Analytical and Biological Evaluation of Chromium Complex with Organic Detector NTADBrP: Stability, Calibration, and Inhibition Studies ^†

Abstract

This study provides a comprehensive investigation into the formation, characteristics, and biological activity of a chromium complex with the organic reagent NTADBrP. The article presents detailed insights into the H1-NMR spectrum of the organic reagent and examines the optimal conditions for chromium complex formation, considering the effects of time, pH, and temperature. The stoichiometry and stability constant of the complex are determined using specific methods, leading to the calculation of a significant stability constant. Additionally, a calibration curve for the chromium ion is derived, and the complex’s biological activity against Escherichia coli and Staphylococcus bacteria is studied. These findings contribute to the understanding of chromium complex behavior and open new avenues for applications in analytical chemistry and pharmaceutical research.

1. Introduction

Thiazole compounds and their derivatives are characterized by high molecular weights and limited solubility in water, attributes stemming from their covalent bonds [1,2,3]. These compounds have various applications, notably in spectrophotometric and fluorescence assessments of positive ions and as precipitants due to their chromophoric groups [4,5,6]. Chromium, a key element in the VIB family of the periodic table, offers distinct properties such as a small atomic size, high melting point, and resistance to corrosion [7,8,9,10,11]. It is a transition metal with multiple oxidation states, forming complex ions and participating in vital biological functions [12,13,14]. Its different valences have varying toxicity levels, with Cr(VI) being toxic and carcinogenic [15]. The exploration of thiazole derivatives and chromium in antibacterial treatments forms the focus of this research. Previous studies have highlighted the antibacterial potency of thiazole derivatives and their complexes with transition metals [16,17]. However, the understanding of the specific inhibitory effects on particular bacterial strains and the underlying mechanisms is limited. In this context, the present research investigates a complex consisting of chromium (iii) and the thiazole derivative 2-2-(5-nitro thiazolyl) azo-4,6-dibromo phenol (NTADBrP). The study centers on the ability of this complex to inhibit Escherichia coli and Staphylococcus bacteria [18]. The objective is to uncover the underlying properties and mechanisms that enable the chromium–thiazole complex to suppress specific bacterial activity, thus contributing to the potential development of novel antibacterial agents. The significance of this work lies in its unique perspective on the synergistic effects of thiazole derivatives and chromium, as well as its potential applications in healthcare.

The article is organized into sections detailing the materials and methods, results, discussion, and conclusions, offering insights that may benefit both the scientific community and industries seeking innovative antibacterial solutions.

2. Materials and Methods

2.1. Chemicals and Materials

Ethanol absolute 99.9%, glacial acetic acid, and nitric acid 70% were all supplied by Germany. Sodium hydroxide 97%, 2,4-dibromophenol 97%, sodium nitrite 96%, and chromium chloride hexahydrate 99% were procured from Alpha Chemika. Hydrochloric acid 37% was supplied by J.T. Baker, dimethyl sulphoxide 98% by CARLO ERBA, and 2-amino 5-nitro thiazole 99% by Glanthum. All chemicals were of analytical grade and were used without further purification. The specific roles and characteristics of these chemicals were aligned with the study’s procedures, ensuring consistency and accuracy in the experimental process.

2.2. Preparation of the Organic Reagent: 2-[2-(5-Nitro thiazolyl) azo]-4,6-dibromo Phenol (NTADBrP)

The organic reagent (NTADBrP) was synthesized following the method proposed by Nafaa, M.R [19]. The preparation began by mixing a thiazole derivative (0.01 mol) with 9 mL of concentrated hydrochloric acid and cooling the mixture to 3 °C. A sodium nitrite solution (0.01 mol) was subsequently added at the same temperature, with constant stirring, to form the diazonium salt. This was followed by the gradual addition of 10% sodium hydroxide solution (measurements may need to be clarified) with continued stirring, resulting in a dark red solution. After two hours, chilled water was added, and the medium was adjusted to a pH of 6.5–7. The mixture was left to stand for 24 h, allowing the reagent to deposit. It was then filtered and washed with distilled water, followed by recrystallization with absolute ethanol. The prepared NTADBrP was stored in an opaque bottle, ready for use in the experiments under study. The chemical process behind the synthesis of NTADBrP is described through a series of equations as shown in Figure 1 below. This figure illustrates the step-by-step reactions involved in the formation of the organic reagent, providing a visual representation of the synthesis process.

