ADAM F. LEE

1.1 INTRODUCTION

Sustainability, in essence the development of methodologies to meet the needs of the present without compromising those of future generations has become a watchword for modern society, with developed and devel­oping nations and multinational corporations promoting international re­search programmes into sustainable food, energy, materials and even city planning. In the context of energy and materials (specifically synthetic chemicals), despite significant growth in proven and predicted fossil fuel reserves over the next two decades, notably heavy crude oil, tar sands, deepwater wells, and shale oil and gas, there are great uncertainties in the economics of their exploitation via current extraction methodologies,

Catalysing Sustainable Fuel and Chemical Synthesis. © Lee AF. Applied Petrochemical Research 4,1 (2014), doi: 10.1007/s13203-014-0056-z. Licensed under Creative Commons Attribution License, http://creativecommons. org/licenses/by/3.0.

and crucially, an increasing proportion of such carbon resources (estimates vary between 65 and 80 % [1-3]) cannot be burned without breaching the UNFCC targets for a 2°C increase in mean global temperature relative to the pre-industrial level [4, 5]. There is clearly a tightrope to walk between meeting rising energy demands, predicted to rise 50 % globally by 2040 [6] and the requirement to mitigate current CO2 emissions and hence cli­mate change. The quest for sustainable resources to meet the demands of a rapidly rising global population represents one of this century’s grand challenges [7, 8].

While many alternative sources of renewable energy have the poten­tial to meet future energy demands for stationary power generation, bio­mass offers the most readily implemented, low cost solution to a drop-in transportation fuel for blending with/replacing conventional diesel [9] via carbohydrate hydrodeoxygenation (HDO) or lipid transesterification illus­trated in Scheme 1. First generation bio-based fuels derived from edible plant materials received much criticism over the attendant competition be­tween land usage for fuel crops versus traditional agricultural cultivation [10]. Deforestation practices, notably in Indonesia, wherein vast tracts of rainforest and peat land are being cleared to support palm oil plantations have also provoked controversy [11]. To be considered sustainable, sec­ond generation bio-based fuels and chemicals are sought that use biomass sourced from non-edible components of crops, such as stems, leaves and husks or cellulose from agricultural or forestry waste. Alternative non-food crops such as switchgrass or Jatropha curcas [12], which require minimal cultivation and do not compete with traditional arable land or drive de­forestation, are other potential candidate biofuel feedstocks. There is also growing interest in extracting bio-oils from aquatic biomass, which can yield 80-180 times the annual volume of oil per hectare than that obtained from plants [13]. Approximately 9 % of transportation energy needs are predicted to be met via liquid bio-fuels by 2030 [14]. While the abundance of land and aquatic biomass, and particularly of agricultural, forestry and industrial waste, is driving the search for technologies to transform ligno — cellulose into fuels and chemical, energy and atom-efficient processes to isolate lignin and hemicellulose from the more tractable cellulose compo­nent, remain to be identified [15]. Thermal pyrolysis offers one avenue by which to obtain transportation fuels, and wherein catalysis will undoubt-

SCHEME 1: Chemical conversion routes for the co-production of chemicals and transportation fuels from biomass edly play a significant role in both pyrolysis of raw biomass and subse­quent upgrading of bio-oils via deoxygenation and carbon chain growth. Catalytic depolymerisation of lignin may also unlock opportunities for the production of phenolics and related aromatic compounds for fine chemical and pharmaceutical applications [16].

Biodiesel is a clean burning and biodegradable fuel which, when de­rived from non-food plant or algal oils or animal fats, is viewed as a viable alternative (or additive) to current petroleum-derived diesel [17]. Com­mercial biodiesel is currently synthesised via liquid base-catalysed trans­esterification of C14-C20 triacylglyceride (TAG) components of lipids with C1-C2 alcohols [18-21] into fatty acid methyl esters (FAMEs) which con­stitute biodiesel as shown in Scheme 2, alongside glycerol as a potentially valuable by-product [22]. While the use of higher (e. g. C4) alcohols is also possible [23], and advantageous in respect of producing a less polar and corrosive FAME [24] with reduced cloud and pour points [25], the current high cost of longer chain alcohols, and difficulties associated with sepa­rating the heavier FAME product from unreacted alcohol and glycerol, remain problematic. Unfortunately, homogeneous acid and base catalysts can corrode reactors and engine manifolds, and their removal from the resulting biofuel is particularly problematic and energy intensive, requir­ing aqueous quench and neutralisation steps which result in the formation of stable emulsions and soaps [9, 26, 27]. Such homogeneous approaches also yield the glycerine by-product, of significant potential value to the pharmaceutical and cosmetic industries, in a dilute aqueous phase con­taminated by inorganic salts. Heterogeneous catalysis has a rich history of facilitating energy efficient selective molecular transformations and con­tributes to 90 % of chemical manufacturing processes and to more than 20 % of all industrial products [28, 29]. While catalysis has long played a piv­otal role in petroleum refining and petrochemistry, in a post-petroleum era, it will face new challenges as an enabling technology to overcoming the engineering and scientific barriers to economically feasible routes to bio­fuels. The utility of solid base and acid catalysts for biodiesel production has been extensively reviewed [20, 30-33], wherein they offer improved process efficiency by eliminating the need for quenching steps, allowing continuous operation [34], and enhancing the purity of the glycerol by­product. Technical advances in catalyst and reactor design remain essen­tial to utilise non-food based feedstocks and thereby ensure that biodiesel remains a key player in the renewable energy sector for the 21st century. Select pertinent developments in tailoring the nanostructure of solid acid and base catalysts for TAG transesterification to FAMEs and the related esterification of free fatty acid (FFAs) impurities common in bio-oil feed­stocks are therefore discussed herein.

