To analyse the structural changes of EB involved in conductance of different cations, we continued with a tBLM system in which EB was incorporated in the presence of Na + (after at least 1 h of incorporation) and exchanged ions to K + , Cs + , Li + , Mg 2+ and Ca 2+ (Figure ). Ion exchange with Na + , K + and Cs + showed a reversible behaviour (Figure ), as apparent by an approximately constant (or increased) SEIRA intensity, so that these experiments were performed on the same tBLM. However, Li + , Mg 2+ and Ca 2+ showed irreversible changes (Figure ) that decreased the absorbance of EB and led to much less defined spectral features than in Na + , K + and Cs + (see enlarged spectra in Figure ). EB+Na + and EB+K + SEIRA spectra from ion exchange experiments show same peak positions (1746/1658 cm -1 and 1747/1665 cm -1 , respectively) and line shapes as during the incorporation (Figure ) pointing to a completed replacement of these ions in EBn:M + complexes in the membrane (the same is true for Cs + , see Figure ). At the same time, the absolute SEIRA intensities are inverted. EB incorporation in presence of K + led to 3 times lower intensities than in Na + , whereas here exchange of ions from Na + to K + induces an increase in absorbance by 1.4 of both ν(COester) and ν(COamide) modes. This effect can be ascribed primarily to an increasing surface enhancement of the IR signal with proximity to the SEIRA Au surface, and as such to a relocation of EB towards the electrode-facing side of the tBLM, or possibly to a more favourable orientation of the ester and amide C=O groups with the surface selection rule. Interestingly, this change of intensity is in line with the previously reported preference of binding K + over Na + by a factor of 1.4 and thus may directly reflect the extent of binding (note that the similarity of both numbers is coincidental). Exchange to Cs + only leads to minor changes in frequency (1746/1659 cm -1 ), more clearly demonstrated in the difference spectrum, and increases intensity by 1.2 in comparison to the spectrum in presence of Na + . The associated spectral changes are more clearly visible in the difference spectra when exchanging from Na + to K + or Cs + , where the difference peak of the amide mode displays a positive lobe at slightly higher frequencies of 1666 or 1660 cm -1 , respectively. The v(COester) shows a slight blueshift and an increase in intensity. Previously, Ovchinnikov et al. have determined IR spectra of EBn:M + complexes to identify specific complexes in acetonitrile as solvent. Surprisingly, in their work IR spectra of Figure . SEIRA spectra of membrane-incorporated EB after buffer exchange from Na + to K + , Cs + , Li + , Mg 2+ , and Ca 2+ (from top to bottom in both A and B). A: Black lines show spectra after ion exchange; grey filled spectra represent the difference spectra vs. the spectrum in presence of Na + . Exchange between Na + , K + , and Cs + was reversible, such that spectra from a single experiment are depicted (see Figure ). Due irreversible behavior in Li + , Mg 2+ , and Ca 2+ , spectra were obtained from individual experiments and are scaled such that the spectrum in Na + prior to buffer exchange had similar intensities. B: Peak fit to spectra in A with red lines showing the overall fits, and red, blue and orange filled features depicting band shapes (red, blue and yellow peaks are assigned via comparison to DFT-based spectra to the species shown in Figure ). EB with Na + , K + and Cs + did not show any considerable difference (1746-1744/1666-1665 cm -1 ) while the very similar beauvericin (with phenylalanine instead of valine) displayed much more significant changes. Apart from a solvatochromic effect due to different environments, it is obvious that EB behaves differently when incorporated into a membrane than in an organic solvent, so that the structural assignment to complexes of different stoichiometry must be revisited.