**3. Results**

### *3.1. Selection of the Solvent*

The 41 target phenols present a high polarity range, as can be seen in Table 1, with log KOW values ranging between 0.2 and 3.5. Therefore, their chromatographic separation and response are expected to be highly dependent on the dilution solvent. Different aqueousbased solvents were tested since one of the objectives of this work is the development of a green methodology, reduced usage of toxic solvents.

Experiments were performed employing standard solutions containing the 41 target phenols at 200 μg L−<sup>1</sup> prepared in: methanol (MeOH), acidified water with 0.1% formic acid (AW) and acidified water (0.1% formic acid)/methanol (80:20, *v*/*v*) (AW/MeOH). Results for some target phenols from high polar to low polar ones are shown in Figure 2. As can be seen, both aqueous based- solvents provided the highest chromatographic response for most compounds, especially for the highest polar ones, such as cinnamic- and benzoic- acids derivatives (gallic-, caftaric-, gentisic-, chlorogenic- acid, etc.). In contrast, the use of methanol to prepare the standard solutions for these compounds, resulted in chromatographic responses up to three times lower than those obtained with the aqueousbased solvents. Regarding medium polarity compounds, lower differences were observed for some compounds (α-resorcylic acid, umbelliferone, veratraldehyde, etc.) between the responses for the three tested solvents, whereas others achieved worse response for MeOH (epicatechin, p-coumaric acid, etc.). On the other hand, higher responses were obtained with AW/MeOH and MeOH for the low polar compounds, such as the flavonols quercetin and kaempferol and the flavones apigenin and chrysin.

**Figure 2.** Chromatographic response for some phenols standard solutions (200 μg <sup>L</sup>−1) prepared in AW: acidified water (0.1% formic acid); AW/MeOH: acidified water (0.1% formic)/methanol (80:20, *v*/*v*); MeOH: methanol. \* Divided/10.

As it is well known, the solvent not only affects the chromatographic response (abundance) of the analytes, but also highly affects the retention efficiency and thus, the chromatographic peak shape. Figure 3 shows the comparison between the chromatographic peaks for protocatechualdehyde (Figure 3a) and chlorogenic acid (Figure 3b) (200 μg <sup>L</sup>−1) prepared in AW, AW/MeOH and MeOH.

As can be seen, the standard prepared in methanol presented the worst peak shape, whereas standard prepared in AW and AW/MeOH showed in both cases satisfactory peak resolution. This behaviour was similar for most compounds, especially for those eluting first. Therefore, in view of these results, both aqueous based-solvent solutions were selected for further experiments.