Biomass also offers the only non-fossil fuel route to organic molecules for the manufacture of bulk, fine and speciality chemicals and polymers [35] required to meet societal demands for advanced materials [8, 36]. The production of such highly functional molecules, whether derived from petroleum feedstocks, requires chemoselective transformations in which e. g. specific heteroatoms or functional groups are incorporated or removed without compromising the underpinning molecular properties. The selective oxidation (selox) of alcohols, carbohydrates and related a, P-unsaturated substrates represent an important class of reactions that

SCHEME 2: Carbon cycle for biodiesel production from renewable bio-oils via catalytic transesterification

underpin the synthesis of valuable chemical intermediates [37, 38]. The scientific, technological and commercial importance of green chemistry presents a significant challenge to traditional selox methods, which pre­viously employed hazardous and toxic stoichiometric oxidants including permanganates, chromates and peroxides, with concomitant poor atom ef­ficiencies and requiring energy-intensive separation steps to obtain the de­sired carbonyl or acid product. Alternative heterogeneous catalysts utilis­ing oxygen or air as the oxidant offer vastly improved activity, selectivity and overall atom efficiency in alcohol selox (Scheme 3), but are particu­larly demanding due to the requirement to activate molecular oxygen and C-O bonds in close proximity at a surface in a solid-liquid-gas environ­ment [39-41], and must also be scalable in terms of both catalyst synthe­sis and implementation. For example, continuous flow microreactors have been implemented in both homogeneous and heterogeneous aerobic selox, providing facile catalyst recovery from feedstreams for the latter [42, 43],
but their scale-up/out requires complex manifolding to ensure adequate oxygen dissolution uniform reactant mixing and delivery [44, 45]. Ef­forts to overcome mass transport and solubility issues inherent to 3-phase catalysed oxidations have centred around the use of supercritical carbon dioxide to facilitate rapid diffusion of substrates to and products from the active catalyst site at modest temperatures [46] affording enhanced turn­over frequencies (TOFs), selectivity and on-stream performance versus conventional batch operation in liquid organic solvents [47-51].

The past decade has seen significant progress in understanding the fun­damental mode of action of Platinum Group Metal heterogeneous cata­lysts for aerobic selox and the associated reaction pathways and deactiva­tion processes [41]. This insight has been aided by advances in analytical methodologies, notably the development of in situ or operando (under working conditions) spectroscopic [52-54] /microscopic [55-58] tools able to provide quantitative, spatio-temporal information on structure — function relations of solid catalysts in the liquid and vapour phase. Parallel improvements in inorganic synthetic protocols offer finer control over pre­parative methods to direct the nanostructure (composition, morphology, size, valence and support architecture) of palladium catalysts [59-61] and thereby enhance activity, selectivity and lifetime in an informed manner.

Ultimately, heterogeneous catalysts may offer significant advantages over homogeneous analogues in respect of initial catalyst cost, product separa­tion, and metal recovery and recyclability [62]. Catalyst development can thus no longer be considered simply a matter of reaction kinetics, but as a clean technology wherein all aspects of process design, such as solvent selection, batch/flow operation, catalyst recovery and waste production and disposal are balanced [63]. The efficacy of Platinum Group Metals (PGMs) surfaces towards the liquid phase oxidation of alcohols has been known for over 50 years [64], and the development of heterogeneous plati­num selox catalysts (and more recently coinage metals such as gold [65, 66]) the subject of recent reviews [39, 67-69] hence only palladium selox catalysis is described herein.

When relating gas chromatography to catalytic transformations of bio­mass, it can be stated that GC (Figure 2) provides qualitative and quantita­tive determination of organic components such as extractives, hemicel-

lulose building blocks, organic acids, etc. The derivatized and vaporized products are introduced to the column for separation and identified in a detector, whose response is recorded as a chromatogram. Capillary col­umns made of fused silica with a stationary phase as a thin film of liq­uid or gum polymer on the inside of the tube are mainly used. The most commonly utilized stationary phases are siloxane polymer gums with dif­ferent substituents providing different polarity. The polymers are usually cross-linked in the column by photolytic or free-radical reactions, bringing strength to the polymer films. Wall-coated open-tubular columns with a liquid phase coated directly on the inner walls, as well as support-coated open-tubular columns are applied. In the latter case a stationary phase is coated on fine particles deposited on the inner walls. Among non-polar columns, HP-1, DB-1, etc., based on dimethyl, polysiloxane could be mentioned. HP-5 with 95% dimethyl polysiloxane and 5% phenyl groups is slightly more polar. Still more polar columns employ polyoxyethylene or polyester liquid phases.

Capillary columns are available in a wide range of internal diameters, lengths and liquid film thicknesses (Figure 2). Although longer columns provide better separation, they have an increased analysis time which is usually undesired. In addition, longer columns lead to higher pressure and thus to problems with the injection. Columns with thicker films have higher capacity, but usually require higher temperature, while thin-film columns are suited for large molecules with low volatility. In principle, analysis of components with up to 60 carbon atoms is possible.

Different types of injection systems are used in GC. Split mode, where the injected material after evaporation is split between the column and an outlet, affords rapid volatilization and homogeneous mixing with the car­rier gas. Most of the sample will pass out through the split vent and only a small proportion will flow into the column. Splitless systems provide a more reliable quantification allowing analysis of even such high-molec­ular mass compounds as triglycerides and steryl esters. Flame ionization detectors, which are of destructive nature, have high sensitivity to hydro­carbons, but are not able to detect water. On-line coupling of capillary columns with mass spectrometers is routine nowadays and enables conve­nient structure identification.

An important but sometimes forgotten issue is the fact that the sen­sitivity for different compounds is varying for a detector; thus, different peak areas are in proportion to the weight concentration. Knowledge of re­sponse factors is therefore necessary and calibration for components espe­cially with various functional groups should be properly done. Commonly, internal standard compounds are applied, e. g., compounds which are not present in the sample itself are purposely added. Chemically they should be similar to the sample compounds with close retention time, however, with no peak overlapping (Figure 3).

In addition to such advantages of GC as accurate quantification based on internal standards, a possibility to be combined with a mass spectrom­eter and complete automation regarding injection and analytical runs, the very high resolution should also be mentioned. On the other hand only molecules up to about 1000 mass units can be analyzed, as they should be stable at high temperatures. Therefore, sometimes samples should be processed before the analysis. The last point is important for polar com­pounds, like for example acids, which should be derivatized. GC and GC-MS analysis in the vapour phase require volatile derivatives that do not adsorb onto the column wall. Different derivatizations for different substances are recommended, e. g., silylation or methylation for extrac­tives, methanolysis and silylation for carbohydrates. Silyl derivatives of R-O-Si(CH3)3 type containing a trimethylsilyl group (TMS) are formed by the displacement of the active proton in — OH, — NH and — SH groups. Thus, protic sites are blocked, which decreases dipole-dipole interactions and increases volatility. Common silylation reagents are listed in Figure 4.

