UV-VIS Spectrophotometric Determination of Selected Heavy Metals (Pb, Cr, Cd and As) in Soil, Water and Biological Samples with Acetylpyridine-2,4-Dinitrophenyl Hydrazone as the Chromogenic Reagent

methods. The results are expected to contribute to the development of a low-cost, sensitive, and field- deployable analytical protocol for monitoring toxic metals, which can be particularly beneficial in resource-limited or remote regions. Materials Used: All the regents and materials used in the synthesis and characterization of the phenyl hydrazones were of analytical grade, sourced from accredited vendors and were used without further purification, unless stated otherwise. Samples Collection and Treatment: The samples (waste water, water and soil) in triplicates were collected from Challawa Industrial Area of Kumbotso Local Government Area of Kano State Nigeria West Africa into polythene bags and transported to the laboratory of Federal College of Agricultural Produce Technology Kano for analysis. The waste water samples collected (500.00 cm3) were filtered using a filter paper (Whatman No.41) and then each filtered water sample was evaporated nearly to dryness with a mixture of 5.00 cm3 of concentrated H2SO4 and 10.00 cm3 concentrated HNO3 in a fume cupboard and then cooled to room temperature. In order to dissolve the salts, the residue was then heated with 15.00 cm3 of distilled water. After cooling the solution was neutralized with dilute NH4OH solution and the obtained solution was filtered into 50.00 cm3 standard flask and made up to the mark with deionized water [1], 5, 7 and 21. Before digestion of the soil samples, each was dried at 65 °C for 48 hours. All samples were performed in triplicates. Five grams (5.00 g) of the samples in crucibles was placed in a preheated muffle furnace at 200-250 °C for 30 minutes and ashed for eight hours at 500-550 °C. Then, the sample was removed from the furnace and cooled down. 2.00 cm3 of 5.00 mol/dm3 of HNO3 was added and evapourated to dryness on a sand bath. The samples were then placed in a furnace and heated to 550 °C for 15 minutes. The samples were removed from the furnace, cooled and moistened with four drops of distilled water. Then 2.00 cm3 of concentrated HCl was added into the sample was evaporated to dryness, removed, and 5.00 cm3 2.00 mol/dm3 HCl was again added and the crucible. The solution was filtered through Whatman No. 41 filter paper and the transferred quantitatively to a 50.00 cm3 flask by making it up with deionized water [5] and 21. The biological samples (liver, blood and flesh from animals) and plants samples were collected from Challawa industrial of Kano State Nigeria West Africa. The blood samples of 5.00 cm3, the liver, flesh and plants samples were oven dried and 5.00 g were transferred into crucibles. The samples were then ashed in the muffle furnace at 500-550 oC for 8.0 hours in the presence of 10.00 cm3 nitric acid. The contents of each crucible were cooled to room temperature, and 1.50 cm3 of concentrated hydrochloric acid was added and warmed slightly. The content of each crucible was filtered. The solutions were then transferred quantitatively into 50.0 cm3 calibrated flask and made up to the mark with deionized water [5], 6 and 21. A suitable aliquot of the final solutions was pipetted and the selected heavy metals levels determined as described under procedures using EDTA as the masking agent. The same elements in the samples were also determined with Atomic Absorption spectrophotometric method as the reference methods and the results tabulated [6]. , Samples Collected and their Codes: F = Animal flesh, L = Animal liver, P1 = Spinach, P2 = Cabbage, B = Animal blood, IW = Irrigation water, SW = Waste water, WS = Farm land Soil, WW = Well water. Effect of Interference or Foreign Ions: The effects of foreign ions (cations; Co, Cu, Al, Fe, Hg, Zn, Ca and anions like SO42-, NO3-) on the determination