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    13 views22 pages

    Bio Ethanol

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    Bio Ethanol

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    Denar on Bioethanol: | if Production and Utilization ann, oo = a, g HM ere Charles von Wea Applied Energy Technology Series CHAPTER 18 METHODS FOR THE CHEMICAL ANALYSIS OF BIOMASS PROCESS STREAMS, Christine I. Ehrman ‘The accurate compositional analysis of biomass is crucial to advancing and commer- cializing the technology for biomass-to-fuels conversion. Researchers need reliable ‘methods to analyze feedstocks, process intermediates, and end products from biomass conversion. Standard methods for lignocellulosic compositional analysis, including ‘methods for extracting samples and for characterizing and quantifying moisture, lignin (acid-insoluble, acid-soluble, and total lignin), carbohydrates (sugars, uronic acids, and hydrolysis degradation products), extractives (volatile and nonvolatile), ash, and metals are reviewed. The methods now used extensively to chemically analyze bio- ‘mass, or having the greatest potential for future use, are emphasized, 18.1. INTRODUCTION Wet-chemical procedures and a growing number of instrumental methods are avail- abieto chemically analyze lignocellulosic materials. Ideally, methods use to analyze biomass shouldbe accurate, robust, ime ecient, and applicable forall types of bio- mass feedstocks as well as for process samples generated during biomass-to-fuel conversion. Considerable emphasis is now placed on the ability to close the mass bal- ance by providing reliable and complete analysis of all components present in the sample. Successful interpretation of the current biomass-to-ethanol conversion tech- nology requires compet analysis of complex process streams. The methods now used extensively to chemically analyze biomass, or having the greatest potential for use, are emphasized in tis chapter. Procedural details for each technique can be ‘obtained from the references. ‘The classic wet-chemical procedures are based on fractionating the sample into gross chemical components, such as lignin or holocellulose (te cellulose plus hemicellulose faction), with the resulting facons frequently being described in terms Ba BIS 386 HANDBOOK ON BIOETHANOL: PRODUCTION AND UTILIZATION of their method of isolation (Such as Klason lignin), rather than by their exact chemical structure. Researchers have not yet separated the uncontaminated major constituents ‘of biomass without adulterating them, because lignin and carbohydrates bond readily {1). Despite this limitation, these wet-chemical separation and analysis techniques offer considerable information about the chemical composition of complex lignocellu- losic samples, Instrumental methods that greatly enhance the specificity and convenience of chemical analysis of lignocellulosics have been introduced and rapidly developed. ‘In many cases they have expanded the breadth and number of components quantified, improved sensitivity, increased time efficiency, allowed some analysis steps to be automated, and required less sample than classical wet-chemistry techniques. 18.2 SAMPLE PREPARATION Lignocellulosic samples frequently require particle size reduction before they can be ‘chemically analyzed. A number of approaches have been used, the most common of ‘which involves milling a dried sample through a 40-mesh (0.40 mm) screen. Regrind- ‘ng or discarding fines is not recommended because the fines differ in composition and. reactivity from the larger particles [1]. For certain analyses, interfering extractives may also need to be removed from the sample before itis analyzed. Consecutive extractions with 1:2 ethanol-benzene, 95% ethanol, and distilled water are used 10 prepare extractive-free material (2]. The protocol described in ASTM Test Method 1105 [3] deletes the 95% ethanol extraction step. Waxes, fats, and, to some degree, resins and wood gums, are extracted by the ethanol-benzene mixture. Tannins, gums, sugars, starches, and coloring matter are removed during the hot water step. ‘Ultrasonic treatment or a combined homogenization/extraction using 80% ‘aqueous ethanol is an effective, nontoxic alternative to extractions using benzene. ‘This protocol effectively removes hydrophilic and lipophilic extractives [4]. Ultrason- ication is used primarily to remove extractives from dried lignocellulose materials, ‘whereas homogenization/extraction is reserved for leaves, grasses, and other fresh ‘materials with high moisture content. Soxhlet extraction with 95% ethanol is a sut- able alternative to ultrasonication, uses less solvent, and is less labor intensive (5). retreated biomass has been successfully analyzed without solvent extrac- tions although this type of process sample often requires rinsing with distilled water toremove residual pretreatment liquor 6]. Fermentation residues require extensive ‘water washing to remove protein and other compounds that may interfere with fermen- tation solids analysis (7). With both classes of process samples, particle size reduction ‘will be required ifthe material forms clumps as it dries. ‘The amount of material extracted is quantified by evaporating the solvent containing the extractives and weighing the residue. The chemical characterization ‘and quantification of the extractives are covered later