2.3. Preparation of Standard Solutions Used in the Experiments

2.3.1. Chromium Solution

The preparation of the chromium (III) solution was a key step in the experimental setup. A stock solution of Cr(III) with a concentration of 1000 $\mathsf{\mu }$g/mL was meticulously prepared by dissolving 1.2812 g of CrCl₃·6H₂O in 250 mL of deionized water. Care was taken to ensure complete dissolution.

Subsequent dilutions of this stock solution were made with distilled water to create various concentrations for different experimental conditions. The solutions were stored under controlled conditions to prevent oxidation or other alterations.

2.3.2. (NTADBrP) Solution

A standard solution of the organic reagent NTADBrP was prepared at a concentration of $3\times {10}^{-4}$ M. A precise amount of 0.01224 g was weighed and dissolved in 100 mL of absolute ethanol. The solution was continuously stirred to ensure uniformity and kept in a sealed container to prevent evaporation or contamination. The accuracy of the concentration was validated through various analytical techniques, ensuring the solution’s suitability for subsequent experiments.

2.3.3. Preparation of Culture Medium

The Mueller–Hinton agar culture medium was prepared following the specific instructions provided by the manufacturer. Initially, 38 g of culture medium powder was precisely weighed and dissolved in 1 L of distilled water within a conical flask [20]. This solution was then heated in a water bath, with continuous stirring until the agar dissolved entirely. Sterilization was performed using an autoclave, where the culture medium was exposed to 121 °C and 1 atmosphere of pressure [21]. After cooling, the medium was dispensed into dishes, and the prepared bacteria were evenly spread. Incubation was performed at 37 °C for 24 h, creating optimal growth conditions for the bacterial culture [22]. In all preparation procedures, quality control measures were applied to ensure the accuracy and reproducibility of the solutions and culture media. Proper safety protocols were followed, including the use of personal protective equipment, to ensure the safety of laboratory personnel. Calibration and validation procedures were applied to all equipment and materials used in the preparation processes.

3. Results and Discussion

3.1. Absorption Spectra

By studying the electronic transitions in the UV-Vis absorption spectra of both the organic reagent (NTADBrP) and the chromium complex before and after applying the optimal conditions, several important observations were made. The highest peak of the organic reagent’s absorption spectrum was at ${\lambda }_{\max }=390\mathrm{nm}$. In contrast, the chromium complex exhibited its highest absorption peak at ${\lambda }_{\max }=235\mathrm{nm}$, indicating a shift towards shorter wavelengths, commonly referred to as the blue shift. This phenomenon is attributed to the charge transfer between the metal and the reagent. In addition, a small absorption peak appeared in the spectrum of the complex at a higher wavelength due to d–d transitions.

As shown in Figure 2, the absorption spectra provide insight into the electronic properties and interactions between the organic reagent and the chromium complex, enhancing our understanding of their chemical behavior.

3.2. FTIR Spectrum

The FT-IR spectrum of the organic reagent is depicted in Figure 2, where several significant features can be identified. A broad absorption band corresponding to the O–H group appeared at a wavelength of $3332.76{\mathrm{cm}}^{-1}$. Two peaks at wavelengths $2839.18{\mathrm{cm}}^{-1}$ and $2815.88{\mathrm{cm}}^{-1}$ are attributed to the aromatic and aliphatic C–H groups, respectively. The thiazole ring C=N showed an absorption at $1679.24{\mathrm{cm}}^{-1}$, while for the azo group N=N, the absorption spectrum exhibited a frequency within the range of $1564.37{\mathrm{cm}}^{-1}$ to $1458.08{\mathrm{cm}}^{-1}$ [23]. The C–S group of the thiazole ring displayed absorption spectra at $1265.22{\mathrm{cm}}^{-1}$ and $1211.21{\mathrm{cm}}^{-1}$, and the absorption frequency at $840.91{\mathrm{cm}}^{-1}$ corresponds to the C–Br bond.