Methylation relies on the following reactions: utilization of diazo- metane (CH2N2): R-COOH + CH2N2 = RCOOMe + N2; acid-catalyzed esterification: R-COOH + ROH => RCOOR’, as well as on-column es­terification using tetra — methyl ammonium salts R-COOH + N+Me4OH — => RCOOMe.

One of the variants of GC is associated with coupling pyrolysis to it (Figure 5). In this arrangement the sample is thermally degraded in an inert atmosphere. The degradation products are introduced to GC or GC — MS for separation and identification allowing qualitative and quantitative determination of semi-volatile and non-volatile components, such as ex­tractives, polymers, paper chemicals, and lignin, etc.

STUART M. LECKIE, GAVIN J. HARKNESS, and MATTHEW L. CLARKE

The production of cellulose-derived chemicals is significantly more com­mercially attractive if economic value can be obtained from the lignin fraction of ligno-cellulose. There is currently great interest in research­ing the conversion of lignin to aromatics and alkanes. [1] These studies generally focus on the possible production of fuels, bulk, or commodity chemicals. The reactions used are depolymerisation of lignin, and hydro­deoxygenation reactions i. e. the replacement of the C-O bond with inert C-H bonds. [2,3]

We considered a new challenge in this field of renewable chemistry; if a small portion of lignin-derived bio-oils can be converted into one or more higher value fine chemicals, prior to hydro-deoxygenation, then extra eco-

Catalytic Constructive Deoxygenation of Lignin-Derived Phenols: New C-C Bond Formation Pro­cesses from Imidazole-Sulfonates and Ether Cleavage Reactions. © Leckie SM, Harkness GJ, and Clarke ML. Chemical Communications 50 (2014), DOI: 10.1039/C4CC04939J. Licensed under Cre­ative Commons Attribution 3.0 Unported License, http://creativecommons. org/licenses/by/3.0/.

nomic value can be derived from this lignin fraction. The research to find efficient lignin depolymerisation methods is still very much an expanding effort. None-the-less in the research published so far, 2-methoxyphenol (guaiacol) is a very common major component in lignin-derived bio-oils.3 2-Methoxyphenol is somewhat more volatile than some aromatic compo­nents, and can also be converted during processing to catechol, which may be possible to separate due to its acidity. While other building blocks may become viable in the future, it seems likely that 2-methoxyphenol and cat­echol will be produced from lignin feedstocks. [2b,3] Another possibility is that catalysis chemistry could be developed to selectively remove guaia­col or other monomers from lignin. [4] A further speculative possibility is to functionalise specific monomers in a bio-oil mixture, to give new fine chemicals that are readily separated from the rest of the bio-oil.

In order to give a larger range of possible target fine chemicals, new catalytic chemistry needs to be developed to convert chemicals like 2-methoxyphenol into less oxygenated, but still functionalised aromatic compounds, i. e. the challenge of catalytic constructive deoxygenation (Scheme 1). Longer term requirements are likely to be heavily focused on cost, so while improving the economics of the catalytic processes needs to be addressed in due course, certain aspects such as the reagents used to activate C-O bonds and the processes chosen to study need to consid­ered now. This actually leads to some interesting problems for catalysis chemists to study. Here we show the first studies on this concept and re­port new protocols to replace one or both C-O bonds in 2-methoxyphenol with C-C bonds.

Lignin

depolymerisation

MeO

Bio-oil ^

[PdCl2(dppf)]: 7% product, 84% consumption of sulfonate starting material (1 h)

[PdCl2((S)-Xyl-Phanephos)]:

94% product, >99% consumption of 1 (4 h)

SCHEME 2: Catalytic reactions of 2-methoxyphenyl-1H-imidazole-1-sulfonate with Grignards, nitromethane and benzoxazole.

The conversion of phenolic derivatives to activated compounds, fol­lowed by cross-coupling reactions is, of course, known methodology in a general sense. However, specific methods need to be developed for 2-me — thoxyphenol that do not use expensive triflates or other incompatible or expensive reagents.

We have focused on the coupling of the imidazole-sulfonate of 2-me- thoxyphenol, 1 (Scheme 2) since this is a reasonably cheap leaving group that is also claimed to give less toxic waste streams relative to triflates and their derivatives. [5] For the cross-coupling partners, we have assessed a range of suitable possibilities, but here we have studied Grignard reagents, nitromethane, heteroaromatic compounds and the cyanide anion since these are economic coupling partners.

The Kumada cross-coupling of Grignard reagents with imidazole-sul­fonates had not been reported, but our starting point was procedures that work well for aryl halides. The use of [PdCl2(dppf)] (dppf = 1,1′-bisdiphe- nylphosphino-ferrocene) in methyl-THF has previously been found to be an excellent procedure for Grignard cross-coupling, even under very con­centrated conditions. [6] However, none of the desired product was formed. Changing solvent to tert-amyl methyl ether enabled the cross coupling to proceed, although very unselectively, and very slowly using [PdCl2(dppf)] (see ESIf). We were pleased to find that the use of [PdCl2((S)-Xyl-phane- phos)] [7] as catalyst is much more active and selective (Scheme 2, eqn (1)).

Excellent results were obtained using this previously unexplored Gri­gnard coupling catalyst. In the ESIf a further table of results comparing Pd/dppf and Pd/Xyl-phanephos for aryl halides shows that PdCl2(Xyl-pha — nephos) is a more active catalyst than the widely applied Pd/dppf catalyst for this reaction. We used the expensive enantiomerically pure catalyst, but the racemic analogue would be a relatively economic ligand to use in achiral C-C bond forming reactions. Xyl-phanephos has a larger bite angle than dppf; this is generally associated with more efficient reductive elimination, but a lower propensity to other off-cycle events using this system is also possible.

