Rhamnolipids Characteristics, Production, Applications, and Analysis - Biosurfactants: Research Trends and Applications - page 92

For separating the congeners in the mixture, Koch et al. (1991) used a mixture of
methanol, water, and trifluoroacetic acid with ratios of 90, 10, and 0.25, respectively.
With their method, rhamnolipid congeners with different lengths of alkyl chains
were separated. After that, with the orcinol test, using reagents such as the ceric
dip, they can be analyzed further (Abdel-Mawgoud et al., 2011). The original TLC
method has been evolved to a new generation of chromatography method, the high-
performance thin layer chromatography.
Liquid Chromatography
Liquid chromatography (LC) is another chromatographic method that uses a mobile
phase (solvent) moving through the stationary phase, which could be silica gel par-
ticles in a column or a flat surface. The advantage of this method is accuracy, and it
needs only a small amount of sample (Allwooda and Goodacre, 2010; Ardrey, 2003).
The more advanced generation of this method is the high-pressure liquid chroma-
tography (HPLC), which has been used extensively in laboratories around the world.
High-Pressure Liquid Chromatography
High-pressure liquid chromatography or high-performance liquid chromatography
(HPLC) is one of the most used analytical methods in the laboratory today. The
accuracy, the ease of use, and its ability to perform analysis with a small amount of
sample make it one of the most useful tools in laboratories. In HPLC, the sample and
mobile phase are moved with high pressure through the stationary phase column, a
column packed with spherical particles. In reversed-phase liquid chromatography,
mobile phase is a polar solvent like water and a less polar solvent like methanol
and stationary phase is made by a column packed with octadecylsilyl (C 18 ). In the
normal-phase liquid chromatography, the mobile phase is a less polar solvent such as
toluene, and the stationary phase column is a column packed with spherical particles
such as silica gel particles. Through penetration of the mobile phase through station-
ary phase, molecules in the samples are separated based on their hydrophobicity.
For example, nonpolar functional groups are attracted by the silica particles and are
bonding with them (Ardrey, 2003; TDMU, 2012).
HPLC is one of the most useful methods for analyzing rhamnolipids. Mata-
Sandoval et al. (1999) reported that they developed an HPLC method for quantifying
rhamnolipid species. According to Abdel-Mawgoud et al. (2011), for analyzing the
rhamnolipids with HPLC, a column packed with C 8 or C 18 was used as the stationary
phase and water and acetonitrile as the mobile phase or para-bromoacetophenone.
The detection was at a wavelength of 265 nm. Mata-Sandoval et al. (1999) stated that
to confirm the data from the HPLC, they used MS for further analysis.
Liquid Chromatography Coupled to Mass Spectrometry
According to Van Bramer (1997), MS is an accurate technique for analyzing the
molecular structure of materials. This technique helps in accurately identifying and
quantifying the component of a mixture. In MS, sample is under vacuum in the
source region, where they are ionized. Charged particles of the sample pass through
a magnetic field to be separated based on their mass-to-charge ratio. After separating
ions, they go through a detector and then data are sent for analysis.