solution of tetraethylammonium chloride monohydrate ((C₂H₅)₄NCl.H₂O, 0.98 g in 50 mL DW). The redox potential activity of the adduct samples was evaluated under the following voltammetric conditions: Working electrode: Glassy carbon electrode (GCE); Reference electrode: Ag/AgCl; and Counter electrode: Platinum wire. Scanned potential range: −2.5 V to + 2.5 V, and Current measurement range: 100 nA to 10 mA.

2.3.7. Mass spectrometry (MS) and elemental analysis

The molecular weights of the isolated compounds were determined using a Waters SQD2 mass spectrometer for low-resolution mass spectrometry (LRMS) and a Waters Q-TOF micro mass spectrometer for high-resolution mass spectrometry (HRMS). Mass spectra were acquired in the m/z format. Elemental composition (CHNS) was analyzed using a vario EL CHNS elemental analyzer (Serial No. 11086109).

2.3.8. Computational Methodology

All quantum chemical calculations were conducted using the Gaussian 09 software. Full geometric optimizations for all four structures were performed employing density functional theory (DFT) at the B3LYP/6-311 + G(d,p) level, incorporating an implicit solvation model to simulate aqueous conditions. A frontier molecular orbital (FMO) analysis was then carried out to evaluate the HOMO and LUMO energy levels, HOMO–LUMO energy gap, orbital symmetries, and dipole moments.

3. Results and Discussion

3.1. Characterization of VCA: FT-IR, ¹H NMR, and ¹³C NMR.

The identity of the synthesized ascorbic acid adducts 3(a–f) was confirmed using multiple spectroscopic techniques, including FT-IR, ¹H NMR, and ¹³C NMR spectroscopy. FT-IR spectral analysis (Fig. 2) focused on the key functional groups present in the adducts and was categorized into two main diagnostic regions. The first region, between 1800 and 1600 cm⁻¹, corresponds to carbonyl (C = O) stretching vibrations, clearly indicating the presence of all three carbonyl groups in each adduct. The second region, spanning 2800 to 3600 cm⁻¹, includes N–H, O–H, and C–H stretching vibrations. The presence and alignment of absorption bands in these regions collectively confirm the successful formation of the ascorbic acid adducts.

Fig. 2

FT-IR spectra of compounds 3(a-f).

The proton configurations of the synthesized adducts were determined from their ¹H NMR spectra, as shown in Fig. 3A. The chemical shift signals for all hydrogen atoms in each ascorbic acid adduct were successfully identified and categorized into three main regions. The first region (4–6 ppm) corresponds to the five lactonic protons, observed consistently in the upfield area for all adducts. The second region (6–8 ppm) represents aromatic protons, while the most downfield region includes exchangeable protons such as N–H and O–H. Notably, in compound 3(b), the hydrazineyl protons (–NH–NH–) appear significantly more deshielded, with chemical shifts at 11.42 and 9.45 ppm. This pronounced downfield shift suggests stronger hydrogen bonding or electronic effects, and the result was confirmed through repeated measurements using identical concentrations of the adducts. The ¹³C NMR spectra of all synthesized adducts are shown in Fig. 3B. The carbon signals were classified into three distinct regions: the upfield region (68–77 ppm), corresponding to the three sp³-hybridized carbons in the lactone ring; the aromatic region (110–155 ppm), representing the aromatic carbon atoms; and the downfield region (above 156 ppm), assigned to carbonyl (C = O) carbons.

Fig. 3

FT-NMR spectra of compounds 3(a-f). (A): ¹H NMR; (B): ¹³C NMR.

3.2. UV–Visible Absorption study

UV-visible spectroscopy is a powerful analytical technique for precisely determining the absorption maxima of acid-base indicators and tracking absorption shifts associated with protonation and solvatochromism. It enables the accurate calculation of pKa values under various conditions. Furthermore, UV-visible spectroscopy provides valuable insights into essential physicochemical and optical properties, including band gap energy and refractive index.[55], [74], [75]

3.2.1. Influence of Substituents: Scope and Limitations

The effect of substituents on pH sensitivity was initially assessed by evaluating the deprotonation behavior of the hydrazinyl group in adducts 3(a–f). The synthesized ascorbic acid adducts were examined both visually and spectroscopically, with pH responsiveness assessed through observable color changes and UV–visible absorption spectra (Figs. 4A and 4B). Among the series, only the nitro-substituted derivatives 3(b-d) exhibited clear pH- and concentration-dependent behavior. In contrast, the meta-nitro adduct 3(c) displayed minimal color change across varying pH levels. This limited observation aligns with the well-documented role of nitro groups as chromophoric and strongly electron-withdrawing substituents. The position of the nitro group within the aromatic ring significantly influences π-electron delocalization across the conjugated system, which is critical in modulating the optical properties of organic acid–base indicators. [37], [41]–[46]

Fig. 4

UV-Vis spectra of adducts 3(a-f). (A) In aqueous solutions at pH 4; (B) In aqueous KOH solutions at pH 11.

3.2.2. Solvatochromism and intermolecular charge transfer (ICT)

Solvatochromism describes the shifts in the position of a UV-Vis absorption band resulting from changes in the polarity of the surrounding solvent. Taking into account the main criteria for good solvents in UV-Vis spectroscopy—such as the ability to dissolve the sample without reacting with it, not absorbing UV-Vis radiation, providing a clear spectrum, and stabilizing either the ground or excited states of the solutes—a range of solvents with varying polarities were used (arranged from most polar to least polar, see Figs. 5A and 5B). All solvents employed are transparent in the UV-Vis range, with relatively low cutoff values. Except for the non-polar hydrocarbon (cyclohexane), all solvents demonstrated good solvation capabilities and produced relatively clear and well-defined UV-Vis spectra. Except aqueous KOH and trimethylamine (Et₃N), which react with dyes 3(b) and 3(d), altering their chemical composition, all other solvents remained inert toward the solutes. Both adducts exhibited a bathochromic shift (red shift) in polar protic solvents such as H₂O, ethanol (EtOH), and 2-propanol (see Table 1). Notably, the ortho-NO₂ adduct 3(b) displayed significant absorption in the visible region, resulting in a distinct yellow coloration across all solvents used. Intramolecular charge transfer (ICT) plays a crucial role in solvent-solute interactions and governs the appearance of UV-Vis absorption bands. ICT occurs when an electron donor (e.g., -N = N-, -NH₂, -OH) and an electron acceptor (e.g., -NO₂, -C = O) coexist within the same molecular system, facilitating the redistribution of electron density from the donor to the acceptor upon excitation. This charge transfer process significantly impacts a molecule’s electronic and optical properties, particularly in solvatochromic behavior, where solvent polarity modulates the extent of charge delocalization, leading to shifts in absorption maxima. To assess the solvatochromic behavior (positive or negative) of compounds 3(b) and 3(d), the empirical ET(30) solvent polarity scale, which represents molar electronic transition energies, was employed. The solvatochromic shifts of both dyes were evaluated in various solvents using the mathematical relationship given in Eq. (1) and summarized in Table 1.