The coupling of nitromethane with imidazole-sulfonates was not known, but there had been a report of coupling aryl halides with nitro- methane. [8] This protocol makes use of Pd/XPhos as the leading catalyst, although none of these operates at low catalyst loading. Our initial screen­ing (see ESIf and Scheme 2, eqn (2)) revealed that the combination used for aryl halides was ineffective, but Pd/TrixiePhos proved to be the only reasonably effective catalyst for coupling nitromethane with 1 to give de­sired product 3. Further research on more active nitromethylation catalysts in general would be worthwhile. It seems likely a mono-ligated Pd centre is desirable given the very bulky ligands required for any conversion in this study. Another pro-nucleophile with relatively acidic C-H bonds is benzoxazole. In this case, there had already been a report of C-H function­alisation using a range of phenols activated as their imidazole sulfonates, including compound 1. [9] We therefore used this procedure here (Scheme 2, eqn (3)), although again note that improvements in catalytic turnover are desirable in the future.

We next studied cyanation using cheap and non-toxic K4Fe(CN)6. This cyanide source, introduced in a seminal paper by Beller and co-workers, has been studied in the coupling with aryl halides and aryl mesylates, but not imidazole-sulfonates. [10] A more extensive screening of conditions and many different catalysts can be found in the ESI;f most catalysts are not sufficiently active, and 1 slowly hydrolyses to guaiacol, 6. Table 1 shows that combinations of either X-Phos or triphenylphosphine com­bined with Pd(II) pre-catalysts were effective. The more economic triphe — nylphosphine based system was found to give the optimal results for the production of 5 (Table 1, entry 7).

The cross-coupling processes shown above suggest that, beyond the realm of this specific project, it should be possible to carry out effective cyanation, Grignard cross-coupling and nitromethylation reactions using phenol-imidazolesulfonates and the new procedures identified here. More­over, these studies show that, with further research, it should be feasible to develop scalable methods for the C-C bond forming reaction using 1. This should be useful for making various phenolic compounds containing only one aromatic C-O bond. To increase the potential scope of this build­ing block, it would be desirable to be able to swap the remaining aromatic C-O bond for a C-C bond. [11] There are some important fine chemicals that could be produced effectively using this type of route, [12] but at this early stage, we wanted to map out what was possible.

TABLE 1: Selected examples from the optimisation of the cyanation of 2-methoxyphenyl — 1H-imidazole-1-sulfonate

As already noted, it is convenient to produce 4, using C-H activation coupling of benzoxazole with 1, so we considered modifying the Mey­ers reaction towards this class of substrate. The Meyers reaction normally uses certain oxazolines as activating groups for ether cleavage, [13] and to the best of our knowledge, there are not any examples of Meyers coupling using this type of benzoxazole. We were pleased to find that these reac­tions proceed well at near ambient temperatures using a range of aromatic, alkenyl and alkyl Grignards. Scheme 3 lists the products 7a-7h produced and reaction conditions.

ArMgX = PhMgCl ArMgX = 4-MeOC6H4mGCl (3.3 equiv.) (4.2 equiv.)

2-MeTHF, 40°C, 48 h 2-MeTHF, 40°C, 48 h

SCHEME 3: Modified Meyers reaction of anisyl-benzoxazoles with Grignard reagents.

In summary, some cross-coupling reactions that use relatively econom­ic nucleophilic partners and the imidazole-sulfonate of 2-methoxyphenol, 1 have been studied. It is proposed that this type of catalysis might be useful for creaming off some high value products from bio-oil mixtures, or bio-oil derived 2-methoxyphenol. In this case, we have identified sev­eral new protocols for cross-coupling imidazole sulfonate derivatives with Grignards, nitromethane and a non-toxic cyanide source. Modified Mey­ers reactions on benzoxazoles are also reported. These discoveries should prove enabling to those needing new organic methodology, in addition to presenting the first steps towards constructive deoxygenation reactions of renewables.

We thank the EPSRC for funding, and all the technical staff in the School of Chemistry for their assistance.

As the molecules included in the various types of liquid biomass can be relatively large and complicated, cracking reactions are desired to convert them into molecules of the size and boiling point range of conventional fuels, mainly gasoline, kerosene and diesel. A characteristic reaction that occurs during catalytic hydrotreating of oils / fats is the cracking of tri­glycerides into its consisting fatty acids (carboxylic acids) and propane as shown in Scheme 1 [5][6]. This reaction is critical as it converts the initial large triglycerides molecules of boiling point over 600°C into mid­distillate range molecules (naphtha, kerosene and diesel).

Other cracking reactions may take place however such as those de­scribed in Schemes 2 and 3, depending on the type of molecules pres­ent in the feedstock. For example Scheme 2 is a cracking reaction which may occur during catalytic hydrotreatment of pyrolysis oil which includes polyaromatic and aromatic compounds. Alternatively Scheme 3 may fol­low deoxygenation of carboxylic acids on the produced long chain paraf­finic molecules, leading to smaller chain paraffins, during the upgrading of Fischer-Tropsch wax.

R-R’ + H2 — ► R-H + R‘-H

SCHEME 3.

Lignocellulosic biomass can be treated under inert atmosphere at tempera­tures of 648-800 K in a process called pyrolysis. At these conditions, solid biomass undergoes a number of processes including depolymerization, de­hydration and C-C bond breaking reactions which lead to the formation of reactive vapor species. [35] Upon subsequent cooling, the vapor products condense generating a dark viscous liquid referred to as bio-oil. This bio­oil is a complex mixture of more than 400 highly oxygenated compounds, including acids, alcohols, aldehydes, esters, ketones and aromatic species, along with some remnants of polymeric carbohydrates and lignin frag­ments. [63,64] Consequently, once separated into their components, bio­oil could serve as a source of chemicals. The final composition of the bio-oil depends on a large number of factors (e. g., feedstock type, reaction conditions, alkali content of the feedstock, storage conditions). Biooils typically contain about 25 wt% water (derived from the initial water of the feedstock and from the pyrolysis process itself), and retain up to 70% of the energy stored in the biomass feedstock, [52] thereby allowing for concentration of the energy of biomass in a dense liquid that is more easily transportable. The main advantage of pyrolysis over BTL is its simplic­ity, because it requires only a single reactor and low capital investments, thereby allowing the development of cost-effective processing units on small scale. Thus, small portable pyrolysis reactors (i. e., 50-100 tons of biomass per day) are currently commercially developed to produce liquid biofuels close to the biomass location in countries like the US, Canada and the Netherlands. [65,66]

Even though bio-oils can be used directly in simple boilers and tur­bines for heat and electricity production, their utilization as transporta­tion fuels has multiple shortcomings. The high oxygen content of bio-oils negatively affects the energy density (16-19 MJ kg-1 versus 46 MJ kg-1 of regular gasoline), and it leads to low volatility and poor stability proper­ties of the bio-oil liquid. Furthermore, the high corrosiveness (pH ~ 2.5) and viscosity of bio-oils discourage their utilization in internal combus­tion engines. Since the pyrolysis process does not involve a deep chemi­cal transformation in the feedstock, extensive oxygen removal is required for bio-oils to have hydrocarbon-like properties (e. g., high energy density, high volatility and high thermal stability), and several routes are available in this respect (Fig. 5).

