Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 14, Number 2, October 2025 | Pages: 725-729 | DOI: 10.14421/biomedich.2025.142.725-729 ISSN 2540-9328 (online) Sensitive and Selective Chromatographic Method to Determine Vanadium in Commercial Phosphoric Acid Hassan Pyar1,2*, Abdul Rahman Wahoud3 1Faculty of Environmental Science and Marine Biology, Hadhramout University, Yemen. 2College of Medicine and Health Science, Seiyun University, Yemen. 3Chemistry Department, Faculty of Science, Hadhramout University, Yemen. Corresponding author* hassanpyar@gmail.com Manuscript received: 27 April, 2025. Revision accepted: 07 August, 2025. Published: 01 October, 2025. Abstract The spectrophotometric method based on N-Benzoyl-N-phenylhydroxylamine was executed for ascertaining vanadium in environmental samples. high performance liquid chromatography (HPLC) system equipped with a C18 RP column was used to determine vanadium in wet phosphoric acid and phosphate samples. A rapid pre-column response of vanadium with 5-Br-PADAP was used without prior separation or adding any masking agents. The results showed that, the chelates were detected after being injected through the column by UV- Vis detector at wavelength 585 nm. The molar absorptivity is 5.5 x104 and the retention time is 6.3 minutes. The sensitivity of this reagent is equal to twice as high as the use of (PAR) reagent. The RSD was 1.7% for weak concentrations to 0.22% for high concentrations and the relative error (RE) was between 3.21% and 1.07%. In conclusion the method of the currently study was found highly sensitive and selective and the results show a good accuracy and precision. Keywords: HPLC system; Phosphoric acid; Spectrophotometric method; Vanadium. INTRODUCTION The extraction-spectrophotometric method is utilized with N-Benzoyl-N-phenylhydroxylamine was adopted to evaluate vanadium in environmental samples (Agarwal et al., 1990). Methods based on using of 4-(2-pyridylazo) resorcinol (PAR) which was generally was applied to determine vanadium in biological samples (Bag et al.,1982; Agnihotri et al., 1999), sewage (Buchberger, 2000), silicate rocks (Chakrabarti,1995), petroleum (Fasanmade, 1994) and titanium alloys (Fukasawa et al.,1981; Filik et al., 2008). Vanadium traces in aluminum samples could be determined with a good accuracy. Vanadium was first isolated by organic solvent extraction and then, the absorption of the complex was measured at wavelength 585 nm (Geană, et al., 2019). Many methods that utilize vanadium chelating complexes have been used to determine vanadium such as pyrogallol for soils (George et al., 1990), Hydroxamic acid for steel and rocks (Guangyu et al., 2005; Gustafsson et al., 2019), Crown hydroxamic acid for body fluids (Iki et al., 2001) Phenylfluorone for steel alloys (Jamaluddin et al., 1999), 1,5-diphenylcarbazide for environmental samples and 6- chloro-3-hydroxy-7-methyl-2-(2-thienyl)-4H-chromen-4- 1 for flue dust and waste water samples (Krasiejko et al., 1986; Kubinyi, 1995). These extraction- spectrophotometric methods with different chelating agents were accurate, but they lack selectivity and need prior extraction and separation or masking agent which must be added. Liquid chromatography has an important role in recent years due its use for separation and analysis of various metals such as Mn, Cd, Co, Ni, Zn, and Pb using post-column reaction and UV-V detector at different wavelengths, but they did not mention vanadium in their applications (Matsumiya et al., 2004; Michalski, 2018). Sulfonylcalix[4]arenetetrasulfonate was used and checked for ultra-trace determination of different cations in river and tap water samples by HPLC with UV-Vis detector in an acetic buffer solution (pH4.7) (Pyrzyńska, 2005; Srivastav et al.,1996). Reversed phase liquid chromatography was developed to simultaneously determine of vanadium, copper and cobalt founding in electroplating baths (Vale et al., 2007). This paper purposes a developed method to determine vanadium in phosphate industrial connecting parts by high performance liquid chromatography (HPLC) using the rapid reaction of vanadium with 5-Br-PADAP as a chelating agent in the pre-column system without prior separation or adding masking agents. https://doi.org/10.14421/biomedich.2025.142.725-729 mailto:hassanpyar@gmail.com 726 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 725-729 MATERIALS AND METHODS Commercial wet phosphoric acid samples with 27% wt P2O5 and 1.26 g/cm3 and other samples were supplied by the General Fertilizer Company (GFC) Homs /Syria. Methanol, acetonitrile, absolute ethanol and 2-(5-bromo- 2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) HPLC-grade, his formula as in Fig. 1. All other chemicals employed were of analytical grade. Figure 1. The structural formula of 5-Br-PADAP. Sample preparation 1 ml of 0.1M sodium acetate, 1 ml