**3. Results and Discussion**

#### *3.1. Brazilin Reference and Brazilwood Pigment*

SERS spectra could be readily obtained from the brazilin reference sample, the main constituent in brazilwood dye sources, utilizing the conventional colloidal SERS approach at pH quasi neutral (Figure 2i) and reported in Table 2. In order to obtain a higher quality SERS spectrum from a reference sample of a brazilwood lake pigment, it was instead necessary to routinely pre-treat the sample with HF acid vapors to liberate the main dye constituent from its inorganic substrate, an action that allows for a greater adsorption of dye molecules onto the SERS active substrate (Figure 2ii). On comparison of these spectra, and on account of the differing SERS experimental procedures, spectral behaviors such as frequency shifts and peak intensity fluctuations can be noticed. Such behavior is due to the varied chemisorption of the dye/hydrolyzed components on the silver colloidal surfaces on formation of a complex. This can lead to a modification of the polarizability and vibrational modes and may also enable charge transfer to occur. The change in intensity of bands in the SERS spectra can be attributed to the relative orientation of the analyte with respect to the silver. A broad background remains observable in both spectra due to residual intrinsic fluorescence, impurities or the SERS continuum of undefined physical origin [21].


**Table 2.** Bands observed in the Raman and SERS spectra (in cm−1, excited at 532 nm) and tentative band assignments. Marker bands for the recognition of each material are given in bold.

*ν:* stretching; *δ:* bending; *γ:* out-of-plane bending; as: asymmetric; and sy: symmetric.

The intense broad band at 1562–4 cm−<sup>1</sup> attributed to aromatic ring vibrations with δ(OH) and *ν*(C=C) contributions together with the ring deformations at 466–7 cm−<sup>1</sup> are present in both spectra and represent the diagnostic SERS bands indicative of colorants in brazilwood. The *ν*(C=C(=O)-C=C) system located at 1377 cm−<sup>1</sup> for the brazilwood pigment is attributed to its brazilein content, and it is unobservable for the brazilin reference which instead has a peak at 1352 cm−<sup>1</sup> attributed to the *ν*(C-O), δ(OCC) and δ(CH2) modes. The peak at 1619 cm−1, apparent in the hydrolyzed lake samples, is possibly again due to brazilein and attributed to *ν*(C=C) and *ν*(C=O) and can also be seen as a weak shoulder (marked with an asterisk) in the brazilin reference. This could in fact indicate a small amount of brazilein in the brazilin reference sample due to natural oxidation. It is likely that the brazilwood constituents are attached to the silver colloidal surface via the C=O and OH groups, similar to the flavonoid class of molecules [23].

Further weak peaks (marked by asterisks in Figure 2ii), which appear at 1515 and 1497 cm−<sup>1</sup> in the brazilin reference and which possibly relate to the *ν*(C=C), are replaced by a small shoulder at 1518 cm−<sup>1</sup> in the hydrolyzed lake. The band at 549 cm<sup>−</sup>1, attributed to the δ(ring), appears less intense in the brazilwood pigment. Other spectral regions instead show pronounced frequency shifts possibly governed by changes in the polarizability of the analyte–metal complexes. The δ(CCH) and *ν*(C-C) bands shift from 1168 cm−<sup>1</sup> in the brazilin reference to 1176 cm−<sup>1</sup> for the brazilwood pigment. The weak 715 cm−<sup>1</sup> band for the brazilin, attributed to the γ(CO) + γ(CH), is shifted to 728 cm−<sup>1</sup> in the pigment. The bands at 1219 cm−<sup>1</sup> (*ν*(C-O) + *ν*(C-C)), 1259 cm−<sup>1</sup> (*ν*(C-O) + *ν*(C-C) + δ(CH2)), 1028 cm−<sup>1</sup> (in-plane δ(CH)) and 676 cm−<sup>1</sup> (γ(CH) + δ(CC-O)) are all instead only observed in the brazilin [13,25,26].

**Figure 2.** SERS spectra of (i) reference sample of brazilin reference (regular colloidal SERS) and (ii) a brazilwood pigment (pre-treated with HF). Reproducible peaks which vary widely in intensity are denoted with an asterisk.

## *3.2. Urolithin C Standard*

Although fluorescence was relatively high, adequate classical Raman scattering of the brazilwood marker component, urolithin C, could be recorded using laser excitation at 514 nm as shown in Figure 3a with main bands highlighted in Table 2. Bands resulting from the phenanthrene skeleton can be tentatively assigned to the *ν*(CC) and *ν*(CO) at 1616 cm−<sup>1</sup> and to the *ν*(CC) and *ν*ass(O-C-O) at 1530 cm−<sup>1</sup> [24]. Bands visible at ~1400 cm−<sup>1</sup> may be due to the *ν*(C-C) and δ(HCC) and the out-of-plane CH modes at 719 cm<sup>−</sup>1. SERS measurements of the same micro-sample of urolithin C reveals two predominant spectra as given in Figure 3b and reported in Table 2. Fluctuations for the bands at 1589 and 1569 cm<sup>−</sup>1, related to *ν*(C=C) + δ(OH) modes, could be observed during spectra collection as there was a constant movement of colloidal/urolithin C particles within the examined aqueous droplet. While this can be related to the common blinking phenomenon, the reproducibility of the spectra could actually indicate a changeable interaction of the hydroxyl groups with the silver surface [27–29].

The bands at 1530, 1167 and very weak 990 cm−<sup>1</sup> (ring breathing vibration) visible in the Raman spectrum are the only ones also featured in the SERS spectra without any great shift. Significant differences in the relative intensity and position of peaks can be observed when the spectra shown in Figure 3a,b are compared. These are the result of SERS surface selection rules and are due to a combination of the orientation of the analyte on the colloidal surface and specific Raman mode symmetry, and very likely in the formation of the surface complex where Raman modes in the molecule disappear on interaction with the surface and instead other modes or new modes may be activated [21].

No bands at higher wavenumber than 1600 cm−<sup>1</sup> are visible under SERS conditions, although there is a large enhancement of the 1530 cm−<sup>1</sup> band (*ν*(CC) and tentatively *ν*assym(OCO)), accompanied by a band at 1335 cm<sup>−</sup>1, possibly attributed to the *ν*symm(OCO). The 1167 cm−<sup>1</sup> SERS band results from *ν*(C-C) and δ(CH). Multiple strong bands at low wavenumbers are due to out-of-plane deformation modes of C=C bonds with a visible δ(C=O) in the range 600–660 cm−1. All this data collectively provides information as to the orientation of urolithin C when chemisorbed or in close interaction with the silver. It is possible that the overall behavior of urolithin C, interacting with the silver colloid via both the carbonyl and hydroxyl functional groups, may be related directly to concentration effects. Possibly at lower concentrations urolithin C can initially orient somewhat flatter, while instead at higher concentrations (near to and above monolayer coverage) it is possibly forced to adsorb end-on due to crowding, resulting in the aforementioned signal fluctuations [30].