Hydrodeoxygenation (i. e., treatment of the bio-oil at moderate temper­atures and high hydrogen pressures, HDO) is probably the most common method to achieve oxygen removal from biooils. [67,68] By means of this technology, bio-oil components are completely hydrogenated and oxygen is removed in the form of water, which appears in the reactor as a sepa­rate phase from the hydrocarbon layer. Hydrodeoxygenation is typically carried out over sulfided CoMo and NiMo based catalysts [68] (used in the petrochemical industry to achieve sulfur and nitrogen removal from crude oil). Precious metals such as Pt and Ru [69,70] show higher hydrogenation activities at the expense of low tolerance to sulfur impurities (typically pres­ent in bio-oils). The large amount of hydrogen required for bio-oil deoxy­genation represents the main drawback of this technology, [71] and strate­gies based on steam-reforming of the water-soluble fraction of bio-oils, [72] along with aqueous-phase reforming of biomass-derived sugars, [73,74] have been studied to avoid the need to supply hydrogen from external fossil fuel sources. Bio-oils typically contain significant amounts of lignin-derived phenols which, once transformed into aromatic hydrocarbons, are valuable gasoline components. [75] One of the challenges of the hydrodeoxygenation process is to achieve complete hydrogenation of aliphatic compounds while avoiding unnecessary hydrogen consumption in the reduction of the valu­able aromatic hydrocarbons. However, this control over the extent of the hy­drogenation process is difficult at the elevated hydrogen pressures required for hydrodeoxygenation (e. g., 100-200 bars). In addition, high pressures lead to increases in operational costs of the process.

Bio-oil deoxygenation can be alternatively carried out at milder condi­tions (e. g., 623-773 K, atmospheric pressure) and without external hydro­gen by processing the bio-liquid over acidic zeolites, in a route that resem­bles the catalytic cracking approach used in petroleum refining. [76-78] At these conditions, bio-oil components undergo a number of reactions in­volving dehydration, cracking and aromatization, and oxygen is removed in the form of CO, CO2 and water (Fig. 5). As a result, bio-oil is converted into a mixture of aliphatic and aromatic hydrocarbons, although a large fraction of the organic carbon reacts to form solid carbonaceous depos­its denoted as coke. Thus, hydrocarbon yields are relatively modest and regeneration cycles under air (to burn off the coke) are frequent. Irrevers­ible deactivation, caused by partial de-alumination of zeolite structures at the water contents typically found in bio-oils, is another drawback of this technology, and research is needed on new acidic catalytic materials with better resistance to water. [16] On the other hand, the conditions of pres­sure and temperature at which zeolite upgrading is carried out are similar to those used in pyrolysis, thereby allowing the integration of these two processes in a single reactor, as recently demonstrated by Huber et al. [79] A third route that could help to reduce oxygen content in biooils while leaving the bio-liquid more amenable for subsequent downstream processes is catalytic ketonic decarboxylation or ketonization [80] (Fig. 5). By means of this reaction, 2 molecules of carboxylic acids are condensed into a larger ketone (2n — 1 carbon atoms) with the release of stoichiometric amounts of CO2 and water. This reaction is typically catalyzed by inorganic oxides such as CeO2, TiO2, Al2 O3 and ZrO2 at moderate temperatures (573-698 K) and atmospheric pressure. [81-84] Interestingly, ketonization achieves oxygen removal (in the form of water and CO2) while consuming carboxylic acids, the latter of which represent an important fraction of bio-oils (up to 30 wt%). [85] Moreover, these acids are hydrogen-consuming compounds, and are responsible for unwanted properties of the bio-liquids such as corrosiveness and chemical instability. Consequently, as represented in Fig. 5, a pretreat­ment of the biooil over a ketonization bed would simultaneously reduce oxygen content and acidity, thereby reducing hydrogen consumption and leaving bio-oil more amenable for subsequent hydrodeoxygenation process­ing. Even though ketonization has not been used to process real lignocel — lulosic bio-oils so far, we believe that this route has potential to upgrade bio-liquids enriched in carboxylic acids. Furthermore, ketonization can also condense typical components of bio-oils like esters, [86-88] and, unlike zeolite upgrading, this reaction can be efficiently carried out under moderate amounts of water. [89]

The material containing sulfonic acid groups was stirred for 24 h in 20 g of 2 mol L-1 NaCl. Next, an acid-base titration was achieved with NaOH and phenolphtaleine as indicator.

Even though vegetable oils are the main feedstock for the production of first generation biofuels, soon their production has troubled the public opinion due to their abated sustainability and to their association with the food vs. fuel debate. As a result the technology hasshifted towards the exploitation of both solid and liquid residual biomass. Waste Cooking Oils (WCOs) is a type of residual biomass resulting from frying with typical vegetable fryingoils (e. g. soybean-oil, corn-oil, olive-oil, sesame-oil etc). WCOs have particular problems regarding their disposal. In particular grease may result in coating of pipelines within the residential sewage system and is one of the most common causes of clogs and sewage spills. Furthermore, in the cases that sewage leaks into the environment, WCOs can cause human and environmental health problems because of the patho­gens contained. It has been estimated that by disposing 1 lit of WCO, over 1,000,000 of liters of water can be contaminated, which is estimated as the average demand of a single person for 14 years.