As for the FT-IR spectrum of Cr(III)-Complex, it revealed absorption spectra similar or close to those in the spectrum of the organic reagent [24], with some displacements occurring due to the coordination of the organic reagent with the ion. The weak absorption band at $3320.54{\mathrm{cm}}^{-1}$ is attributed to the meta hydroxyl group, and the two peaks at $2839.18{\mathrm{cm}}^{-1}$ and $2815.88{\mathrm{cm}}^{-1}$ correspond to aromatic and aliphatic C–H, respectively, similar to those in the spectrum of the organic reagent [25]. The thiazole ring C=N showed a lower frequency than the organic reagent at $1631.54{\mathrm{cm}}^{-1}$. The bonding between the silver ion and nitrogen in the thiazole heterocyclic ring, as well as the azo group N=N, resulted in a reduced absorption spectrum in frequency and strength to $1502.95{\mathrm{cm}}^{-1}$ and $1421.80{\mathrm{cm}}^{-1}$ due to the coordination of the detector with the ion as shown in Figure 3 [26].

3.3. H¹-NMR Spectrum for Organic Reagent

The ${\mathrm{H}}^1$-NMR spectrum of the organic reagent (NTADBrP) was obtained using dimethyl sulfur dioxide (DMSO-$d_6$) as the solvent, with TMS as the reference standard. The chemical shift at $\delta =7.32-7.01$ can be attributed to the ring protons. Additionally, the hydroxyl group displayed a single peak at the location $\delta =10.4$. The chemical shift appearing at $\delta =2.32-2.22$ is due to the solvent [25,27]. Figure 4 illustrates the NMR spectrum of the organic reagent.

3.4. Select Optimal Conditions of Chromium Complex

3.4.1. Effect of Time

The stability of the chromium complex with time was studied, with the absorption values plotted against time. The complex remained approximately stable within 24 h from the onset of formation, confirming the possibility of using the organic reagent to determine the chromium ion [26] as shown in Figure 5.

3.4.2. Effect of pH on Chromium Complex Formation

Figure 6 shows the effect of the pH function on the formation of the chromium complex. The results indicate an increase in the absorption of the chromium complex solution with increasing acidity until it reached pH = 8, at which point the absorption value was highest. The complex’s absorption value then began to decrease at higher pH values, likely due to the precipitation of chromium ion or the formation of unstable complexes [28,29,30].

3.4.3. Effect of the Temperature

The effect of temperature on the absorption of a chromium complex solution was studied. The best absorption value of the chromium complex was within the temperature range of 10–30, where it reached its peak and gave the best color intensity. At higher temperatures, the absorbance decreased, possibly due to a decrease in stability or dissociation of the complex at high temperatures [31,32,33] as shown in Figure 7.

3.5. Determination of Stoichiometry of the Chromium Complex and Calculating the Stability Constant

To determine the ratio of the organic reagent with the chromium ion, the molar ratios method was used, as well as the continuous changes method (Jopp’s method). In the molar ratios method, a fixed concentration of chromium ion $(3.846\times {10}^{-4}\mathrm{M})$ was used, along with varying, increasing, and proportional concentrations of the organic reagent, ranging between $0.8\times {10}^{-4}$ and $0.75\times {10}^{-5}\mathrm{M}$ as shown in Figure 8 [34].

The method of continuous variation included mixing different volumes and equal concentrations of both the metal solution and the reagent such that the final volume of the mixture was $10\mathrm{mL}$ as shown in Figure 9. The results show that the ratio of the complex is 1:2.