of the heavy metals were studied using the methods of [6] and [7] by measuring the absorbance of the metal-ligand complex containing 1 μg mL-1 of the metal of interest. The criterion for interference is an absorbance value varying by more than ±5% from the expected value for the metal under observation alone 5 and 21. Statistical Analysis: The data collected were statistically analyzed using the Math Lab 2014 version with a significance level of P<0.05. All collected data carried out were subjected to one-way analysis of variance (1 way-ANOVA). Table 1: Results of Lead in Soil, Water and Biological Samples with the Developed Method and AAS as reference method Serial Sample Concentration of Pb (μg g-1) ± SD p value Relative Error (%) No Code AAS APDH AAS / APDH Methods 1. F 0.007 ± 0.001 0.0069 ± 0.001 0.2297 -1.42 2. L 0.001 ± 0.001 0.001 ± 0.000 0.0000 0.00 3. P1 1.018 ± 0.001 1.019 ± 0.002 0.5878 +0.09 4. P2 1.014 ± 0.001 1.015 ± 0.001 0.2297 +0.09 5. B 0.001 ± 0.000 0.001 ± 0.000 0.0000 0.00 6. IW 0.810 ± 0.001 0.811 ± 0.001 0.2991 +0.12 7. SW 1.924 ± 0.002 1.926 ± 0.002 0.2297 +0.10 8. WS 1.173 ± 0.003 1.172 ± 0.003 0.5242 -0.08 9. WW 0.877 ± 0.002 0.878 ± 0.002 0.75041 +0.11 Table 2: Results of Cadmium in Soil, Water and Biological Samples with the Developed Method and AAS as the Reference Method. Serial Sample Concentration of Cd (μg g-1) ± SD p value Relative Error (%) No Code AAS APDH AAS /APDH Methods 1. F 0.000 ± 0.000 0.000 ± 0.000 0.00 0.00 2. L 0.020 ± 0.001 0.0199 ± 0.001 0.2297 -0.5 3. P1 0.014 ± 0.003 0.014 ± 0.002 0.75041 0.00 4. P2 0.021 ± 0.003 0.021 ± 0.002 1.0000 0.00 5. B 0.000 ± 0.000 0.000 ± 0.000 0.00 0.00 6. IW 0.031± 0.004 0.032 ± 0.001 0.509224 +3.22 7. SW 0.055 ±0.004 0.054 ± 0.002 0.814812 -1.81 8. WS 0.008 ± 0.004 0.009 ± 0.001 0.791764 +1.50 9. WW 0.009 ± 0.002 0.008 ± 0.002 0.668422 -1.11 , Table 3: Results of Chromium in Soil, Water and Biological Samples with the Developed Method and AAS as the Reference Method. Serial Sample Concentration of Cr (μg g-1) ± SD p value Relative Error (%) No Code AAS APDH AAS / APDH Methods 1. F 0.033 ± 0.001 0.034 ± 0.002 0.45826 +3.03 2. L 0.010 ± 0.002 0.009 ± 0.003 0.43902 -1.00 3. P1 3.474 ± 0.015 3.476 ± 0.003 0.9392 +0.05 4. P2 2.042 ± 0.005 2.042± 0.002 0.8779 0.00 5. B 0.000 ± 0.000 0.000 ± 0.000 0.0000 0.00 6. IW 1.988 ± 0.001 1.989 ± 0.002 0.1237 +0.05 7. SW 2.248 ± 0.003 2.250 ± 0.003 0.2438 +0.08 8. WS 2.616 ± 0.004 2.618 ± 0.002 0.2710 +0.07 9. WW 1.038 ± 0.001 1.040 ± 0.003 0.2926 +0.19 Table 4: Results of Arsenic in Soil, Water and Biological Samples with the Developed Methods and AAS as the Reference Method Serial Sample Concentration of As (μg g-1) ± SD p value Relative Error (%) No Code AAS APDH AAS / APDH Methods 1. F 0.000 ± 0.000 0.000 ± 0.000 0.00 0.00 2. L 0.000 ± 0.000 0.000 ± 0.000 0.00 0.00 3. P1 0.012 ± 0.001 0.012 ± 0.002 0.00 0.00 4. P2 0.014 ± 0.002 0.015 ± 0.003 0.3415 0.71 5. B 0.000 ± 0.000 0.000 ± 0.000 0.00 0.00 6. IW 0.011 ± 0.001 0.010 ± 0.002 0.2297 -0.9 7. SW 0.010 ± 0.002 0.011 ± 0.001 0.5878 +1.0 8. WS 0.009 ± 0.004 0.010 ± 0.001 0.5878 +1.11 9 WW 0.028 ± 0.002 0.027 ± 0.003 0.8000 -3.5 Table 5: Evaluation of accuracy of Cadmium (Cd) for the developed methods Methods Sample Pb added APDH methods Found (μg/ml) Recovery (%) ± SD F - ND - 50.0 49.99 99.98±0.2 100.0 99.98 99.98±0.1 500.0 499.94 99.98±0.4 L - ND - 50.0 49.98 99.96±0.3 100.0 99.97 99.97±.02 500.0 499.95 99.99±0.3 , P1 - ND - 50.0 49.98 99.96±0.3 100.0 99.98 99.98±0.2 500.0 499.99 99.80±0.2 P2 - ND - 50.0 49.97 99.94±0.3 100.0 99.97 99.97±0.3 500.0 499.97 99.99±0.1 B - ND - 50.0 49.99 99.98±0.2 100.0 99.97 99.97±0.3 500.0 499.98 99.99±0.1 IW - ND - 50.0 49.97 99.94±0.3 100.0 99.97 99.97±0.3 500.0 499.97 99.97±0.4 SW - ND - 50.0 49.97 99.94±0.2 100.0 99.96 99.96±0.3 500.0 499.93 99.98±0.4 WS - ND - 50.0 49.93 99.86±0.4 100.0 99.97 99.97±0.3 500.0 499.92 99.98±0.4 WW - ND - 50.0 49.97 99.94±0.3 100.0 99.97 