Catalytic hydroprocessing of WCO was studied as an alternative ap­proach of producing 2nd generation biofuels [20-24]. Initially catalytic hydrocracking was investigated over commercial hydrocracking catalysts leading not only to biodiesel but also to lighter products such as biogaso­line [20], employing a continuous-flow catalytic hydroprocessing pilot — plant with afixed-bed reactor. During this study several parameters were considered including hydrocracking temperature (350-390°C) and liquid hourly space velocity or LHSV (0.5-2.5 hr-1) under high pressure (140 bar), revealing that the conversion is favoured by high reaction temper­ature and low LHSV. Lower and medium temperatures, however, were more suitable for biodiesel production while higher temperatures offered better selectivity for biogasolineproduction. Furthermore, heteroatom re­moval (S, N and particularly O) was increased while saturation of double bonds was decreased with increasing hydrocracking temperature, indicat­ing the necessity of a pre-treatment step.

However catalytic hydrotreatment was later examined in more detail as a more promising technology particularly for paraffinic biodiesel produc­tion (Figure 1). The same team has studied the effect of temperature (330- 398°C) on the product yields and heteroatom removal[21]. The study was conducted in the same pilot plant utilizing a commercial NiMo/Al2O3hy — drotreating catalyst over lower pressure (80 bar). According to this study, the hydrotreatingtemperature is the key operating parameter which defines the catalyst effectiveness and life. In fact lower temperatures (330°C) fa­vour diesel production and selectivity. Sulfur and nitrogen removal were equally effective at all temperatures, while oxygen removal and satura — tionof double bonds were favoured by hydrotreating temperature. The same team also studied the effect of the other three operating parameters i. e. pressure, LHSV and H2/WCOratio [22]. Moreover they also studied the hydrocarbon content of the products [23] qualitatively via two-dimen­sional chromatography and quantitatively via Gas Chromatographywith Flame Ionization Detector (GC-FID), which indicated the presence of C15-C18 paraffins. Interestingly this study showed that as hydrotreating temperature increases, the contentof normal paraffins decreases while of iso-paraffins increases, revealing that isomerization reactions are favoured by temperature.

The total liquid product of WCO catalytic hydrotreatment was further investigated in terms of its percentage that contains paraffins within the diesel boiling point range (220-360°C)[24]. The properties of WCO, hy­drotreated WCO (total liquid product) and the diesel fractionof the hy­drotreated WCO are presented in Table 3. Based on this study the overall yieldof the WCO catalytic hydrotreatment technology was estimated over 92%v/v. The properties of the new 2nd generation paraffinic diesel prod­uct indicated a high-quality diesel with highheating value (49MJ/kg) and high cetane index (77) which is double of the one of fossil diesel. An ad­ditional advantage of the new biodiesel is its oxidation stability (exceeding 22hrs) and negligible acidity, rendering it as a safe biofuel, suitable for use in all engines. The properties and potential of the new biodiesel were further studied [25], for evaluating different fractions of the total liquid product and their suitability as an alternative diesel fuel.

TABLE 4: Properties of different pyrolysis oils according to literature Types of Pyrolysis Biooils Properties Test Methods [26] [27] [28] [29] [30] [31] [32] pH pHmeter 2.2 2.5 2~3 2.5 Density 15C (Kg/L) ASTM D4052 1.207 1.2 1.15-1.2 1.192 1.2 1.19 HHV (MJ/Kg) DIN51900 17.57 LHV (MJ/Kg) DIN51900 15.83 Solids Content (%wt) Insolubles in Ethanol 0.06 Ash content (%wt) ASTM D482 0.0034 <0.1 0.1 0.15 0-0.2 Pour point ASTM D97 -30 -30 Flash point ASTM D93 48 40-65 40-65 51 Viscosity (cP) @ 40C 40 40-100 40-100 43-1510 Viscosity 20°C 9mm2/s) ASTM D445 47.18 Viscosity 50°C (mm2/s) ASTM D445 9.726 Carbon (%wt) ASTM D5291 42.64 40.1 51.1 ~52 39.17 54-58 39.4-46.7 Hydrogen (wt%) ASTM D5291 5.83 7.6 7.3 ~6.4 8.04 5.5-7 7.2-7.9 Nitrogen (wt%) ASTM D5291 0.1 0.1 ~0.2 0.05 0-0.2 0.2 Sulphur (%wt) ASTM 0.01 0.032 Clorine (%wt) ASTM 0.012 AlkaliMetals (%wt) ICP <0.003 Oxygen (wt%) 52.1 41.6 ~40 52.74 35-40 45.7-52.7

A complicated but clear reaction pathway is postulated and shown in Fig­ure 2 based on the detailed product analyses and discussions mentioned above. Under acid-catalyzed conditions, olefin protonation and subsequent proton loss and reprotonation steps generated the isomerized olefins and their cation intermediates. Simultaneously, a series of competing reactions occur, where bio-oil’s components (water, carboxylic acids, phenols and alcohols) and the added olefins add to these cations. This leads to hydra­tion, esterification, O-alkylation, etherification and oligomerization which forms alcohols, esters, phenol O-alkylates, ethers and olefin oligomers, respectively. Moreover, diene intermolecular cyclizations and branched olefin cracking into small fragments as well as reoligomerization of these small fragments occurred. Similarly, under acid catalyzed conditions, protonation of alcohols and subsequent dehydration of these protonated products occurred generating carbocations. Meanwhile, additional com­peting reactions among carboxylic acids, aldehydes, alcohols, phenols and levulinic acid with these carbocations occurred, generating esters, acetals, ethers, phenol O-alkylates and alkyl levulinates, respectively. O-Alkylated phenols isomerized to the thermodynamic C-alkylated phenol via a Frie — del Crafts mechanism. Further addition of carbocations to mono-alkylated phenols generated bis-alkylated phenols.

In addition to reactions between bio-oil components and olefin/alco- hol reagents, the added alcohol adds across olefins to give intermolecu­lar etherification. Self-etherification of the added alcohol reagent also occurs. These reactions occur simultaneously, generating corresponding ethers. Acid-catalyzed dehydration of D-glucose to levulinic acid [28] occurred. First, dehydration of D-glucose gives 5-hydroxymethy furfu­ral (HMF). Then, HMF hydration to its hemiacetal occurred followed by sequential rehydration, ring-opening, loss of both water and formic acid generating levulinic acid. Also, acid-catalyzed 2-hydroxymethylfuran re­hydration and subsequent ring opening, dehydration and tautomerization formed levulinic acid [29]. Levulinic acid, in turn, is converted to alkyl levulinates by alcohols. Independently, polymerization of 2-hydroxymeth — ylfuran via electrophilic aromatic substitution proceeded jointly with loss of formaldehyde to form oligomeric products. Simultaneous dimerization and isomerization of hydroxyacetone occurred, forming cyclic hydroxy — acetone dimers and propionic acid along with some 2-hydroxy-3-meth — ylcyclopent-2-enone. This latter product was likely formed by aldol con­densation of hydroxyacetone to an open hydroxyacetone dimer and its subsequent dehydration and cyclodehydration reactions.

