Supercritical fluid chromatography (SFC), also called ultra-performance convergence chromatography (UPC2), working with supercritical carbon dioxide as the eluant for compound separation, in combination with q-ToF mass spectrometry for compound identification, is used for the analysis of extractives from all types of biomass, lignocellulosic samples and products. The technique is advantageously combined with supercritical fluid extraction beforehand, in a way that scCO2 acts both as solvent for extraction and eluant for the subsequent chromatographic separation and mass spectrometric structure determination.
In the ALICE core facility, supercritical fluid chromatography (SFC) is available as particularly powerful separation technique. Introduced by Waters® in 2012, UPC2 is able to use subcritical or supercritical CO2 as the main eluant which can – according to polarity requirements – be modified by small amount of polar organic modifiers (co-solvents), such as methanol or acetonitrile. Our supercritical fluid chromatography (SFC) unit is hyphenated to two different detectors: a photodiode array detector (PDA) for UV-visible active molecules and a high-resolution quadrupole time-of-flight mass spectrometer (HR-QToF-MS). The QTof system is able to ionize molecules coming from ultra-high performance supercritical fluid chromatography without precipitation of the analytes when CO2 is released at atmospheric pressure. Electrospray ionization (ESI) in positive and negative mode permits the ionization of more polar molecules solvated by the eluant used in the chromatographic gradient or auxiliary solvent added in a second stage by an isocratic pump. Corona pin in the ESI-source can be installed for simultaneous recording of atmospheric pressure chemical ionization (APCI) chromatogram together with ESI, in the so-called ESCi® mode. Other ionization modes are available such atmospheric pressure photo ionization (APPI) and desorption electrospray ionization (DESI).
The advantages to use SFC are manifold. Simplicity, similarity and orthogonality together with elimination of large volume of toxic organic solvent to be disposed (up to 75 times less solvent compared to a standard chromatographic HPLC system) and reduction of time of analysis up to 30 times, increased selectivity, higher reliability, robustness technique and last but not least 100 times lower cost of operation. The separation power equals that of a normal-phase medium, with the same ease of use of a reversed phase.
Compound separation and – in particular – identification is supported by a broad varieties of compounds collected in a huge and quite unique home-made library of lignocellulosic extractives (up to 5000 compounds). The work flow of sample preparation by extraction with scCO2, dilution with scCO2, injection into the UPC2 system working with the scCO2 eluant followed by ionization and detection of the molecules of interested by ESCi® (or only ESI) supported by open-source software (MZMine 2) for data-managing is an especially powerful sequence (see the figure lelow). Method optimization based on different stationary phases and combination of co-solvent and additives for optimum ionization is readily perfomed. Putative molecular structures are identified either by the monoisopic masses (error < 5 ppm) and confirmed by fragmentation of parental peaks by MS/MS.