Aniline is a core raw material for manufacturing dyes and pharmaceuticals, yet it exhibits significant toxicity: upon entering the human body, it may cause acute poisoning, leading to methemoglobinemia and damage to the liver, kidneys, and skin. Long-term exposure to low concentrations can also result in chronic poisoning, inducing toxic liver disease. Currently, common methods for determining these two substances include spectrophotometry, fluorophotometry, variable-angle fluorometry, and high performance liquid chromatography with ultraviolet detection (HPLC-UV). In this study, a method combining HPLC separation and high-selectivity fluorescence detection was adopted. By optimizing separation conditions and integrating wavelength-time switching technology for detection, simultaneous determination of aniline and phenol was achieved without sample pretreatment. This method, which has not been previously reported, can be directly applied to the simultaneous analysis of aniline and phenol in environmental water bodies.
- Experimental Section
1.1 Instruments and Reagents
(1) Instruments: LC600A Series High Performance Liquid Chromatograph (Nanjing Kejie Detection Technology Development Co., Ltd.); solvent filtration system; ultrasonic generator.
(2) Reagents: Methanol (HPLC grade); aniline, phenol, potassium dihydrogen phosphate (KH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄) and other reagents (all of analytical reagent grade, AR); deionized double-distilled water was used as experimental water.
1.2 Experimental Methods
1.2.1 Preparation of Standard Solutions of Aniline and Phenol
Freshly distilled aniline and phenol were separately dissolved in deionized double-distilled water to prepare standard stock solutions with a mass concentration of 1.0 mg/mL, which were stored in a refrigerator under light-proof conditions. For use, two dilution methods could be selected: ① Dilute the single-standard stock solutions separately to the target concentrations; ② First prepare a 1:1 (v/v, volume ratio) mixed standard stock solution, then dilute it to the required mass concentration.
1.2.2 Sample Determination
Aliquots of the 1.00 mg/mL aniline and phenol standard stock solutions were pipetted to prepare a series of mixed standard solutions with mass concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 μg/mL. Under the selected chromatographic conditions, the standard solutions were injected for determination, and the standard curves of aniline and phenol were plotted respectively.
For water samples, suspended solids were first removed (if the water sample was turbid, it was centrifuged and the supernatant was collected) before direct injection for determination. The external standard method was used for quantification, with an injection volume of 20 μL for all samples.
- Results and Discussion
2.1 Selection of Separation Conditions
The fluorescence intensities of aniline and phenol showed significant differences under different pH conditions, with the core reason being that their chemical structures and dissociation properties directly affect their fluorescence characteristics. To optimize the separation effect, multiple mobile phases were screened and their pH values were adjusted in the experiments. Finally, it was determined that when pH 6.87 phosphate buffer-methanol (50:50, v/v) was used as the mobile phase, both aniline and phenol could generate strong and stable fluorescence signals.
A comparative analysis was conducted between the Eclipse XDB-C8 column and the conventional ODS column. The results showed that when the Eclipse XDB-C8 column was used, the resolution of aniline and phenol was higher, and the symmetry of chromatographic peaks was better, which could meet the requirements of simultaneous detection. The chromatograms of standard samples and industrial wastewater samples are shown in Figure 1.
2.2 Water Sample Analysis
In accordance with the above experimental method, aniline and phenol in three types of actual water samples (tap water, urban river water, and untreated wastewater from a chemical plant) were determined. The results were compared with the determination data obtained by the traditional fluorophotometry to verify the accuracy of the proposed method. The specific determination results are shown in Table 1.