99.97±0.3 500.0 499.96 99.99±0.2 Average of five replicate determinations of each Sample (n = 5), ND = Not Detected, SD = standard deviation Table 6: Evaluation of accuracy of Lead (Pb) for the developed methods Sample Pb added APDH Method Found (μg/ml) Recovery (%) ± SD F - ND - 50.0 49.97 99.94±0.3 100.0 99.99 99.99±0.2 500.0 499.94 99.98±0.3 L - ND - 50.0 49.94 99.88±0.4 100.0 99.97 99.97±0.3 500.0 499.94 99.98±0.3 , P1 - ND - 50.0 49.96 99.92±0.3 100.0 99.96 99.96±0.4 500.0 499.93 99.98±0.4 P2 - ND - 50.0 49.97 99.94±0.3 100.0 99.98 99.98±0.2 500.0 499.92 99.98±0.4 B - ND - 50.0 49.97 99.94±0.4 100.0 99.97 99.97±0.4 500.0 499.98 99.99±0.2 IW - ND - 50.0 49.96 99.92±0.4 100.0 99.98 99.98±0.2 500.0 499.98 99.99±0.2 SW - ND - 50.0 49.97 99.94±0.3 100.0 99.98 99.98±0.2 500.0 499.03 99.98±0.4 WS - ND - 50.0 49.95 99.90±0.4 100.0 99.97 99.97±0.3 500.0 499.95 99.98±0.4 WW - ND - 50.0 49.97 99.94±0.3 100.0 99.97 99.97±0.3 500.0 499.98 99.99±0.4 Average of five replicate determinations of each Sample(n=5), ND = Not Detected, SD = Standard Deviation. Table 7: Evaluation of accuracy of Chromium (Cr) for the developed methods . Sample Pb added APDH Method Found (μg/ml) Recovery (%) ± SD F - ND - 50.0 49.98 99.99±0.3 100.0 99.98 99.98±0.2 500.0 499.97 99.98±0.3 L - ND 50.0 49.99 99.99±0.2 100.0 99.99 99.99±0.3 500.0 499.94 99.98±0.3 P1 - ND - , 50.0 49.99 99.99±0.2 100.0 99.97 99.97±0.3 500.0 499.98 99.99±0.2 P2 - ND - 50.0 49.96 99.92±0.4 100.0 99.97 99.97±0.3 500.0 499.98 99.99±0.2 B - ND - 50.0 49.99 99.99±0.2 100.0 99.97 99.97±0.4 500.0 499.94 99.96±0.3 IW - ND - 50.0 49.96 99.92±0.4 100.0 99.97 99.97±0.4 500.0 499.95 99.97±0.3 SW - ND - 50.0 49.98 99.98±0.3 100.0 99.96 99.96±0.3 500.0 499.03 99.80±0.4 WS - ND - 50.0 49.96 99.92±0.4 100.0 99.97 99.97±0.3 500.0 499.95 99.99±0.2 WW - ND - 50.0 49.99 99.98±0.2 100.0 99.99 99.99±0.3 500.0 499.99 99.99±0.2 Average of five replicate determinations of each Sample (n = 5), ND = Not Detected, SD = Standard Deviation. Table 8: Evaluation of accuracy of Arsenic (As) for the methods Sample Pb added APDH Method Found (μg/ml) Recovery (%) ± SD F - ND - 50.0 49.95 99.90±0.4 100.0 99.97 99.97±0.3 500.0 499.95 99.99±0.2 L - ND 50.0 49.99 99.99±0.2 100.0 99.96 99.96±0.3 500.0 499.94 99.99±0.3 P1 - ND - 50.0 49.99 99.99±0.2 , 100.0 99.96 99.96±0.3 500.0 499.94 99.99±0.2 P2 - ND - 50.0 49.96 99.92±0.4 100.0 99.98 99.98±0.2 500.0 499.97 99.99±0.2 B - ND - 50.0 49.99 99.99±0.1 100.0 99.96 99.96±0.3 500.0 499.97 99.99±0.2 IW - ND - 50.0 49.96 99.92±0.3 100.0 99.95 99.95±0.4 500.0 499.94 99.99±0.3 SW - ND - 50.0 49.97 99.94±0.3 100.0 99.93 99.93±0.4 500.0 499.03 99.99±0.2 WS - ND - 50.0 49.96 99.92±0.3 100.0 99.97 99.97±0.2 500.0 499.96 99.99±0.2 WW - ND - 50.0 49.99 99.99±0.1 100.0 99.97 99.97±0.2 500.0 499.98 99.99±0.1 Average of five replicate determinations of each Sample (n = 5), ND = Not Detected, SD = Standard Deviation. Discussion and Interpretation of Analytical Results: The present study aimed to develop and validate a UV-Vis spectrophotometric method using acetylpyridine-2,4-dinitrophenyl hydrazone as a chromogenic reagent for the determination of heavy metals (Pb, Cd, Cr, and As) in environmental and biological matrices. Tables 1 to 4 present a comparative analysis between the APDH method and the reference Atomic Absorption Spectrophotometry , while Tables 5 to 8 evaluate the recovery and accuracy of the developed method. The quantitative determination of the selected heavy metals (Cd, Pb, Cr and As) in the soil, water and biological samples was based on the measurement of increase and reduction of the absorbance of the [Metal-APDH], [8]. The effect of solvent used for the formation of the reaction complex was studied. Of the various organic solvent used; ethanol, chloroform, 1,4- dioxane, dimethysulfoxide and dimethylformide, dimethylformide was found to be the most suitable solvent for the system with the highest maximum absorbance at the same