These chromatographic methods use liquids such as water or organic sol­vents as the mobile phase. Silica or organic polymers as well as anion-ex­change resins are used as stationary phase. Separation is performed either at atmospheric pressure or at high pressure generated by pumps. The last version is often called high-performance liquid chromatography (HPLC) with solvent velocity controlled by high-pressure pumps, giving a constant flow rate of the solvents. Solvents are used not only as single solvents but they can also be mixed in programmed proportions. In fact, even gradient elution could be applied with increasing amounts of one solvent added to another, creating a continuous gradient and allowing a sufficiently rapid elution of all components.

The most commonly used columns contain small silica particles (3-10 pm) coated with a nonpolar monomolecular layer.

For lipophilic (low-polar) compounds the mobile phase is an organic solvent, while reversed phase HPLC employs mixtures of water and ace­tonitrile or water and methanol as eluents and is applied for non-ionized compounds soluble in polar solvents. As examples, such columns (Figure 6) could be mentioned as Agilent Zorbax SB-Aq (4.6×250 mm, 5 qm) allowing the use of highly aqueous mobile phases working in a pH range from 1 to 8 and affording reproducible retention and resolution for polar compounds. Another example is HypercarbTM (4.6×100 mm, 5 qm) with 100% porous graphitic carbon as a stationary phase, which operates in the pH range 0-14 and can resolve highly polar compounds with closely relat­ed structures (e. g., geometric isomers, diastereomers, oligosaccharides). CarboPac PA1 (polymer based) column can be used in mono-, oligo — and polysaccharide analysis by high-performance anion-exchange chromatog­raphy combined at high pH with pulsed amperometric detection.

UV-Vis (Figure 7) and diode-array detectors enabling recording of UV — Vis spectra, for example every second, are common nowadays. They can be used for the analysis of conjugated and aromatic compounds, such as phe­nols. Another popular detector is based on refractive index (RI) monitor­ing and is well suited, for example, for carbohydrates. High-performance anion-exchange chromatography with pulsed amperometric detection is a common technique for analyzing sugars in wood and pulp hydrolysates.

Another important form of HPLC is size-exclusion chromatography (Figure 8), which is widely applied for the determination of molecular — mass distributions of dissolved lignin and hemicelluloses, and even for cellulose dissolved in ionic liquids. The same method can be used for the analysis of extractives and their derivatives, for instance dimers and tri­mers of fatty acids [20]. In SEC, solutes in the mobile phase (for example THF) are separated according to their molecular size. Smaller molecules penetrate far into the porous column packing material and thus elute later than larger ones.

The non-destructive character as well as the absence of derivatization could be mentioned among the advantages of LC. This technique can han­dle both small and large amounts and it can be used also for preparative isolation of compounds from mixtures. Contrary to GC there are almost no, or at least much fewer, limitations in terms of the molecular size. In addition, LC can be combined with mass spectrometry, once again with­out derivatization. Thermally unstable and polar compounds can thus be analyzed as such, and the molecular mass in triple quadrupole or ion-trap LC-MS can be up to m/z 3000, while time-of-flight versions allow even up to 16,000.

LC-MS provides better sensitivity and selectivity than GC-MS and is excellent for the quantification of selected substances in complex mix­tures. On the other hand, this technique is not very suitable for rapid and reliable identification of unknown compounds mainly because fragmenta­tion is sparse as the conditions of ionization are mild. Furthermore, spec­tra libraries enabling identification are not available. Other shortcomings of LC-MS are the rather low sensitivity of the detectors for certain com­pounds. Moreover, it may be difficult to obtain constant pressure, which in turn influences retention; clean, degassed solvents are needed and, finally, it might be challenging to find the optimum solvent mixture.

Nevertheless, there is a large potential in the application of LC-MS toward analysis of oligosaccharides, lignans and oligolignans, flavonoids, stilbenes and tannins, and even fragments of lignin [21].

One form of LC, which is still used in organic synthesis and was popular until the 1960s in the analysis of monosaccharides obtained by hydrolysis of wood, is the so-called planar chromatography or thin-layer chromatography (TLC), where the separation is done in paper sheets or on particle layers deposited on glass, plastic or aluminium plates. Although these times of analysis of carbohydrates are long gone, TLC is an excellent technique for small scale preparative separation of fractions to be further analyzed by GC or LC. During analysis an eluent and the analytes rise in the stationary phase due to capillary forces. The analytes are separated ac­cording to their affinity to the stationary phase, which is most commonly silica (Figure 9).

3.3.2 SPECTROMETRIC METHODS

Besides chromatography a wide variety of other techniques are available, such as capillary electrophoresis (CE), Infra Red spectrometry (IR), Nu­clear Magnetic Resonance (NMR), Raman, Near Infra Red Spectrometry (NIR) and Ultra Violet-Visual Light Spectrometry (UV-Vis). Electropho­resis is a separation technique based on the differential transportation of charged species in an electric field through a conductive medium. Capillary electrophoresis (CE) was designed to separate species depending on their size to charge ratio in the interior of a small capillary filled with an electro­lyte and can be used for analyzing oligosaccharide and monosaccharide re­action products. In the current review we focus mainly on chromatographic methods although the spectrometric methods listed above are certainly of great importance. For instance, UV spectrometry can be used for the deter­mination of lignins in solutions. Colorimetric methods based on selective complexation with special reagents, which can be determined by spectro — metric measurements in the UV-Vis range, are applied for the determination of metal ions, hemicelluloses and pectins. IR is a possibility to identify such functional groups as hydroxyls, carbonyls, carboxyls and amines.

For example, the analysis of products in rapeseed oil hydrogenation was conducted by IR [22]. However, IR spectra of large biomolecules are com­plex, moreover spectra of component mixtures could be difficult to interpret. An advantage of Raman spectroscopy for the transformation of biomass oc­curring often in water solutions is the easy detection of double and triple car­bon-carbon bonds while the adsorption of water is weak. Thus, in contrast to FTIR, wet pulp and wood samples can be analyzed with signals related to extractives, lignin and carbon hydrogen bonds of the polysaccharides, while in FTIR signals of the hydroxyl groups of wood polysaccharides are dominating. NMR is an important method as it provides structural informa­tion about complex molecules, therefore it is frequently used for structural analysis of lignins and even hemicelluloses. Crystalline cellulose requires the application of solid-state NMR, as utilized for instance recently in the hydrolytic hydrogenation of cellulose [17].