of 1% sodium dodecyl sulfate, 1 ml of 6 mM of (5-Br-PADAP) and three drops of 1% hydrogen peroxide were added to the sample and standard solutions. Procedures and apparatus A HPLC system connected to the C18 RP column from Shimadzu/Japan was used. The eluent solution consists of methanol, acetonitrile and water with a ratio of 84:12:4 respectively adjusted to pH=5.5 and degassed before use. As a comparison method the samples were diluted and analyzed by atomic absorption spectroscopy (AAS / Varian). The relative standard deviation (RSD%) and relative error (RE%) calculated as below: 𝑅𝑆𝐷% = (100 ∗ 𝑆𝐷)/ 𝑋 𝑅𝐸% = (𝑋 − 𝑋0) ∗ 100/ 𝑋0 Where: SD : standard deviation. X : mean value. X0 : reference value. RESULTS AND DISCUSSION Standard calibration curve: To create standard calibration curve standard solutions, from one to 25 ppm in 40% pure phosphoric acid were pretreated as described in the experimental and injected through the column. The results are plotted in Fig. 2. in the form of the area versus concentration. The relationship is represented by straight line over studied range of concentration. The correlation factor is R= 0.9998. The correlation is represented by straight line equation: Area =188290 C + 3E+06 Figure 2. Standard calibration curve. To determine the relative standard deviation (RSD), four samples of 40% pure phosphoric acid with predetermined concentration of vanadium (10- 25- 50- 100 ppm) were taken. Each sample was measured five times. The results are presented in Table 1. The RSD was of the order of 1.7 % for weak concentrations to 0.22% for high concentrations. The relative error (RE) was between 3.21% and 1.07%. This indicates that it is possible to use the standard calibration curve to determine vanadium concentration in phosphate derivative samples with an acceptable error. This result was similar to Filik et al. (2008). y = 188290x + 3E+06 R² = 0,9998 Area = 188290 C + 3E+06 R² = 0.9998 A re a C ppm Pyar & Wahoud – Sensitive and Selective Chromatographic Method to Determine Vanadium … 727 Table 1. Evaluations of relative standard deviation. Reference value [V] ppm Measured value [V] ppm X  SD (ppm) RE (%) RSD (%) 10 9.78 10.43 10.51 9.88 10.25 10.17  0.33 1.7 3.21 25 25.67 25.54 24.77 25.89 24.23 25.22  0.69 0.88 2.76 50 49.21 50.76 49.51 50.93 50.34 50.15  0.76 0.3 1.52 100 100.98 99.21 100.76 98.91 101.25 100.23  1.08 0.22 1.07 The samples were also analyzed using the atomic absorption spectroscopy method for comparison. The results are presented in Table 2 showing a close approximation between the results of both methods. These results were in agreement with others (Krasiejko et al., 1986; Chakrabarti, 1995; Iki et al., 2001). An Attempt was made to inject samples containing metals such as Zn, Cd, Cr, Fe, Cu, Mn, Co, and U without vanadium (Pyrzyńska, 2005; Wen-yan et al., 2018). No peaks were obtained for chelates for these metals with 5- Br-PADAP because the chelates were unstable in the chromatographic system used in this study and they were decomposed in the mobile phase (Zaki, et al.,1991). Table 2: Vanadium concentrations in phosphate industrial derivatives. Sample HPLC value (ppm) Spectrometric value (ppm) Strong green phosphoric acid 123.7 125 Weak green phosphoric acid 86.5 88.2 Phosphate 110.8 111.5 Determination of vanadium in phosphate industrial derivatives Phosphate and green phosphoric acid samples were pretreated as described in the experiment and injected through the column. A typical chromatogram for these samples has two sharp and resolved peaks (Fig. 3). This result was in the same line with Zhao and Fu (Zhao et al., 1991). The first peak showed at retention time of 6.3 minutes which indicates to vanadium chelate and another peak appeared at retention time of 10.3 minutes which indicates 5-Br-PADAP reagent (Zhao et al.,1991). Figure 3. Typical sample chromatogram. 728 Biology, Medicine, & Natural Product Chemistry 14 (2), 2025: 725-729 CONCLUSIONS The proposal method was applied for a wide range of concentrations of phosphate industrial derivatives and different aqueous media without any need to use masking agent or prior separation of metal ions. The method doesn't consume rather expensive post column reagents like the other method but the sample needs only simple pretreatment. Clearly, proposed method is practical and selective for vanadium, and the sensitivity is double as high as in the use of (PAR) reagents. The results show good accuracy and high precision. Acknowledgment: The authors are indebted to Hadhramout University for their valuable help, also would like to express their thanks and appreciation to General Fertilizer Company (GFC) for supplying catalyst samples. Competing Interests: All authors state that there are no competing interests. REFERENCES Agarwal C., M. Deb & Mishra, R. )1990). 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