working conditions with the other organic solvent, hence DMF was used as the choice for solvent as recommended by [6] and [9]. The composition and stability of the complexes were determined by mole ratio method. The mole ratio showed that the reagent complex [Metal-APDH] had 1: 1. The reaction temperature for the metal-reagent complex was studied. The reactions were carried out at room temperature of 37.00 ± 2 oC to the maximum absorbance without the , addition of heating of the reaction mixtures to completion within ten (10) minutes. The volume of solvent required for complete solubility of the reagent complex was an important factor for optimal performance. The colour formation and development were instantly. To avoid precipitation of the DMF, DMSO and 1, 4-dioxane volume of the final solution, the solvents used must not be below 40.00 % total volume as recommended by [10] and [11]. Method validation: under the optimized conditions, the calibration curves were constructed by plotting the absorbance signal against the concentrations of each analyte subjected according to the general procedure [11]. The solution was transferred into the optical cell of 1.00 cm3 for the measurement of each metal ion spectrophotometrically at the respective absorption maximum against a reagent blank prepared under similar conditions. The calibration graphs follow a straight-line equation Y= ac + b; where c is the concentration of the solution, Y is the measured absorbance, a and b are the constants. The effect of pH on formation the metal- reagent complex was studied based on the method reported by [12], In a series of 10.00 cm3 volumetric flaks, 2.00 cm3 solution (1.00 x10-4 mol/dm3), of the salt of metal of interest, 3.00 cm3 of the hydrazone solution (1.00 x 10-4 mol/dm3) and 4.00 cm3 of the buffer of varying pH were added and made up to the mark with deionized water and the absorbances were measured against the ligand blank at the scanned and recorded wavelength. A plot was then made between absorbance and pH in the UV-Vis range. The pH with the highest absorbance was determined as the working pH for each metal of interest. It was noted that the metal-ligand complexes had higher absorbances at pH between 6.5 - 7.5 which was slightly acidic to almost neutral and was resolved to be the working pH for the study. Comparative Assessment of APDH and AAS Methods (Tables 1–4) The results demonstrate a high degree of correlation between the APDH method and the AAS across all sample types and metals. For lead (Table 1), the measured concentrations via APDH closely matched AAS results, with relative errors ranging from -1.42% to +0.12% and p-values generally above 0.05, indicating no statistically significant difference the values are similar to 1, 2, 5, 6, 9, 11 12 and 21. Similar trends were observed for cadmium (Table 2), chromium (Table 3), and arsenic (Table 4), with relative errors well within the acceptable analytical range (±3.5%) as reported by 1, 5, 8, 11, 14, 15, 19, 21 and 22. The low standard deviations and consistent reproducibility across different matrices (soil, water, plant, and animal tissues) demonstrate the method's robustness and matrix independence. Notably, the APDH method effectively detected low concentrations of heavy metals, such as 0.008 μg/g Cd in well water and 0.010 μg/g As in irrigation water, affirming its sensitivity and potential for trace-level environmental monitoring as observed by 1, 4, 8, 12, 13, 15, 16, 21 and 22. Accuracy and Recovery Studies (Tables 5–8) Recovery studies were conducted by spiking known quantities of each metal (50, 100, and 500 μg/mL) into various sample matrices. The APDH method achieved exceptionally high recoveries ranging from 99.80% to 99.99%, with standard deviations between ±0.1 and ±0.4. These results highlight the method's accuracy and suitability for quantitative analysis as reported by 6, 8, 12, 17, 18, 20 and 21. Recovery across all