The constants in Eq. (1) are defined as follows: h (Planck’s constant), c (speed of light), NA (Avogadro’s number), and λₘₐₓ (wavelength of maximum absorbance of the solute in a given solvent). By plotting ET(30) values against the reciprocal of λₘₐₓ, a polarity scale was constructed (see Fig. 5C), allowing direct comparison with the theoretical polarity ranking of the solvents used. The results indicate that both organic dyes, 3(b) and 3(d), exhibit positive solvatochromism, as an increase in solvent polarity correlates with an increase in λₘₐₓ, leading to a bathochromic (red) shift. This phenomenon arises from the greater stabilization of the first excited state relative to the ground state in more polar solvents. The enhanced stabilization effectively reduces the HOMO-LUMO energy gap, thereby shifting the absorption to longer wavelengths. Notably, 3(b) exhibited minimal solvation effects on λₘₐₓ, with most values appearing around 396 nm, compared to 3(d). This suggests that 3(b) has enhanced structural stability and a more solvent-independent optical behavior, making it a reliable and versatile pH indicator.

Table 1

E_T(30) values and corresponding λ_max of nitro-adduct isomers 3(b) & 3(d) in different solvents.
Solvents	λ_max ^{(3b, and 3d)}	1/ λ_max^{(3b, and 3d)}	ET(30) ^{(3b, and 3d)} / (kcal/mol)
H₂O	396, 360	0.0025316, 0.0027777	72.382, 79.419
MeCN	384, 342	0.002604, 0.002923	74.455, 83.599
Ethanol	389, 351	0.002570, 0.002849	73.122, 81.455
2-Propanol	391, 351	0.002557, 0.002849	75.041, 81.455
Acetone	387, 337	0.002583, 0.002967	73.878, 84.839
DCM	386, 323	0.002590, 0.003095	73.498, 88.517
EtOAc	385, 339	0.002597, 0.002949	74.262, 84.339
Cyclohexane	-	-	-
Et₃N	492, 417	0.002032, 0.002398	58.111, 68.563
Aq. KOH Solution	534, 472	0.001872, 0.002118	53.541, 60.574

Fig. 5

Solvatochromic Analysis. (A) UV-Vis absorption spectra of compound 3(b); (B) UV-Vis absorption spectra of compound 3(d); (C) E_T(30) plot versus 1/λ_max for adducts 3(b) and 3(d) in various solvents at room temperature.

3.2.3. Halochromism and dissociation constant determination

The pH-sensitive behaviors of the halochromic compounds 3(b) and 3(d) were further investigated in aqueous solution across a range of acidic and basic pH values (Figs. 6A and 6B). The observed color changes with varying pH are attributed to a reversible protonation–deprotonation mechanism. The appearance of dye 3(b) as a stable yellow solution across the pH range of 1–9, along with its lowest pKa value and highest absorption maximum (λₘₐₓ = 396 nm), is attributed to the strong mesomeric effect of the nitro group, which reinforces the electron-accepting nature of the nitrophenyl ring within the conjugated system. Moreover, the nitro group engages in intramolecular hydrogen bonding with the adjacent NH group of the hydrazinyl moiety, which promotes greater molecular planarity and further electronic stabilization. Evidence for this intramolecular hydrogen bonding is provided by ¹H NMR spectroscopy, where the NH protons in compound 3(b) display the most downfield chemical shifts (11.42 and 9.42 ppm) among all synthesized adducts 3(a–f), indicating significant deshielding due to hydrogen bond formation. In contrast, dye 3(d) exhibited a faint yellow to nearly colorless appearance in aqueous solution over the pH range of 1–10, with a maximum absorption (λₘₐₓ = 361 nm), indicating a different electronic environment and potentially reduced conjugation relative to 3(b).

Fig. 6

Halochromatic behavior and pKa determination. (A) and (B) UV-Vis spectra of adducts 3(b) and 3(d), respectively, in aqueous solution across varying pH levels. (C) and (D) pKa analysis of adducts 3(b) and 3(d), respectively.

Upon deprotonation of dye 3(b) at pH values above 9 using aqueous KOH, a pronounced bathochromic shift of 138 nm was observed, with λₘₐₓ shifting from 396 nm to 534 nm. This spectral change was accompanied by a visible and sharp color transformation to dark purple, indicating extensive extension of the conjugation and reorganization of the electronic structure in the deprotonated form. Similarly, dye 3(d) underwent deprotonation at pH values above 10, also using aqueous KOH, resulting in a bathochromic shift of 111 nm (λₘₐₓ = 361 → 472 nm) and a noticeable color change from colorless to dark rosewood, readily detectable by the naked eye.

Furthermore, the UV–vis spectra of both 3(b) and 3(d) showed two well-defined isosbestic points at 342 and 438 nm for 3(b), and at 305 and 400 nm for 3(d). The presence of these isosbestic points confirms the existence of two distinct chemical species in equilibrium and supports the reversible and pH-dependent halochromic behavior of both compounds.

The acid dissociation constants (pKa) of adducts 3(b) and 3(d) were determined spectroscopically following the method described by L. E. V. Salgado et al.[76] Absorbance measurements were taken across a range of pH values at both the lower and higher λₘₐₓ characteristic of each dye. To estimate the pKa values, absorbance was plotted against pH, and linear regression (y = a + bx) was applied to pairs of adjacent points near the inflection regions of the resulting curves. The intersection points of these fitted lines provided the estimated pKa values: 8.99 for 3(b) and 10.68 for 3(d), as shown in Figs. 6C and 6D.

3.3. Investigating Electronic Transitions and Proton Transfer: An Integrated Electrochemical, Optical, DFT Calculations, and Biochemical Study.

3.3.1. Electrochemical Activity and Band Gap Determination via Cyclic Voltammetry

The adduct dyes 3(b) and 3(d) were investigated for their electrochemical behavior using cyclic voltammetry (CV). The corresponding voltammograms (Figs. 7A and 7B) were obtained by applying a linearly varying potential (potential sweep) to the respective electrochemical systems. The resulting current, generated by the redox activity of the dyes, was measured at the working electrode— specifically, a glassy carbon electrode (GCE). This current response was used to determine the associated redox potentials. Notably, the voltammetric profiles exhibited quasi-reversible redox behavior and well-defined electron transfer characteristics, both of which are essential for evaluating the dyes’ electrochemical stability and mechanistic pathways.

A distinct voltammetric peak associated with the oxidation of the hydrazineyl group (–NH–NH–) to the azo form (–N = N–) was observed for both dyes 3(b) and 3(d). In acidic medium (pH 4), the oxidation potentials were recorded at 0.061 V for 3(b) and − 0.204 V for 3(d), versus the Ag/AgCl reference electrode. In contrast, these oxidation peaks were absent in basic medium (pH 11), even at elevated dye concentrations. This disappearance is attributed to deprotonation of the hydrazineyl group, leading to spontaneous formation of the azo structure. The resulting increase in conjugation yields more intensely colored solutions.

