The time evolution of both ester and amide signals during EB incorporation is shown in Figure . Comparing both traces one can observe major differences between EB's behaviour with Na + and K + . Accordingly, after an initial burst of binding that decays rapidly, a plateau is reached immediately or develops gradually in the presence of Na + or K + , respectively. However, after ca. 30 min only in presence of Na + an unexpected and pronounced sigmoidal increase occurs, which provides evidence for a distinctly different mechanism of EB incorporation. After a constant amide intensity was reached at ca. 1.3 mOD and 0.7 mOD (or 60⋅10 -3 and 25⋅10 -3 according to integrated intensity as shown in Figure ) with Na + and K + , respectively, the buffer was replaced to remove residual EB in the bulk, which led to a decrease of the EB signals to roughly 65 % of the intensity before the wash in both cases. This observation indicates a substantial amount of surface-bound EB on the membrane interface during incubation. At the same time, the washing step leads to slight changes of both bands in presence of Na + , where the v(COamide) shifts from 1659 cm -1 to 1658 cm -1 as appreciable more clearly in the difference spectra (grey spectra; after scaling to similar absolute intensities). Since the amide C=O groups are more likely to coordinate the ions, this shift indicates the existence of a mixture of different EBn:Na + species when covering or incorporated into the membrane. In the case of K + no changes are observed to the amide band, indicating only one species or a mixture of species with maintained composition. Less pronounced changes are detectable only for the ester mode with both ions. The difference band centered around 1740 cm -1 can be assigned either to minor changes of the lipid's C=O stretches or can originate directly from solvatochromic shift (hydrophobic membrane core vs. polar head groups) of EB's v(COester) modes upon removal of solvent exposed EB:K + complexes. Incorporation in the presence of Cs + showed a similar behaviour to K + and is discussed in the SI (Figure ).