matrices—animal flesh, liver, blood, plant samples, soil, and water—was consistent, indicating minimal matrix effects. Such matrix independence is crucial for field applicability in diverse environmental and biological samples. The linear response across the concentration range further supports the method's reliability and analytical performance 16,17, 18, 20, 21 and 23. , Analytical Significance and Environmental Implications The APDH-based spectrophotometric method offers several advantages, including cost- effectiveness, operational simplicity, and reduced need for sophisticated instrumentation, making it highly appropriate for use in developing regions or in routine monitoring where AAS or ICP-MS may not be feasible 1, 6, 8, 12, 17, 18, 20, 21 and 23. The elevated levels of lead and chromium in industrial wastewater and soil samples underscore significant environmental pollution, particularly in the Challawa Industrial Area. These findings validate the method's application in environmental risk assessment and contamination monitoring 2, 6, 8, 11, 13, 15, 16, 17, 18, 20, 21 and 23. Applications of the developed methods in the determination of Cd, Pb, Cr and As in soil samples was carried out and reported. Prior to the digestion of the soil samples, each sample was dried at 65°C for 48 hours in the oven. All samples were performed in triplicates. Five grams (5.00 g) of the samples in crucibles was placed in a pre-heated muffle furnace at 200- 250 °C for 30 minutes, and the ashed for four hours at 500-550 °C. Then, the sample was removed from the furnace and cooled down. 2.00 cm3 of 5.0 M of HNO3 was added and evapourated to dryness on a sand bath. Next, about the samples were placed in a furnace and heated to 400 °C for 15 minutes. The samples were removed from the furnace, cooled and moistened with four drops of distilled water. Next, 2.0 cm3 of concentrated HCl was added and the sample was evaporated to dryness, removed, and the 5.0 cm3 2.0 M HCl was again added and the tube was swirled. The solution was filtered through Whatman No. 42 filter paper and the transferred quantitatively to a 50.0 cm3 flask by making it up with distilled water [4]. The Cd, Pb, Cr and As present in the soil samples were determined from the calibrated plots (Beer’s law plot). The results were checked with parallel determination by AAS as the reference method. Statistical analysis of the results by t-test shows that, there is no significant difference in the accuracy and precision of the proposed and reference method [1], [2],[3], [11], [19], [5], [6], [21], [22], [13], [23] and [24]. Conclusion: In conclusion, the present method was applied for the determination of Cd, Pb, Cr and As in different samples. The real samples were water, biological and environmental samples. Satisfactory results were obtained in the analysis. The present method is simple, sensitive, inexpensive, reasonable selective and rapid without the need of heating and extraction. Although many sophisticated techniques such as pulse polarography, High Performance Liquid Chromatography , Atomic absorption spectroscopy , ICP- AES, and ICP-MS are available for the determination of cadmium, lead, chromium and arsenic in numerous real sample materials, factors such as the low cost of the equipment, easy handling, lack of requirement for consumables and almost no maintenance have caused Uv-vis spectrophotometry to remain a popular analytical technique, particularly in laboratories of developing countries with limited budgets. The sensitivity in terms of molar absorptivity and precision in terms of standard deviation of the present methods are very reliable for the determination of Cd, Pb, Cr and As in real samples down to nano-gram per gram (ηg g-1) levels in aqueous medium at room temperature. The comparative and recovery data confirm its equivalence with the AAS reference method. 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