Fig. 7

Band gap determination for adducts 3(b) and 3(d). (A, B) Electronic band gap (E_{g(electronic)}) calculated from cyclic voltammetry (CV) at pH 4 and 11. (C, D) Optical band gap (E_g(optical)) determined from absorption spectra at pH 4 and 11.

The electrochemical band gap, E_{g(electronic)}, was determined directly from voltammetric data as a quantitative measure of the electronic structure. This was achieved by estimating the highest occupied and lowest unoccupied molecular orbital (HOMO and LUMO) energy levels from the respective onset potentials (

$\:{E}_{onset}^{ox}$

and

$\:{E}_{onset}^{red}$

) at pH 11, using established empirical relations (Equations 2–4). The value of E_{g(electronic)} defines the energy of the lowest-energy electronic transition and is thus a fundamental descriptor of molecular optoelectronic characteristics.[77]

E _(LUMO)= −(

$\:{E}_{onset}^{red}$

+ 4.4) eV (2)

E _(HOMO)= −(

$\:{E}_{onset}^{ox}$

+ 4.4) eV (3)

E _{g(electronic)} = E_(LUMO) − E_(HOMO) eV (4)

3.3.2. Nature of Electronic Transitions: Band Gap Analysis and DFT Calculation

The optical properties and electronic transitions of dyes 3(b) and 3(d) were investigated using UV-Vis absorption spectroscopy in acidic (pH 4) and alkaline (pH 11) media. The optical band gap, E_g(optical), was determined from the absorption edge wavelength (λ_offset) using the relationship E_g(optical) = hc/λ_offset,[78] as depicted in Fig. 7C-D. In acidic medium, E_g(optical) values were 2.352 eV for 3(b) and 2.90 eV for 3(d). A significant reduction in the band gap was observed under alkaline conditions, with values decreasing to 1.71 eV for 3(b) and 2.10 eV for 3(d) and shows good agreement with corresponding electrochemical band gaps (1.91 eV and 2.05 eV, respectively). This substantial narrowing (ΔE_g(optical) = − 0.633 eV and − 0.802 eV, respectively) aligns with enhanced conjugation upon formation of the azo chromophore.

The nature of the electronic transitions was further probed using Tauc’s analysis. The model is defined by the equation (αhν)ⁿ=B(hν − E_g), where α is the absorption coefficient, hν is the photon energy, B is a material-dependent constant, and the exponent n denotes the type of electronic transition. The Tauc band gap, E_g(Tauc), was determined by extrapolating the linear region of plots of (αhν)ⁿ versus hν, as shown in Fig. 8(A-D). The value of “n” for each dye was identified as the one for which the resulting E_g(Tauc) matched the experimentally determined E_g(optical) and E_{g(electronic)} values.[55], [63], [79]

For dye 3(b) at pH 4, the optimal linear fit was obtained with n = 1/2, yielding a Tauc band gap E_g(Tauc) = 2.35 eV, which is indicative of an indirect allowed transition. In contrast, the data for dye 3(d) under the same acidic conditions were best fitted with n = 2/3 (E_g(Tauc) = 2.94 eV), characteristic of a direct forbidden transition. Interestingly, under alkaline media (pH 11), the electronic behavior converged, with both 3(b) and 3(d) exhibiting a direct allowed transition (n = 2), yielding band gaps of 1.91 eV and 2.18 eV, respectively. This pronounced shift in transition type underscores the profound influence of the proton transfer mechanism and subsequent azo formation on the dyes' electronic structures.

From a theoretical perspective, the nature of the observed electronic transitions can be interpreted in the context of quantum mechanical selection rules and density functional theory (DFT) calculations as explained in Fig. 8. The strong absorptions of the more stable E-isomers for both adducts 3(b) and 3(d) at pH 11 are consistent with symmetry-allowed direct transitions. These are attributed to π→π* excitations with pronounced intramolecular charge transfer (ICT) character, herein designated as (π, π*)-ICT.[80] In the deprotonated state, the intact –N = N– azo bridge facilitates complete electronic coupling, creating an efficient push-pull architecture[81], [82] (Fig. 8, Scheme 2). Within this system, electron density is transferred from donor groups (green) to acceptor groups (maroon, Fig. 8), in a delocalization pathway that originates at the carbonyl functionalities of adduct and extends across the conjugated nitro-aromatic π-system. This electronic arrangement raises the energy of the HOMO (π-bonding) and lowers the energy of the LUMO (π*-antibonding), collectively reducing the HOMO-LUMO band gap (E_g(DFT) = 2.118 eV for 3(b) and 2.481 eV for 3(d)). This reduced gap results in the observed low-energy absorption. Furthermore, the HOMO and LUMO in this configuration possess different spatial symmetries, generating a larger transition dipole moment (µ = 8.519 D for 3(b) and 8.699 D for 3(d)), along the molecular axis that fully satisfies electric dipole selection rules and resulting in the observed intense absorption and bathochromic shift. This satisfies the electric dipole selection rules and results in the high transition probability, manifesting as intense absorption with a bathochromic shift. The significant ground-state dipole moments calculated for 3(b) and 3(d) quantitatively reflect the strength of this push-pull effect, confirming its correlation with the enhanced red shift observed in basic media.[83]–[85]

Fig. 8

Analysis of band gap and electronic transitions using Tauc plot methodology. (A) and (B) illustrate compound 3(b) at pH 4 and pH 11, respectively, while (C) and (D) depict compound 3(d) at pH 4 and pH 11.

Conversely, the transitions observed at pH 4 are symmetry-forbidden but exhibit weak absorption features, attributable to secondary intensity-gaining mechanisms. For compound 3(b), the transition is likely an n→π* excitation (λ = 557 nm, see Fig. 7C), which is formally forbidden by symmetry but becomes indirectly allowed through vibronic coupling—a process in which molecular vibrations facilitate the mixing of electronic states, thereby relaxing selection rules and enabling otherwise forbidden transitions.[86] In the case of 3(d), the electronic transition at pH 4 is classified as a symmetry-forbidden direct excitation. Analysis of the frontier molecular orbitals (Fig. 8) reveals that the HOMO is predominantly localized on the NO₂-substituted aromatic moiety, which exhibits a partially conjugated system across the aromatic backbone, while the LUMO is primarily situated on the ascorbic acid moiety. This significant spatial separation of electron density leads to a relatively small transition dipole moment (µ = 7.08 D) and, consequently, a very low transition probability. This contrasting behavior underscores the profound sensitivity of the electronic transitions to protonation state and structural changes. The formation or disruption of the azo bridge's conjugation directly modulates orbital symmetry and overlap, thereby governing transition probabilities and the resulting optical properties. Experimentally derived optical band gaps (E_g(optical), E_g(Tauc)) and the electronic band gap E_{g(electronic)} are comprised for comparison with the calculated DFT gap E_g(DFT). The calculated E_g(DFT) values show close agreement with the experimental trends, exhibiting the expected small positive bias characteristic of the B3LYP functional (See Fig. 8).[77], [87]–[89]

Fig. 9

DFT/B3LYP calculated frontier molecular orbitals (HOMO and LUMO) for adducts 3(b) and (3)d at their minimum energy geometries under acidic (pH 4, hydrazineyl form) and alkaline (pH 11, azo form, most stable E-isomers) conditions, highlighting the push-pull electronic system. The green and maroon iso-surfaces represent electron density associated with donor and acceptor groups, respectively.