Clearly, bio-oil upgrading by simultaneous reactions with olefin/alco — hol over solid acids is complex, involving many simultaneous equilibria and competing reactions. However, the key reason for the success of this upgrading process is the role of acid-catalyzed olefin hydration. Olefin hydration removes water. As water concentration drops, esterification and acetal formation equilibria shift toward ester and acetal products.

5.3 EXPERIMENTAL SECTION

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), and used without further purification unless otherwise noted.

1.2.1 SOLID ACID CATALYSED BIODIESEL SYNTHESIS

A wide range of inorganic and polymeric solid acids are commercially available, however, their application for the transesterification of oils into biodiesel has only been recently explored, in part reflecting their lower activity compared with base-catalysed routes [27], in turn necessitating higher reaction temperatures to deliver suitable conversions. While their activities are generally low, solid acids have the advantage that they are less sensitive to FFA contaminants than their solid base analogues, and hence can operate with unrefined feedstocks containing 3-6 wt% FFAs [27]. In contrast to solid bases which require feedstock pretreatment to re­move fatty acid impurities, solid acids are able to esterify FFAs through to FAME in parallel with transesterification major TAG components without soap formation and thus reduce the number of processing steps to bio­diesel [70-72].

Mesoporous silicas from the SBA family [73] have been examined for biodiesel synthesis, and include materials grafted with sulfonic acid groups [74, 75] or SO4/ZrO2 surface coatings [76]. Phenyl and propyl sulfonic acid SBA-15 catalysts are particularly attractive materials with activities com­parable to Nafion and Amberlyst resins in palmitic acid esterification [77].

Phenylsulfonic acid functionalised silica is reportedly more active than their corresponding propyl analogues, in line with their respective acid strengths but is more difficult to prepare. Unfortunately, conventionally syn­thesised sulfonic acid functionalised SBA-15 silicas with pore sizes below ~6 nm possess long, isolated parallel channels and suffer correspondingly slow in-pore diffusion and catalytic turnover in FFA esterification. How­ever, poragens such as trimethylbenzene [78] triethylbenzene or triisopro­pylbenzene [79] can induce swelling of the Pluronic P123 micelles used to produce SBA-15, enabling ordered mesoporous silicas with diameters spanning 5-30 nm, and indeed ultra-large-pores with a BJH pore diameter as much as 34 nm [79]. This methodology was recently applied to prepare a range of large pore SBA-15 materials employing trimethylbenzene as the poragen, resulting in the formation of highly ordered periodic meso — structures with pore diameters of ~6, 8 and 14 nm [80]. These silicas were subsequently functionalised by mercaptopropyl trimethoxysilane (MPTS) and oxidised with H2O2 to yield expanded PrSO3-SBA-15 catalysts which were effective in both palmitic acid esterification with methanol and trica — prylin and triolein transesterification with methanol under mild conditions.

FIGURE 2: Superior performance of interconnected, mesoporous propylsulfonic acid KIT-6 catalysts for biodiesel synthesis via FFA esterification with methanol versus non-interconnected mesoporous SBA-15 analogue. Adapted from reference [82]. Copyright 2012 American Chemical Society

For both reactions, turnover frequencies dramatically increased with pore diameter, and all sulfonic acid heterogeneous catalysts significantly out­performed a commercial Amberlyst resin (Fig. 1). These rate enhance­ments are attributed to superior mass transport of the bulky FFA and tri­glycerides within the expanded PrSO3-SBA-15. Similar observations have been made over Poly(styrenesulfonic acid)-functionalised ultra-large pore SBA-15 in the esterification of oleic acid with butanol [81].

Improving pore interconnectivity, for example through swapping the p6 mm architecture of SBA-15 for the Ia3d of KIT-6 was subsequently explored as an alternative means to enhance in-pore active site accessi­bility (Scheme 1) for FFA esterification [82]. KIT-6 mesoporous materi­als exhibit improved characteristics for biomolecule immobilisation [83] reflecting superior diffusion within the interconnected cubic structure. A family of pore-expanded propylsulfonic acid KIT-6 analogues were pre­pared via MPTS grafting and oxidation and screened for FFA esterification with methanol as a function of alkyl chain length under mild conditions. As-synthesised PrSO3H-KIT-6 exhibited respective 40 and 70 % TOF en­hancements toward propanoic and hexanoic acid esterification compared with a PrSO3H-SBA-15 analogue of comparable (5 nm) pore diameter as a consequence of the improved mesopore interconnectivity. However, pore accessibility remained rate-limiting for esterification of the longer chain lauric and palmitic acids. Hydrothermal aging protocols facilitated expan­sion of the KIT-6 mesopore up to 7 nm, with consequent doubling of TOFs for lauric and palmitic acid esterification versus PrSO3H-SBA-15 (Fig. 2).