The stability constant was also calculated for the complex formed in the acidic medium $(\mathrm{pH}=8)$ and, using the equations below, the stability constant was found to be $(7.755\times {10}^7)$:

$$\begin{array}{cc}\hfill K_{\mathrm{st}}& =\frac{1}{K_{\mathrm{ins}}}\hfill \end{array}$$

(1)

$$\begin{array}{cc}\hfill K_{\mathrm{ins}}& =\frac{\left(\alpha C\right){\left(n\alpha C\right)}^2}{C(1-\alpha )}.\alpha =\frac{A_{\mathrm{m}}-A_{\mathrm{s}}}{A_{\mathrm{m}}}\hfill \end{array}$$

(2)

where:

• $\alpha$ = degree of complex dissociation;
• C = concentration;
• n = mole ratio;
• $A_{\mathrm{m}}$ = absorption of the complex at its greatest value;
• $A_{\mathrm{s}}$ = absorption of the complex at the equivalence point.

3.6. Characteristics of Analysis

Calibration Curve for Chromium Complex

The absorbance was plotted against the concentration of chromium ion in units of ppm to find the calibration curve for the ternary chromium ion under optimal conditions. It is clear from the results and through

Table 1. Analytical data and some analytical parameters for the proposed Cr(III) determination technique.

Analytical Parameter	Cr(III)/ppm
${\lambda }_{\max }$	235 nm
Regression Equation	$Y=0.018x+0.0374$
Molar Absorptivity (L·mol−1·cm−1)	899.64
Sandell Sensitivity	0.0578 L−1·gm·cm
Correlation Coefficient $\left(r\right)$	0.9899
Detection Limit (ppm)	0.100
Percent Relative Error %	−0.115%
Percent Recovery %	99.885
Composition of Complex (M:L)	1:2
Linear Dynamic Range (ppm)	(1–60)
Standard Deviation	0.000577
Relative Standard Deviation %	0.167%

that this method used has high sensitivity and can be used to estimate the triple chromium ion within low concentrations [35]. Figure 10 shows the linearity of the calibration curve.

3.7. Biological Activity

The Mueller–Hinton agar (MHA) culture medium was prepared following the instructions provided by the supplying company as detailed in experimental part of this research. Subsequently, the culture medium was sterilized in an autoclave at a temperature of 121 °C and a pressure of 1 atmosphere for 15 min. After cooling, the prepared bacteria, namely Escherichia coli and Staphylococcus, were spread on the culture medium. Separate dishes were prepared for each type of bacteria, and the dishes were incubated at a temperature of 37 °C for 24 h to allow for bacterial growth.

Dimethyl sulfoxide was utilized as a solvent, and aliquots of the complex solution were placed on the medium [36]. The results demonstrated the complex’s efficacy in inhibiting the activity of both types of bacteria under study. Specifically, inhibition ratios of 0.3–0.7 cm were observed for Escherichia coli and 1–1.5 cm for Staphylococcus bacteria. The effect of the chromium complex on the activity of these bacteria is illustrated in Figure 11.

4. Conclusions

The comprehensive investigation into the chromium complex with the organic reagent NTADBrP led to several important findings. The characteristics of the H1-NMR spectrum of the organic reagent were detailed, revealing specific chemical displacements and confirming the structure of the organic reagent. The study further explored the optimal conditions for the chromium complex formation, analyzing the effects of time, pH, and temperature on the absorption of the complex. It was found that the complex remains stable within 24 h of formation, with the highest absorption value at pH = 8, and an optimal temperature range between 10 °C and 30 °C. The stoichiometry of the chromium complex was determined using the molar ratios method and the continuous changes method (Jopp’s method). The stability constant for the complex was calculated, yielding a value of $7.755\times {10}^7$. Analytical techniques were employed to derive a calibration curve for the ternary chromium ion, demonstrating high sensitivity and applicability within low concentration ranges. Additionally, the complex exhibited biological activity against Escherichia coli and Staphylococcus bacteria, indicating potential applications in antibacterial treatments. The findings of this study contribute to the understanding of chromium complex formation and behavior under different conditions.

The analytical techniques and biological activities revealed in this investigation may have broader implications in the fields of analytical chemistry and pharmaceutical research. The results also prompt further inquiry into other potential applications and behaviors of chromium complexes, thus opening avenues for future research and technological advancements.