Spectroscopy / Chromatography / Plant Cell Cultures / Synthesis

Spectroscopy:

While we use UV/vis and fluorescence spectroscopy mostly as detection tools for liquid chromatography separations and less often for compound identification, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are used for both identification and quantitation. Our mass spectrometers are coupled to liquid and gas chromatographs allowing for the identification and quantification of volatile and non-volatile compounds. Regarding our liquid chromatography-MS couplings, a linear ion trap mass spectrometer is mostly used for characterization purposes (MSⁿ) of oligosaccharides and other natural products, whereas a triple quadrupole mass spectrometer is used for quantitation purposes, often in the multiple reaction monitoring mode, also applying stable isotope dilution techniques. Our gas chromatographs are coupled to single quadrupole mass analyzers. Different from MS, NMR spectroscopy allows for unambiguous, often independent structural characterization. One- and several two-dimensional (e.g. H,H-COSY, NOESY, TOCSY, HSQC, HSQC-TOCSY, HMBC) (and, if necessary, three-dimensional) NMR-experiments are ideal tools for a detailed structural characterization of both cell wall polymers such as non-starch polysaccharides and lignins and low-molecular weight phytochemicals. In addition, we strive to apply and develop NMR approaches for (semi) quantitative purposes in food chemistry and phytochemistry.

Chromatography:

We use a wide range of available chromatography techniques for the separation of our target molecules: reversed-phase (RP) and normal phase (NP) chromatography, hydrophilic interaction chromatography (HILIC), ion chromatography (IC), size exclusion/gel permeation chromatography (SEC/GPC), and gas chromatography (GC). RP-high-performance liquid chromatography (HPLC) using mostly standard stationary phases is used for the separation of most phytochemicals of interest on both analytical and preparative scale. Specific (more polar) phytochemicals may require the application of HILIC. Our routine carbohydrate analysis (for example, monomers of cell wall polysaccharides) is performed by applying high-performance anion exchange chromatography (HPAEC) coupled to a pulsed amperometric detector (PAD). SEC and GPC (low and medium pressure) is often but not exclusively used for the separation of oligosaccharides and polysaccharides as well as hydroxycinnamic acid oligomers etc. Standard applications of our NP-flash chromatography system include the purification of synthetic raw products. GC is most often used after analyte derivatization, for example to analyze partially methylated alditol acetates (PMAAs) as diagnostic end products of the methylation analysis to study glycosidic linkages. In addition, we use more specialized stationary phases such as porous graphitic carbon for selected applications. Our liquid chromatographs are coupled to various detectors such as photodiode array, fluorescence, evaporative light scattering, refractive index, and PAD detectors as well as to mass spectrometers.

Plant Cell Cultures:

Plant cell cultures are widely used for entirely different purposes such as the large scale production of phytochemicals to be used as, for example, pharmaceuticals, for plant genetic engineering or to study biochemical pathways in the plant. Because cultured plant cells proliferate indefinetly in an in-vitro system and because biological processes can be studied under reproducible and easy to manipulate conditions plant cell cultures are an ideal resource in plant chemistry research. Callus cultures, which represent an amorphous mass of parenchyma cells growing on the surface of solid culture media, grow slowly an are used in our lab to maintain our plant cell lines for long period of time. Cell suspension cultures, represented by suspensions of rapidly dividing cells in a liquid medium, are initiated of a callus culture and used for our studies. In the past, we have used suspension cell cultures, for example, to modify plant cell walls and to study the conjugation of mycotoxins. 

Synthesis:

Many natural products are not commercially available as standard compounds for analytical methods or to perform biological and other studies. Independent synthesis of identical standard compounds or of standard compounds with specific structural elements also supports unambiguous structural characterization of phytochemicals or of individual structural elements within polymers. Incorporation of stable labels such as ¹³C or deuterium into standard compounds provides the basis for stable isotope dilution techniques.