# Infrared Spectroscopy for Studying Structure and Aging Effects in Rhamolipid Biosurfactants
## 3 Results
Four features highlighting spectral differences are marked by arrows in Figure 2. The most distinguishable signatures that are present only or at least significantly stronger in R1 and R2 can be found at 1592, 1484, 1380, 1241, and 850 cm\({}^{-1}\). The band at 1592 cm\({}^{-1}\) is likely due to the anti-symmetric stretching of deprotonated carboxylate groups. While R1 and R2 exhibit a distinct peak, there are weak shoulder bands at this position. The same observation is made at 1380 cm\({}^{-1}\), which can be attributed to the corresponding symmetric mode. The other bands that appear differently for R1 and R2 and R3-5 are related with CH deformation vibrations and hence it is difficult to assign them unambiguously to specific structural differences without involving further analytical techniques. Further differences between R1 and R2 and R3-5 are found at high wavenumber. The O-H stretching band around 3300 cm\({}^{-1}\) is stronger in R3-5. The latter indicates that the R1 and R2 samples contain a lower number of rhamnose units. Probably, mono-rhammolipids are dominating here while di-rhammolipids are mainly present in R3-5. The presence of mainly di-rhammolipids was also suggested by reference measurements with mass spectrometry, see Table 2. The enhanced IR signatures at around 1600 and 1240 cm\({}^{-1}\) in the R1 and R2 samples can be attributed to ester groups indicating that these samples contain more fatty acids than R3-5. Hence, we can conclude that the IR spectrum is a suitable indicator of the structure and of the number of rhamnose units. \begin{table}
\begin{tabular}{c c c c c c} \hline
**R1** & **R2** & **R3** & **R4** & **R5** & **Assignment** \\ \hline  & \multicolumn{4}{c}{\(\sur\)cm\({}^{-1}\)} & \\
981 & 981 & 983 & 983 & 983 & \\
964 & 964 & 965 & 965 & 966 & \\
954s & 954s & - & - & - & \\
918 & 918 & 917 & 917 & 918 & \\
849 & 849 & 849 & 850s & 850s & \\
818 & 818 & 818s & 818s & 818s & \\
809 & 809 & 809 & 809 & 809 & \\
752 & 753 & 755 & 756 & 756 & residual CHCl\({}_{3}\) \\ - & 735s & - & - & - & CH rock \\
724s & 723s & 724 & 724 & 724 & CH rock \\
700 & 701 & 701 & 701 & 701 & CH rock \\ \hline \end{tabular}
\end{table} 
Table 1: _Cont._  
Figure 2: IR spectra of the five rhamnpled samples. The arrows mark spectral position where significant changes can be observed. In order to analyze whether or not ATR-IR spectroscopy can be used to study aging effects as well, a second set of spectra was recorded after five months. During this period, the samples were kept at room temperature (298 K) but in darkness to avoid additional effects due to photo bleaching. Figure 3 shows the difference spectra between the initial and aged samples. Consequently, positive contributions mean that the signature reduced in absorbance over time; negative contributions mean the opposite. As there are clear changes in the spectra, it can be concluded that aging takes place. On the other hand, there are only a few signatures in the difference spectra that are similar in all five cases, e.g., the positive double peak in the CH stretching region. The initial spectra exhibit four main peaks in this range: 2954, 2924, 2871, and 2854 cm\({}^{-1}\). The two peaks in the difference spectra are at 2915 and 2850 cm\({}^{-1}\). The 2954 and 2924 cm\({}^{-1}\) peaks can be assigned to the anti-symmetric stretching modes of the CH\({}_{3}\) and CH\({}_{2}\) groups, respectively. The peaks at 2871 and 2854 cm\({}^{-1}\) are due to the corresponding symmetric modes. The observed behavior indicates a decrease in the number of CH\({}_{2}\) groups. The dissociation of the fatty acid chains can explain this. Further support for the hypothesis that the fatty acid part of the rhamnoids is mainly affected during the aging is provided by the negative contribution around 1700 cm\({}^{-1}\). In this region, the C=O stretching modes of the ester groups can be found. The spectra suggest that these groups are increasing during aging. This could be interpreted in terms of an ongoing esterification reaction.