3.3.3. Biochemical Study of VCA: Assessing Positive Charges via Simple Gram Staining

Further investigations were conducted to evaluate the presence of positive charges on the supposed molecular structures of the hydrazinyl and azo forms of the promising synthetic dyes 3(b) and 3(d) during protonation and

deprotonation processes. Gram staining is a well-established technique used to differentiate bacterial species based on the composition of their cell walls, enabling accurate identification of bacterial shape, size, and structural characteristics. When basic (cationic) dyes such as crystal violet are applied, their positively charged molecules bind to negatively charged components—primarily the abundant phosphate groups—in the thick peptidoglycan layer of Gram-positive bacterial cell walls. This strong electrostatic interaction enables Gram-positive bacteria to retain the purple crystal violet stain, even after alcohol decolorization.[90]–[94]

The biochemical activity of dye solutions 3(b) and 3(d) was evaluated under both acidic and basic conditions using a simplified Gram staining procedure, omitting the decolorization step. This assay was performed on heat-fixed, rod-shaped, freshly cultured Bacillus cereus, a well-characterized Gram-positive bacterium.[95] Microbiological visualization of dye–bacteria interactions showed that only the aqueous solution of the standard crystal violet dye produced a strong Gram-positive staining response in B. cereus. In contrast, aqueous solutions of dyes 3(b) and 3(d) displayed neutral behavior across different pH conditions, suggesting minimal to no electrostatic interaction with the bacterial cell wall, likely due to the absence of significant positive charge. These findings are summarized in Table 2. Based on the combined optical, electrochemical, theoretical, and biochemical evidence, a proposed protonation–deprotonation mechanism is illustrated in Scheme 2.

Scheme 2

Deprotonation/ protonation and push–pull system of nitro adducts 3(b-d).

Table 2

Staining Response of Gram-Positive B. cereus to VCAs and Crystal Violet.
Entries	Organic Dyes/ Solvents/ pH	Staining Outcome
1	3(b)/ H₂O/ 6	No staining
2	3(b)/ Aqueous KOH/ 11	No staining
3	3(d)/ H₂O/ 6	No staining
4	3(d)/ Aqueous KOH/ 11	No staining
5	Crystal Violet/ H₂O/ 6	Deep staining

3.4. Thermal stability: TGA & DTA

The thermal stability of the ascorbic acid adducts 3(b–d) was evaluated using thermogravimetric analysis (TGA) and differential thermal analysis (DTA). The corresponding thermograms, shown in Figs. 10A–C, were recorded under an inert nitrogen atmosphere over a temperature range of ambient to 970°C, with a constant heating rate of 10°C/min. Overall, the thermal decomposition of the vitamin C adducts proceeded in three distinct stages. The first stage, attributed to moisture evaporation from the samples, occurred within the temperature range of 25–112°C for compounds 3(b–d), resulting in initial weight losses of 6.78%, 3.1%, and 5.59%, respectively.

Fig. 10

TGA and DTA thermograms of the compounds. (A–C) correspond to compounds 3(b), 3(c), and 3(d), respectively.

The second stage, pyrolysis, marked the onset of substantial decomposition, occurring within an average temperature range of 112–340°C. The corresponding weight losses in this phase were 80.81%, 63.32%, and 52.48% for compounds 3(b), 3(c), and 3(d), respectively. In the final carbonization stage, the thermograms revealed further decomposition of the samples. For compound 3(b), significant degradation was observed at approximately 340°C, resulting in a residual ash content of 8%. A similar pattern was noted for compounds 3(c) and 3(d), though they exhibited slightly lower thermal stability (more details are provided in the ESI). The endothermic DTA curves further supported the TGA findings by providing insights into the physical changes occurring during the heating process, such as water evaporation and melting. Additionally, the lowest points of the peaks in the DTA curves indicated the temperatures at which the samples experienced rapid weight loss.

3.5. Applications: Evaluation of vitamin C adducts as acid-base indicators

The indicator performance of the three synthesized nitro ascorbic acid adducts 3(b-d) was evaluated using a standard acid–base titration system involving a strong acid (HCl), a strong base (KOH), and methyl red (MR) as the reference indicator. Aqueous solutions of each adduct were prepared by dissolving 20 mg of the respective compound in 10 mL of distilled water. In three separate experiments, three drops of each adduct solution were added to 10 mL of freshly prepared KOH solution (0.1 N) in a conical flask, followed by titration with standard HCl solution (0.1 N) from a burette. For adduct 3(b), the basic solution exhibited a sharp color change from dark violet to light yellow at the equivalence point, while adduct 3(d) induced a transition from dark rosewood to colorless—both matching the endpoint observed using methyl red. However, adduct 3(c) did not display a distinct color transition, and the titration endpoint could not be accurately detected, suggesting that 3(c) is unsuitable as an acid–base indicator under these conditions. Moreover, upon the addition of excess KOH to the acidic titrated solutions containing 3(b) and 3(d), the original dark colors were restored, further confirming their reversible and pH-responsive indicator behavior.

4. Conclusion

This study successfully developed a series of ascorbic acid-based adducts and assessed their potential as sustainable acid-base indicators. The ortho- and para-nitro-substituted derivatives, specifically 3(b) and 3(d), exhibited the most pronounced halochromic behavior, characterized by reversible and distinct color changes under acidic and alkaline conditions. In contrast, the other adduct derivatives, including the meta-isomer 3(c), did not demonstrate significant pH responsiveness. The observed pH sensitivity was attributed to a protonation-deprotonation equilibrium, evidenced by clearly defined isosbestic points, with pKa values of 8.99 for 3(b) and 10.68 for 3(d).

The optical properties were characterized by a combination of spectroscopic and computational techniques, which confirmed that the color changes arise from π→π* and intramolecular charge-transfer (ICT) transitions within a push–pull electronic system. A strong correlation was observed among the optical (E_{₍optical₎}, E_₍Tauc₎), electrochemical (E₍_{electronic₎}), and DFT-calculated (E_₍DFT₎) band gaps. The intense bathochromic shifts measured under alkaline conditions of adducts were correlated with the reduced band gaps and increased DFT-derived dipole moments. Frontier molecular orbital analysis further substantiated the donor–acceptor character and confirmed the critical role of the azo bridge in π-electron delocalization.

