# Infrared Spectroscopy for Studying Structure and Aging Effects in Rhamolipid Biosurfactants
## 3 Results  
As natural products, rhamnoids are a class of compounds rather than a pure substance. Key characteristics of a sample are the number of rhamnose and carboxylic acid/ester units. Figure 1 illustrates the structures of four common compounds contained in a rhamnoid biosurfactant. The most common rhamnoids comprise of one or two rhamnose units and one or two fatty acid chains with C10 or C12 chain length. The distinction between mono- and di-rhamnolipids is the most important task for an analytical tool [21; 22]. Figure 2 displays the spectra of the five different samples obtained from different fermentation experiments. Note that the diamond crystal has very low transmission between 1900 and 2300 cm\({}^{-1}\); hence, the spectra exhibit experimental artifacts rather than meaningful signatures in this range. Most of the rest of the spectrum can be interpreted in a straightforward manner and Table 1 summarizes the observed signatures. The broad band between 3700 and 3100 cm\({}^{-1}\) is due to O-H stretching vibrations. The range 3100-2700 cm\({}^{-1}\) contains the C-H stretching modes. The fingerprint region, i.e., below 1750 cm\({}^{-1}\), is very rich in information. At 1707 cm\({}^{-1}\) the C=O stretching mode of the carboxylic acid groups can be found. The high-wavenumber wing of this band shows a shoulder bands at  
-1740 cm\({}^{-1}\), which can be attributed to C=O stretching of the ester groups. At lower wavenumbers, the C-H bending; C-C and C-O stretching; and a multitude of rocking, scissoring, wagging, and twisting modes can be found. A detailed assignment is rather complicated as all these modes can be overlapping with each other, and hence the assignment in Table 1 is a tentative one. However, as aforesaid, the main task is to distinguish between the different classes of compounds. Therefore, we focus on the differences of the spectra. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**R1** & **R2** & **R3** & **R4** & **R5** & **Assignment** \\ \hline \multicolumn{6}{c}{v/cm\({}^{-1}\)} \\
3274 & 3274 & 3288 & 3288 & 3285 & OH str \\
2954s & 2954s & 2954s & 2954s & 2954s & CH\({}_{3}\) asym str \\
2926 & 2926 & 2926 & 2927s & 2926 & CH\({}_{2}\) asym str \\ - & - & - & 2916 & - & CH\({}_{2}\) asym str \\
2871s & 2871s & 2871s & 2871s & 2871s & CH\({}_{3}\) sym str \\
2855 & 2855 & 2855 & 2850 & 2855 & CH\({}_{2}\) sym str \\
1740s & 1740s & 1740s & 1740s & 1740s & C=O str ester \\
1707 & 1707 & 1707 & 1707 & C=O str acid \\
1669 & 1669 & 1647 & 1647 & 1647 & OH bend (residual water) \\
1592 & 1592 & 1590s & 1591s & 1591s & COO asym str \\
1527 & 1527 & 1527 & 1527 & 1527 & Residual CHCl\({}_{3}\) \\
1484 & 1484 & 1484s & - & - & Residual HCCl\({}_{3}\) \\
1442 & 1442 & 1442 & 1442 & 1442 & CH\({}_{2}\) sciss \\
1380 & 1380 & 1380s & 1380s & 1380s & COO sym str \\
1292 & 1292 & 1297s & 1301s & 1301 & CH\({}_{2}\) wag, COH bend \\
1276s & 1277 & 1277 & 1274 & 1273 & CH\({}_{2}\) wag, COH bend \\
1241 & 1242 & 1242s & 1242s & 1242s & \\
1214 & 1214 & 1207 & 1206 & 1206 & Residual CHCl\({}_{3}\) \\
1155 & 1155 & 1155 & 1156 & 1156 & CH rock \\
1120 & 1120 & 1121 & 1121 & 1121 & CO str, CH rock \\
1032 & 1032 & 1031 & 1031 & 1031 & CO str \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Center wavenumbers of observed IR peaks in cm\({}^{-1}\) and their tentative assignment. Some of the bands in the fingerprint region are not assigned due to possible ambiguity. s = shoulder, sym = symmetric, asym = anti-symmetric, str = stretching, bend = bending, sciss = scissoring, wag = wagging, rock = rocking. Figure 1: Chemical structures of four typical rhamnolipid compounds. The spectra of the five samples suggest that there are two groups of spectra exhibiting significant similarities: (1) R1 and R2; and (2) R3, R4, and R5. This visual observation has been confirmed by a principal component analysis.