The discrepancies between our SEIRA data and previous results of EB-cation complexes in acetonitrile in presence of Na + and Cs + (i.e. the frequencies of amide modes at 1658 and 1659 cm -1 vs. 1665 and 1666 cm -1 in our results vs. those of Ovchinnikov , respectively) indicate different species being adopted in membranes compared to the EB2:M + as proposed in acetonitrile. We aim to assign the species observed in our SEIRA spectra by comparison to the set of spectra obtained using DFT. First, we can exclude the presence of EB:Na + in (despite being proposed previously) or free EB in our SEIRA spectra in Na + , based on the absence of noticeable signals between the dominant v(COester) and v(COamide) bands at ~1740 and ~1660 cm -1 (see Figure and Figure ). Further, in Figure and Figure we noted that SEIRA spectra in presence of Na + showed redshifts of ester and amide bands by ~ -1 and ~ -7 cm -1 , respectively, when compared to K + and washing with fresh buffer led to an additional change observed in amide range (Figure , difference spectrum in grey). Satisfying both observations, we can propose the distorted EB2:Na + complex at/in the membrane as a main species (red peaks in Figure and spectra Figure ), which coexists with an additional fraction of EB:Na + out, (blue peaks in Figure and spectra Figure ). This interpretation explains the broader band shape in comparison to the spectra in K + . In Figure , we show that a 2:1 mixture of the DFT-IR-spectra of the distorted EB2:Na + and EB:Na + out can satisfactorily reproduce the SEIRA spectra and the differences with respect to spectra in K + .