The adducts also displayed positive solvatochromism and were thermally stable up to 210°C. In practical acid–base titrations, compounds 3(b) and 3(d) performed with an accuracy comparable to the conventional indicator methyl red. Due to their straightforward synthesis from a benign precursor and their tunable optical properties, these ascorbic acid adducts represent promising, eco-friendly candidates for applications in fields such as food science, cosmetics, and diagnostics.

5. Funding

The author declares that no funds, grants, or other support were received during the preparation of this article.

Data Availability

The datasets supporting the findings of this research are available within the main body of the manuscript and in the accompanying Electronic Supplementary Information (ESI) file.

Electronic Supplementary Material

Below is the link to the electronic supplementary material

Supplementary Material 1

Author Contribution

Dr. Srood Omer Rashid conducted the synthetic experiments, interpreted the experimental results, drafted the manuscript, and supervised the overall project.

7. References

Steinegger, A., Wolfbeis, O. S. & Borisov, S. M. Optical Sensing and Imaging of pH Values: Spectroscopies, Materials, and Applications. Chem. Rev. 120 (22), 12357–12489. 10.1021/acs.chemrev.0c00451 (2020).

Xiong, Y., Pei, K., Wu, Y. & Xiong, Y. Colorimetric ELISA based on glucose oxidase-regulated the color of acid–base indicator for sensitive detection of aflatoxin B1 in corn samples. Food Control. 78, 317–323. 10.1016/j.foodcont.2017.03.002 (2017).

Szadkowski, B. & Marzec, A. Chitosan vs chitin: Comparative study of functional pH bioindicators synthesized from natural red dyes and biopolymers as potential packaging additives, Food Hydrocoll., vol. 150, no. August 2024, (2023). 10.1016/j.foodhyd.2023.109670

Ardila-Leal, L. D., Poutou-Piñales, R. A., Pedroza-Rodríguez, A. M. & Quevedo-Hidalgo, B. E. A brief history of colour, the environmental impact of synthetic dyes and removal by using laccases. Molecules 26 (13). 10.3390/molecules26133813 (2021).

Rasouli, Z., Abdollahi, H. & Maeder, M. Generalized indicator-based determination of solution pH. Anal. Chim. Acta. 1109, 90–97. 10.1016/j.aca.2020.03.004 (May 2020).

Zhou, J. et al. Stable, reusable, and rapid response smart pH-responsive cotton fabric based on covalently immobilized with naphthalimide-rhodamine probe, Sensors Actuators B Chem., vol. 355, no. December p. 131310, 2022, (2021). 10.1016/j.snb.2021.131310

Sri Sruthi, P. et al. Eco-friendly pH detecting paper-based analytical device: Towards process intensification. Anal. Chim. Acta. 1182, 338953. 10.1016/j.aca.2021.338953 (Oct. 2021).

Michalak, M., Kalisz, J., Michalska, A. & Maksymiuk, K. High resolution electrochemical monitoring of small pH changes. Electrochim. Acta. 468, 143147. 10.1016/j.electacta.2023.143147 (Nov. 2023).

Chavan, R. B. Environmentally friendly dyes, in Handbook of Textile and Industrial Dyeing, vol. 1, Elsevier, 515–561. doi: 10.1533/9780857093974.2.515. (2011).

10.

Ben Slama, H. et al. Diversity of synthetic dyes from textile industries, discharge impacts and treatment methods. Appl. Sci. 11, 1–21. 10.3390/app11146255 (2021).

11.

Totland, C. et al. A Broad-Range Fluorescence Lifetime pH Sensing Material Based on a Single Organic Fluorophore, J. Fluoresc., vol. 29, no. 5, pp. 1125–1131, Sep. (2019). 10.1007/s10895-019-02426-9

12.

Do Kim, Y. et al. Synthesis, application and investigation of structure-thermal stability relationships of thermally stable water-soluble azo naphthalene dyes for LCD red color filters. Dye Pigment. 89 (1), 1–8. 10.1016/j.dyepig.2010.07.008 (2011).

13.

Gotor, R., Ashokkumar, P., Hecht, M., Keil, K. & Rurack, K. Optical pH Sensor Covering the Range from pH 0–14 Compatible with Mobile-Device Readout and Based on a Set of Rationally Designed Indicator Dyes. Anal. Chem. 89 (16), 8437–8444. 10.1021/acs.analchem.7b01903 (2017).

14.

Luo, X., Zaitoon, A. & Lim, L. T. A review on colorimetric indicators for monitoring product freshness in intelligent food packaging: Indicator dyes, preparation methods, and applications. Compr. Rev. Food Sci. Food Saf. 21 (3), 2489–2519. 10.1111/1541-4337.12942 (2022).

15.

Almasi, H., Forghani, S. & Moradi, M. Recent advances on intelligent food freshness indicators; an update on natural colorants and methods of preparation, Food Packag. Shelf Life, vol. 32, no. November p. 100839, 2022, (2020). 10.1016/j.fpsl.2022.100839

16.

Liu, D. et al. Recent Advances in pH-Responsive Freshness Indicators Using Natural Food Colorants to Monitor Food Freshness. Foods 11 (13). 10.3390/foods11131884 (2022).

17.

Shahid, M., Mohammad, F. & Shahid-Ul-Islam, and Recent advancements in natural dye applications: A review. J. Clean. Prod. 53, 310–331. 10.1016/j.jclepro.2013.03.031 (2013).

18.

Banerjee, M., Bhosle, A. A., Chatterjee, A. & Saha, S. Mechanochemical Synthesis of Organic Dyes and Fluorophores. J. Org. Chem. 86, 13911–13923. 10.1021/acs.joc.1c01540 (2021).

19.

Gaffer, H. & Khalifa, M. Eco-Friendly Synthesis of Some Thiosemicarbazones and Their Applications as Intermediates for 5-Arylazothiazole Disperse Dyes, Molecules, vol. 20, no. 12, pp. 21982–21991, Dec. (2015). 10.3390/molecules201219820

20.

Alegbe, E. O. & Uthman, T. O. A review of history, properties, classification, applications and challenges of natural and synthetic dyes. Heliyon 10, e33646. 10.1016/j.heliyon.2024.e33646 (2024).

21.

Fried, R., Oprea, I., Fleck, K. & Rudroff, F. Biogenic colourants in the textile industry – a promising and sustainable alternative to synthetic dyes. Green. Chem. 24 (1), 13–35. 10.1039/d1gc02968a (2022).

22.

Zoromba, M. S. Novel and economic acid-base indicator based on (p-toluidine) oligomer: Synthesis; characterization and solvatochromism applications. Spectrochim Acta - Part. Mol. Biomol. Spectrosc. 187, 61–67. 10.1016/j.saa.2017.06.028 (2017).

