*3.2. Extraction Method*

The analysis of the plant extract was done by HPLC-DAD-HRMS. It was possible to find as a major chromophore Lut-7-*O*-glu, and minor compounds, Api-6,8-*C*-glu, Lut-di-*O*glu, Lut-3 ,7-*O*-glu; Api-7-*O*-glu, Chry-gly, Lut-4 -*O*-glu, Lut and Api, see Figure 2. This is in accordance with what has been reported in the literature [6,8,9]. It was confirmed that the chromophores identified by HPLC-HRMS were the same as observed by HPLC-DAD.

For both *WL1* and *WL2* recipes, potassium bicarbonate is added to the plant material' solution, raising the pH to a neutral media (pH ~6). For the rest of the recipes, potassium carbonate was added previously to the weld, also resulting in a neutral media (pH ~6), except for the *WL3* recipe that remained at a basic media. Although the recipes present two different extraction methods, they do not influence the chromophores extracted as the chromatographic profiles are similar, see Figure 2 and Figure S1.

It is very interesting the use of carbonates (KHCO3 and K2CO3) for the extraction of the flavonoids. The use of such extract solutions instead of water was possibly to allow the highest amount of lake pigment. Favaro et al. used fluorimetric titration to characterize the various luteolin species detected within the pH range explored (pH = 2–12) [28]: neutral form (pH < 5), mono-anion (pH ~7), di-anion (pH ~9) and tri-anion (pH ~12), and the successive deprotonations occur in the order 7-OH; 4 -OH; 3 -OH or/and 5-OH [28]. In *WL3*, *WL4* and *WL5* the extraction is carried out in a basic pH, turning neutral after the addition of the plant material. This creates the optimum conditions for the metal chelation through the OH at C<sup>5</sup> and the carbonyl at C4, since the first is deprotonated only at pH ≈ 10.3, as mentioned above.

#### *3.3. Characterization of the Weld Lake Pigments*

A summary of the multi-analytical results of Colorimetry, High-Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) and Fourier Transform Infrared Spectroscopy (FTIR) for the weld lake pigments prepared may be observed in Table 3.

#### 3.3.1. HPLC-DAD Analysis

Other authors have done an extensive analysis of the characterization of weld by HPLC, including quantitation of the chromophores [6,8–10,29,30]. Based on this, in this work, we only compared the chromatographic profiles of the lake pigments using HPLC-DAD, which is preferable to perform a semi-quantification.

When analyzing the HPLC chromatograms of the weld lake pigment extracts, some differences are visible, as shown in Table 3. The two variants of recipe *Yellow from Weld* (*WL1* and *WL2*) present the same chromatographic profile, indicating that the order in which alum and calcium carbonate are added does not affect the chromatographic profile, i.e., the percentage of chromophores present, as seen in Table S2. However, when compared with the extract, in Figure 2, it is possible to see that both lake pigments present a higher percentage of Lut-3 ,7-*O*-glu than the plant extract (15.35–17.25% in the lake pigment when compared with 4.92% of the extract). Considering that the extracts in K2CO3 and KHCO3 presented the same chromatographic profile as in MeOH:H2O (see Figure S1), the difference is not due to different extraction methods, but possibly to a higher preference of complexation for the di-glucoside. Interestingly, this difference is even higher in the lake pigment from *WL3* and *WL5*, where the Lut-3 ,7-*O*-gluc represents 20.91% and 23,05% of the total peak area, while the Lut-7-*O*-glu represents 30.26% and 18.63%, respectively. *WL3* is the only recipe with the addition of alum to an extraction solution of *Reseda luteola* at a basic pH of around 9. Moreover, *WL5* also has a higher percentage of Api-7-*O*-glu, representing

11.22% of the total peak area. *WL5* is the only recipe where alumina is added. Regarding the recipe *WL4*, it has the closest chromatographic profile to that of the extraction, with 13.17% of luteolin 3 ,7-di-*O*-glucoside and 52.551% of luteolin 7-*O*-glucoside.

### 3.3.2. FTIR Analysis

Both yellow lake pigments from the recipe *Yellow from Weld* (*WL1* and *WL2*) have shown similar FTIR results as observed by HPLC-DAD. Notably, the formation of gypsum (CaSO4·2H2O) by FTIR was detected, due to its characteristic absorption bands for νOH at 3405 cm<sup>−</sup>1, νas(SO4 <sup>2</sup>−) at 1132 cm−<sup>1</sup> and δas(SO4 <sup>2</sup>−) at 670 cm−<sup>1</sup> [31], as observed in Table 3. Gypsum was not directly added but was rather a product of the reaction between alum (KAl(SO4)2) and calcium carbonate (CaCO3). The reason why W&N chose to create gypsum through a reaction rather than adding it directly is still unclear at the moment. Further experimentation will be performed using gypsum directly in the recipe to understand the role of this reaction.

As may be seen in Table 3, FTIR analysis of all yellow lake pigments shows bands attributed to flavonoids-metal complexes, which is very clear in the infrared spectra of *WL3* and *WL4*, where only alum was added, plus borax in the latter recipe; the role of this ingredient is also still to be investigated. A more thorough analysis of the infrared data of the flavonoids-metal complexes is offered below. Besides identifying gypsum in *WL1* and *WL2* pigments, it was also detected the presence of hydrated alumina in the *WL5* lake pigment, due to its characteristic absorption bands for δ(H2O) at 1485 cm-1 and the δ(Al-OH) at 852 cm−<sup>1</sup> [31], as shown in Table 3.
