Hp 1100 series liquid chromatograph

The quantification
of the target
pollutants in water samples was performed by liquid chromatography
coupled to a UV detector (HP1100 series liquid chromatograph (Agilent,
Palo Alto, CA), which provides good sensitivity levels as it is indicated
in Table S1. The chromatographic separation
took place on an Agilent Eclipse XDB-C18 column (150 mm × 4.6
mm i.d. × 5.0 μm) (Waltham, MA, USA) using a flow rate
of 1 mL min^–1 for the mobile phase. A gradient elution
mode was used. The initial mobile phase’s composition consisted
of 70% component A (water) and 30% component B (ACN). The percentage
of component B was linearly increased from 30 to 70% (from 4 to 7
min) and then decreased again to 30% (from 7 to 8 min) for column
re-equilibration. 20 μL of solutions was automatically injected
by an autosampler, and the pollutants were measured at 280 nm.

Malic acid, ascorbic acid, and citric acid determination was carried out according the method described by Scherer et al. (15 ). An HP 1100 series liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a degasser, a quaternary pump, an autosampler adjusted to deliver 20 μL injections, an RP-C18 column (5 μm particle size, 250×4.6 mm I.D., kept at 25°C), and a UV-visible diode array detector. The mobile phase consisted of a 25 mM KH₂PO₄ buffer solution (pH=2.60 adjusted with o-phosphoric acid). An isocratic elution procedure with a flow rate of 0.8 mL/min was used. Malic acid and citric acid were detected at 210 nm, and ascorbic acid was detected at 250 nm. Peaks of interest were identified by their retention times. Peaks observed on the UV-visible spectra of each sample were compared to peaks observed on the UV-visible spectra of known standards. A five-point external standard curve was used to quantify each analyte. The concentrations of the standard curves ranged from the quantification limits and 0.25 mg/mL, 0.5 mg/mL, 0.01 mg/mL, and 0.8 mg/mL for malic, ascorbic, and citric acids, respectively. The linearity range of each analyte was evaluated by plotting the peak area, as a function of the known concentration of each standard.