23.

Naime, J. et al. Synthesis, characterization and application of a novel polyazo dye as a universal acid-base indicator. RSC Adv. 12 (43), 28034–28042. 10.1039/d2ra04930a (2022).

24.

Karita, S. & Kaneta, T. Acid–Base Titrations Using Microfluidic Paper-Based Analytical Devices. Anal. Chem. 86 (24), 12108–12114. 10.1021/ac5039384 (Dec. 2014).

25.

Patil, A. R. et al. Synthesis and characterization of acid-base indicator: Exploring pH sensor, photophysical, thermal applications and theoretical study. J. Mol. Struct. 1254, 132363. 10.1016/j.molstruc.2022.132363 (Apr. 2022).

26.

Rashid Al-Jorani, K., Fahmi, R., Abbas & Amer Waheb, A. Synthesis and characterization of a novel benzimidazole derivative: Solvatochromic and acid-base indicator applications, Mater. Today Proc., vol. 42, pp. 1791–1799, (2021). 10.1016/j.matpr.2020.12.184

27.

Han, J. & Burgess, K. Fluorescent indicators for intracellular pH. Chem. Rev. 110 (5), 2709–2728. 10.1021/cr900249z (2010).

28.

Ma, H. et al. Ultrahigh adsorption capacities for anionic and cationic dyes from wastewater using only chitosan, J. Clean. Prod., vol. 214, pp. 89–94, Mar. (2019). 10.1016/j.jclepro.2018.12.217

29.

Shinde, M. N., Khurana, R., Barooah, N., Bhasikuttan, A. C. & Mohanty, J. Metal ion-induced supramolecular pK a tuning and fluorescence regeneration of a p-sulfonatocalixarene encapsulated neutral red dye. Org. Biomol. Chem. 15 (18), 3975–3984. 10.1039/C7OB00506G (2017).

30.

Ooyama, Y. & Harima, Y. Molecular designs and syntheses of organic dyes for dye-sensitized solar cells. Eur. J. Org. Chem. no. 18, 2903–2934. 10.1002/ejoc.200900236 (2009).

31.

Benkhaya, S., rabet, S. M. & El Harfi, A. A review on classifications, recent synthesis and applications of textile dyes. Inorg. Chem. Commun. 115, 107891. 10.1016/j.inoche.2020.107891 (2020). January.

32.

Cai, Y. et al. Organic Dye Based Nanoparticles for Cancer Phototheranostics. Small 14 (25), 1–17. 10.1002/smll.201704247 (2018).

33.

Kim, D. et al. Selective colorimetric sensors and diagnostic microfiber yarn for carbonyl groups via ion-pairing dyes with plural chromatic behaviors. Chem. Eng. J. 498, 155296. 10.1016/j.cej.2024.155296 (Oct. 2024).

34.

Trupp, S. et al. Development of pH-sensitive indicator dyes for the preparation of micro-patterned optical sensor layers. Sens. Actuators B Chem. 150 (1), 206–210. 10.1016/j.snb.2010.07.015 (2010).

35.

De Meyer, T., Hemelsoet, K., Van Speybroeck, V. & De Clerck, K. Substituent effects on absorption spectra of pH indicators: An experimental and computational study of sulfonphthaleine dyes. Dye Pigment. 102, 241–250. 10.1016/j.dyepig.2013.10.048 (2014).

36.

De Meyer, T. et al. Halochromic properties of sulfonphthaleine dyes in a textile environment: The influence of substituents. Dye Pigment. 124, 249–257. 10.1016/j.dyepig.2015.09.007 (2016).

37.

Desai, S. R. et al. Solvatochromism and halochromism in nitro lophine derivatives: Photophysical study, computational calculations and applications as pH sensing material. J. Photochem. Photobiol Chem. 455, 115751. 10.1016/j.jphotochem.2024.115751 (Oct. 2024).

38.

Cardona, M. A., Makuc, D., Szaciłowski, K., Plavec, J. & Magri, D. C. Water-Soluble Colorimetric Amino[ bis (ethanesulfonate)] Azobenzene pH Indicators: A UV–Vis Absorption, DFT, and 1 H– 15 N NMR Spectroscopy Study, ACS Omega, vol. 2, no. 9, pp. 6159–6166, Sep. (2017). 10.1021/acsomega.7b00887

39.

Derkowska-Zielinska, B. et al. Influence of peripheral substituents on the optical properties of heterocyclic azo dyes. Opt. Mater. (Amst). 49, 325–329. 10.1016/j.optmat.2015.10.001 (Nov. 2015).

40.

Wang, R., Deng, L., Zhang, T. & Li, J. Substituent effect on the photophysical properties, electrochemical properties and electroluminescence performance of orange-emitting iridium complexes. Dalt Trans. 41 (22), 6833. 10.1039/c2dt12206e (2012).

41.

Meleshenkova, V. V., Tyurin, V. S., Zamilatskov, I. A. & Kuznetsov, D. N. Halochromic properties of new nitrophenol-based azochromophores. J. Photochem. Photobiol Chem. 444, 114924. 10.1016/j.jphotochem.2023.114924 (Oct. 2023).

42.

Rashid, S. O. Spectrochemical Insights into Aromatic Hydrocarbons in 1,2-Disubstituted Naphthalenes: Investigations Using 1D and 2D NMR Spectroscopy and X-Ray Crystallography Analysis. Polycycl. Aromat. Compd. 45 (3), 524–539. https://doi.org/10.1080/10406638.2024.2411332 (Oct. 2025).

43.

Ehlerova, J., Trevani, L., Sedlbauer, J. & Tremaine, P. Spectrophotometric Determination of the Ionization Constants of Aqueous Nitrophenols at Temperatures up to 225°C, J. Solution Chem., vol. 37, no. 6, pp. 857–874, Jun. (2008). 10.1007/s10953-008-9279-x

44.

Muñoz Resta, I. & Galindo, F. Phenol-based styrylpyrylium dyes for trace water detection via chromogenic and fluorogenic responses. Dye Pigment. 197, 109908. 10.1016/j.dyepig.2021.109908 (Jan. 2022).

45.

Qian, H. F., Geng, J., Xu, D. & Huang, W. Hydrazone to deprotonated azo/azo-enol transformation for isomeric pyrazolone based heterocyclic dyes via metal-ion complexation, Dye. Pigment., vol. 160, no. September pp. 853–862, Jan. 2019, (2018). 10.1016/j.dyepig.2018.09.018

46.

Eom, T. & Khan, A. Dyes and Pigments Push-pull azobenzene chromophores with negative halochromism. Dye Pigment. 188 (2), 109197. 10.1016/j.dyepig.2021.109197 (2021).

47.