While numerous solid acids have been applied for biodiesel synthesis [27, 32, 84], most materials exhibit micro — and/or mesoporosity which, as illustrated above, are not optimal for accommodating bulky C16-C18 TAGs of FFAs. For example, incorporation of a secondary mesoporosity into a microporous H-p-zeolite to create a hierarchical solid acid significantly increased catalytic activity by lowering diffusion barriers [85]. Templated mesporous materials are widely used as catalyst supports [86, 87], with SBA-15 silicas popular candidates for reactions pertinent to biodiesel syn­thesis as previously discussed [75, 77, 88]. However, such surfactant-tem — plated supports possessing long, isolated parallel and narrow channels are ill-suited to efficient in-pore diffusion of bio-oil feedstocks affording poor catalytic turnover. Further improvements in pore architecture are hence required to optimise mass transport of heavier bulky TAGs and FFAs com­monly found in plant and algal oils. Simulations demonstrate that in the Knudsen diffusion regime [89], where reactants/products are able to dif­fuse enter/exit mesopores but experience moderate diffusion limitations, hierarchical pore structures may significantly improve catalyst activity. Materials with interpenetrating, bimodal meso-macropore networks have been prepared using microemulsion [90] or co-surfactant [91] templating routes and are particularly attractive for liquid phase, flow reactors where­in rapid pore diffusion is required. Liquid crystalline (soft) and colloidal polystyrene nanospheres (hard) templating methods have been combined to create highly organised, macro-mesoporous aluminas [92] and ‘SBA — 15 like’ silicas [93] (Scheme 4), in which both macro- and mesopore di­ameters can be independently tuned over the range 200-500 and 5-20 nm, respectively. The resulting hierarchical pore network of a propylsulfonic acid functionalised macro-mesoporous SBA-15 is shown in Fig. 3, where­in macropore incorporation confers a striking enhancement in the rates of tricaprylin transesterification and palmitic acid esterification with metha­nol, attributed to the macropores acting as transport conduits for reactants to rapidly access PrSO3H active sites located within the mesopores.

The hydrophilic nature of polar silica surfaces hinders their application for reactions involving apolar organic molecules. This is problematic for TAG transesterification (or FFA esterification) due to preferential in-pore diffusion and adsorption of alcohol versus fatty acid components. Surface hydroxyl groups also favour H2O adsorption, which if formed during FFA esterification can favour the reverse hydrolysis reaction and consequent low FAME yields. Surface modification via the incorporation of organic functionality into polar oxide surfaces, or dehydroxylation, can lower their polarity and thereby increase initial rates of acid catalysed transformations of liquid phase organic molecules [94]. Surface polarity can also be tuned by incorporating alkyl/aromatic groups directly into the silica framework, for example polysilsesquioxanes can be prepared via the co-condensation of 1,4-bis(triethoxysilyl)benzene (BTEB), or 1,2-bis(trimethoxysilyl)- ethane (BTME), with TEOS and MPTS in the sol-gel process [95, 96] which enhances small molecule esterification [97] and etherification [98]. The incorporation of organic spectator groups (e. g. phenyl, methyl or propyl) during the sol-gel syntheses of SBA-15 [99] and MCM-41 [100]

sulphonic acid silicas is achievable via co-grafting or simple addition of the respective alkyl or aryltrimethoxysilane during co-condensation pro­tocols. An experimental and computational study of sulphonic acid func­tionalised MCM-41 materials was undertaken to evaluate the effect of acid site density and surface hydrophobicity on catalyst acidity and associated performance [101]. MCM-41 was an excellent candidate due to the avail­ability of accurate models for the pore structure from kinetic Monte Carlo simulations [102], and was modified with surface groups to enable dynam­ic simulation of sulphonic acid and octyl groups co-attached within the MCM-41 pores. In parallel experiments, two catalyst series were inves­tigated towards acetic acid esterification with butanol (Scheme 5). In one series, the propylsulphonic acid coverage was varied between 0 (RSO3H) = 0-100 % ML over the bare silica (MCM-SO3H). For the second oc­tyl co-grafted series, both sulfonic acid and octyl coverages were tuned (MCM-Oc-SO3H). These materials allow the effect of lateral interactions between acid head groups and the role of hydrophobic octyl modifiers upon acid strength and activity to be separately probed.

SCHEME 4: Liquid crystal and polystyrene nanosphere dual surfactant/physical templating route to hierarchical macroporous-mesoporous silicas

FIGURE 3: (Left) SEM (a) and low and high magnification TEM (b, c) micrographs of a hierarchical macro-mesoporous Pr-SO3H-SBA-15; (right) corresponding catalytic performance in palmitic acid esterification and tricaprylin transesterification with methanol as a function of macropore density versus a purely mesoporous Pr-SO3H-SBA-15. Adapted from reference [93] with permission from The Royal Society of Chemistry

To avoid diffusion limitations, butanol esterification with acetic acid was selected as a model reaction (Fig. 4). Ammonia calorimetry revealed that the acid strength of polar MCM-SO3H materials increases from 87 to 118 kJ mol-1 with sulphonic acid loading. Co-grafted octyl groups dra­matically enhance the acid strength of MCM-Oc-SO3H for submonolayer SO3H coverages, with _AHads(NH3) rising to 103 kJ mol-1. The per site activity of the MCM-SO3H series in butanol esterification with acetic acid mirrors their acidity, increasing with SO3H content. Octyl surface func­tionalisation promotes esterification for all MCM-Oc-SO3H catalysts, doubling the turnover frequency of the lowest loading SO3H material. Molecular dynamic simulations indicate that the interaction of isolated sulphonic acid moieties with surface silanol groups is the primary cause of the lower acidity and activity of submonolayer samples within the MCM-

Advanced Biofuels: Using Catalytic Routes for Conversion

SO3H series. Lateral interactions with octyl groups help to re-orient sul — phonic acid headgroups into the pore interior, thereby enhancing acid strength and associated esterification activity.

In summary, recent developments in tailoring the structure and sur­face functionality of sulfonic acid silicas have led to a new generation of tunable solid acid catalysts well-suited to the esterification of short and long chain FFAs, and transesterification of diverse TAGs, with methanol under mild reaction conditions. A remaining challenge is to extend the dimensions and types of pore-interconnectivities present within the host silica frameworks, and to find alternative low cost soft and hard templates to facilitate synthetic scale-up of these catalysts for multi-kg production. Surfactant template extraction is typically achieved via energy-intensive solvent reflux, which results in significant volumes of contaminated waste and long processing times, while colloidal templates often require high temperature calcination which prevents template recovery/re-use and re­leases carbon dioxide. Preliminary steps towards the former have been recently taken, employing room temperature ultrasonication in a small sol­vent volume to deliver effective extraction of the P123 Pluronic surfactant used in the preparation of SBA-15 in only 5 min, with a 99.9 % energy saving and 90 % solvent reduction over reflux methods, and without com­promising textural, acidic or catalytic properties of the resultant Pr-SO3H — SBA-15 in hexanoic acid esterification (Fig. 5) [103].

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].