Browne, D. L., Baxendale, I. R. & Ley, S. V. Piecing together the puzzle: understanding a mild, metal free reduction method for the large scale synthesis of hydrazines, Tetrahedron, vol. 67, no. 52, pp. 10296–10303, Dec. (2011). 10.1016/j.tet.2011.09.146

48.

Hu, T., Baxendale, I. & Baumann, M. Exploring Flow Procedures for Diazonium Formation. Molecules 21 (7), 918. 10.3390/molecules21070918 (Jul. 2016).

49.

Marinozzi, M., Marcelli, G., Carotti, A. & Natalini, B. One-pot, telescoped synthesis of N-aryl-5-aminopyrazoles from anilines in environmentally benign conditions. RSC Adv. 4 (14), 7019. 10.1039/c3ra47541g (2014).

50.

Poh, J. S., Browne, D. L. & Ley, S. V. A multistep continuous flow synthesis machine for the preparation of pyrazoles via a metal-free amine-redox process. React. Chem. Eng. 1 (1), 101–105. 10.1039/C5RE00082C (2016).

51.

Schotten, C., Leist, L. G. T., Semrau, A. L. & Browne, D. L. A machine-assisted approach for the preparation of follow-on pharmaceutical compound libraries. React. Chem. Eng. 3 (2), 210–215. 10.1039/C8RE00010G (2018).

52.

Kader, D. A., Rashid, S. O. & Omer, K. M. Green nanocomposite: fabrication, characterization, and photocatalytic application of vitamin C adduct-conjugated ZnO nanoparticles. RSC Adv. 13 (15), 9963–9977. 10.1039/d2ra06575d (2023).

53.

Kader, D. A. & Omer, S. Regioselective direct thiocyanation of anilines and phenols using novel hybrid nanocatalyst (MgONPs @ VCA) under visible light induced photoredox catalysis. Mol. Catal. 547, 113409. 10.1016/j.mcat.2023.113409 (2023).

54.

Yaseen Ahmed, B. & Rashid, S. O. Synthesis, characterization, and application of metal-free sulfonamide-vitamin C adduct to improve the optical properties of PVA polymer. Arab. J. Chem. 15 (10), 104096. 10.1016/j.arabjc.2022.104096 (2022).

55.

Ayad Shamsallah, A. & Omer Rashid, S. Development in gelatin-matrix composite films: The incorporation of vitamin C adducts enhances the optical behaviors of gelatin films†. Arab. J. Chem. 17 (2), 105541. 10.1016/j.arabjc.2023.105541 (2024).

56.

Khanum, R. et al. Recent review on Synthesis, spectral Studies, versatile applications of azo dyes and its metal complexes. Results Chem. 5, 100890. 10.1016/j.rechem.2023.100890 (Jan. 2023).

57.

Selberg, S. et al. February., Non-invasive analysis of natural textile dyes using fluorescence excitation-emission matrices, Talanta, vol. 252, no. p. 123805, Jan. 2023, (2022). 10.1016/j.talanta.2022.123805

58.

Szakács, Z., Hägele, G. & Tyka, R. 1H/31P NMR pH indicator series to eliminate the glass electrode in NMR spectroscopic pKa determinations. Anal. Chim. Acta. 522 (2), 247–258. 10.1016/j.aca.2004.07.005 (Sep. 2004).

59.

Yoo, H., Seo, D., Shin, D. & Ro, C. U. Direct Observation of Particle-To-Particle Variability in Ambient Aerosol pH Using a Novel Analytical Approach Based on Surface-Enhanced Raman Spectroscopy. Environ. Sci. Technol. 58 (18), 7977–7985. 10.1021/acs.est.4c00220 (May 2024).

60.

Millbern, Z., Trettin, A., Wu, R., Demmler, M. & Vinueza, N. R. Synthetic dyes: A mass spectrometry approach and applications, Mass Spectrom. Rev., vol. 43, no. 2, pp. 327–344, Mar. (2024). 10.1002/mas.21818

61.

Barbache, S. et al. Jun., Optical analyses of wool dyeing materials in ancient Moroccan carpets ‘Zarbia(s)’: Combination of UV-vis diffuse reflectance, 3D-fluorescence and Raman spectroscopies, Dye. Pigment., vol. 153, no. January, pp. 256–265, (2018). 10.1016/j.dyepig.2018.02.033

62.

Makuła, P., Pacia, M. & Macyk, W. How To Correctly Determine the Band Gap Energy of Modified Semiconductor Photocatalysts Based on UV–Vis Spectra. J. Phys. Chem. Lett. 9 (23), 6814–6817. 10.1021/acs.jpclett.8b02892 (Dec. 2018).

63.

Sangiorgi, N., Aversa, L., Tatti, R., Verucchi, R. & Sanson, A. Spectrophotometric method for optical band gap and electronic transitions determination of semiconductor materials. Opt. Mater. (Amst). 64, 18–25. 10.1016/j.optmat.2016.11.014 (Feb. 2017).

64.

Adeniyi, A. A., Ngake, T. L. & Conradie, J. Cyclic Voltammetric Study of 2-Hydroxybenzophenone (HBP) Derivatives and the Correspondent Change in the Orbital Energy Levels in Different Solvents, Electroanalysis, vol. 32, no. 12, pp. 2659–2668, Dec. (2020). 10.1002/elan.202060163

65.

Arslan, Ö. et al. 8-Hydroxyquinoline based push-pull azo dye: Novel colorimetric chemosensor for anion detection. J. Mol. Struct. 1149, 499–509. 10.1016/j.molstruc.2017.08.001 (Dec. 2017).

66.

Manzoor, T., Niaz, S. & Pandith, A. H. Exploring the effect of different coumarin donors on the optical and photovoltaic properties of azo-bridged push‐pull systems: A theoretical approach, Int. J. Quantum Chem., vol. 119, no. 18, pp. 1–15, Sep. (2019). 10.1002/qua.25979

67.

Ghanadzadeh Gilani, A., Taghvaei, V., Moradi Rufchahi, E. & Mirzaei, M. Tautomerism, solvatochromism, preferential solvation, and density functional study of some heteroarylazo dyes. J. Mol. Liq. 273, 392–407. 10.1016/j.molliq.2018.10.054 (Jan. 2019).

68.

Hasan, M. M., Tarkhaneh, O., Bungay, S. D., Poirier, R. A. & Islam, S. M. Predicting HOMO–LUMO Gaps Using Hartree–Fock Calculated Data and Machine Learning Models, J. Chem. Inf. Model., vol. 65, no. 18, pp. 9497–9515, Sep. (2025). 10.1021/acs.jcim.5c01412

69.

Hsieh, M. T. & Whang, T. J. Mechanistic investigation on the electropolymerization of phenol red by cyclic voltammetry and the catalytic reactions toward acetaminophen and dopamine using poly(phenol red)-modified GCE, J. Electroanal. Chem., vol. 795, no. April, pp. 130–140, Jun. (2017). 10.1016/j.jelechem.2017.05.001

70.

Momeni, S. & Nematollahi, D. New insights into the electrochemical behavior of acid orange 7: Convergent paired electrochemical synthesis of new aminonaphthol derivatives. Sci. Rep. 7 (1), 41963. 10.1038/srep41963 (Feb. 2017).

71.

Nguyen, T. L. & Saleh, M. A. Thermal degradation of azobenzene dyes. Results Chem. 2, 100085. 10.1016/j.rechem.2020.100085 (Jan. 2020).

72.

Nguyen, T. L., Aparicio, M. & Saleh, M. A. Detection of suspected carcinogen azo dyes in textiles using thermogravimetric analysis. J. Environ. Sci. Heal - Part. Toxic/Hazardous Subst. Environ. Eng. 56 (8), 896–901. 10.1080/10934529.2021.1936989 (2021).

73.

Wilhelm, M. J. et al. Determination of bacterial surface charge density via saturation of adsorbed ions. Biophys. J. 120 (12), 2461–2470. 10.1016/j.bpj.2021.04.018 (Jun. 2021).

74.

Singh, G. & Rani, S. A proficient magnetic nano-platform with covalently assembled methyl red indicator for the dual recognition of pH and Hg2+. Sens. Actuators B Chem. 244, 861–875. 10.1016/j.snb.2017.01.039 (Jun. 2017).

75.

Cardona, M. A. & Magri, D. C. Synthesis and spectrophotometric studies of water-soluble amino[bis(ethanesulfonate)] azobenzene pH indicators. Tetrahedron Lett. 55 (33), 4559–4563. 10.1016/j.tetlet.2014.06.079 (Aug. 2014).

76.

Salgado, L. E. V. & Vargas-Hernández, C. Spectrophotometric Determination of the pKa, Isosbestic Point and Equation of Absorbance vs. pH for a Universal pH Indicator. Am. J. Anal. Chem. 05 (17), 1290–1301. 10.4236/ajac.2014.517135 (2014).

77.

Jewłoszewicz, B. et al. A comprehensive optical and electrical study of unsymmetrical imine with four thiophene rings and their binary and ternary compositions with PTB7 and PC 70 BM towards organic photovoltaics. RSC Adv. 10 (73), 44958–44972. 10.1039/D0RA08330E (2020).

78.

Costa, C. S., Taveira, R. J. S., Lima, C. F. R. A. C. & C, A. M. Optical band gaps of organic semiconductor materials. Opt. Mater. (Amst). 58, 51–60. http://dx.doi.org/10.1016/j.optmat.2016.03.041 (2016).

79.

Li, J. et al. Synthesis of sp 2 -Carbon Covalent Triazine Framework Photocatalysts for Efficient Organic Dye Degradation, Langmuir, vol. 41, no. 40, pp. 27294–27310, Oct. (2025). 10.1021/acs.langmuir.5c03283

80.

Yang, S. et al. Positive Cooperative Protonation of a Metal – Organic Framework: pH-Responsive Fluorescence and Proton Conduction. J. Am. Chem. Soc. 143, 8838–8848. 10.1021/jacs.1c03432 (2021).

81.

Gallardo-Rosas, D. et al. Structure and isomerization behavior relationships of new push–pull azo-pyrrole photoswitches. Org. Biomol. Chem. 22 (20), 4123–4134. 10.1039/D4OB00417E (2024).

82.

Balam-Villarreal, J. A. et al. π-Extended push–pull azo-pyrrole photoswitches: synthesis, solvatochromism and optical band gaps. Org. Biomol. Chem. 18 (8), 1657–1670. 10.1039/C9OB02410G (2020).

83.

Bichot, G., Sallenave, X., Metivier, R. & Peralta, S. Synthesis and characterization of new push-pull azobenzene. Cis → Trans isomerization kinetic study. Dye Pigment. 239 (112774), 1–9. 10.1016/j.dyepig.2025.112774 (2025).

84.

Kundu, R., Kulshreshtha, C. & Advances, R. S. C. RSC Adv., 5, 77460–77468, doi: 10.1039/c5ra13416a. (2015).

85.

Chen, J. & Li, Y. Orbital symmetry matching study on the interactions of flotation reagents with mineral surfaces. Min. Eng. 179 (107469), 1–8. 10.1016/j.mineng.2022.107469 (Mar. 2022).

86.

Mikheev, Y. A. & Ershov, Y. A. STRUCTURE OF MATTER Assignment of the π → π * and n → π * Transitions to the Spectral Bands of Azobenzene and Dimethylaminoazobenzene. Russ J. Phys. Chem. A. 92 (8), 1499–1507. 10.1134/S0036024418080174 (2018).

87.

Aires-de-sousa, J. Machine Learning Methods to Predict Density Functional Theory B3LYP Energies of HOMO and LUMO Orbitals. J. Chem. Inf. Model. 57, 11–21. 10.1021/acs.jcim.6b00340 (2017).

88.

Moore, G. J., Bardagot, O. & Banerji, N. Deep Transfer Learning: A Fast and Accurate Tool to Predict the Energy Levels of Donor Molecules for Organic Photovoltaics. Adv. Theory Simulations. 5, 1–10. 10.1002/adts.202100511 (2022).

89.

Zhang, G. & Musgrave, C. B. Comparison of DFT Methods for Molecular Orbital Eigenvalue Calculations. J. Phys. Chem. A. 111, 1554–1561. 10.1021/jp061633o (2007).

90.

Vollmer, W., Blanot, D. & De Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32 (2), 149–167. 10.1111/j.1574-6976.2007.00094.x (Mar. 2008).

91.

Smith, A., Hussey, M. & Protocols, G. S. Am. Soc. Microbiol., no. September pp. 1–9, 2005, [Online]. (2005). Available: https://www.google.com/url?sa=t&source=web&rct=j&opi=89978449&url=https://asm.org/getattachment/5c95a063-326b-4b2f-98ce-001de9a5ece3/gram-stain-protocol-2886.pdf&ved=2ahUKEwiO5pPn6omFAxUM2DgGHUvMBiwQFnoECCoQAQ&usg=AOvVaw1-NVEcOxHCNBqEerbZUHJO

92.

Rohde, M. The Gram-Positive Bacterial Cell Wall. Microbiol. Spectr. 7 (3). 10.1128/microbiolspec.GPP3-0044-2018 (May 2019).

93.

Thairu, Y., Usman, Y. & Nasir, I. Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-Saharan Afr. J. Med. 1 (4), 168. 10.4103/2384-5147.144725 (2014).

94.

Beveridge, T. J. Use of the gram stain in microbiology. Biotech. Histochem. 76 (3), 111–118. 10.1080/bih.76.3.111.118 (May 2001).

95.

Bottone, E. J. Bacillus cereus, a Volatile Human Pathogen, Clin. Microbiol. Rev., vol. 23, no. 2, pp. 382–398, Apr. (2010). 10.1128/CMR.00073-09

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