in this chapter. [METHODS FOR THE CHEMICAL ANALYSIS OF BIOMASS PROCESS STREAMS 357 18.3 DETERMINATION OF MOISTURE AND TOTAL SOLIDS Depending on the biomass sample type, chemical analyses may be performed on air- dried or oven-dried samples. The results, however, are conventionally reported on a moisture-free (oven-dried) basis. Thus, determining moisture (or total solids) is one ‘ofthe most crucial steps inthe chemical analysis of any biomass sample because this value is used to calculate all subsequent analytical procedures. Any error magnifies certo in other chemical analyses. ‘The most widely accepted method of determining the moisture content of ‘biomass sample involves drying a preweighed sample to constant weight in an oven at 105°C [8-10], An inherent error in any oven-drying procedure is that volatile sub- stances other than water are removed from the sample during drying. Infrared drying typically involves placing the sample on a top-loading bal- ance equipped with an infrared lamp mounted above the pan, then monitoring weight changes. The results are expressed in grams or percentage of moisture lost or as per- cent total solids. If the instrument is operated in automatic mode, the sample weight change rat is monitored. The analysis is terminated when the sample weight changes less than a preset value. The infrared drying approach has the same limitation as the ‘oven-drying method—volatile substances other than water are lost during drying. Microwave ovens have been used to rapidly determine the moisture content ina variety of materials (11]. The oven design typically includes a water loop and heat exchanger. The heat exchanger effectively absorbs and removes excess energy, and avoids inadvertent ignition of samples (such as bark) that absorb microwaves. For samples that become more transparent to microwave radiation as moisture evaporates, ‘amicrowave absorber such as ferrous oxide should be added to the sample. This will ‘make the last trace of moisture easier to remove. As withthe previous two methods, volatile substances other than water may be lost during drying. ‘Azeotropic distillation with water-immiscible solvents, such as toluene and ‘ene, is also used to determine water in biomass samples [12]. Water distills with the solvent and, upon condensation, the water and immiscible solvent are separated sothatthe volume of water can be determined. Azeotropic distillation provides a bet- ter measure of true water content than oven drying, because (1) water can be eom- pletely removed from the sample using this technique; and (2) the results are not affected by volatile substances other than water inthe sample (1, ‘The Karl Fischer method included in ASTM Test Method D1348 (13) has, been used to successfly determine the moisture content of wood and bark (14). Dry ‘methanol displaces the water in a sample during a 20-min extraction. The water is then titrated via the Karl Fischer method. Unlike oven-drying procedures this titration is not adversely aflected by volatile extractives in the sample. Automated Karl Fischer titrators with electrometric end-point detection are available [15). ‘A variety of infrared techniques based on water's ability to absorb in the near infrared have been developed to determine moisture in biomass [16]. Wide-line nuc- lear magnetic resonance (NMR) spectrometers have also been used successfully to 388 HANDBOOK GN BIOETHANOL: PRODUCTION AND UTILIZATION determine moisture in wood samples [17]. Pulsed NMR techniques have been pro- posed as improvernents over the older steady-state NMR methods for measuring water in a variety of biomass materials [18]. Nondestructive methods of determining moisture in lignocellulosic materials. include measurements based on electrical resistivity (19), electric constants [20], attenuation of B- oF y-radiation [1], and microwave power absorption (21). 18.4 DETERMINATION OF LIGNIN 18.4.1 Determination of Acid-Insoluble Lignin ‘The most commonly used method for determining lignin in lignocelluloscs teats the sample with a strong mineral acid that hydrolyzes the polysaccharides, leaving an insoluble residue. The use of uli acid for this purpose (originally proposed by the chemist Peter Klason in 1906) has been incorporated in standard test methods [22- 25}. The residual solid has been termed acid-insoluble lignin ot Klason lignin. Be- ‘case condensation reactions occur during the sulfurie acd treatment, the structure of this isolated lignin is significantly different from the lignin in the original sample. Extractives may need tobe removed before sulfuric acid hydrolysis when ‘working with certin feedstocks, because some extractives (oils, resins, fats, waxes, taanins, gums, and starch) remain insoluble with the lignin. Other types of samples, such as pretreated biomass, may require only drying or milling before they can be hydrolyzed. In most standard test methods for determining Klason lignin, the prepared sample is weated with 720.1% (w/w) sulfuric acid for 2h at 20:1°C, followed by dilution with water to 3% sulfuric acid concentration. After boiling the solution for 4+, the flocculent residue i allowed to setle before itis filtered through a tared cruc- ible. ‘The collected residue is then washed free of acid, dred, and weighed as acid- insoluble ignin. Some protocols cal for correcting the aci-insoluble lignin content for the ash component of the insoluble residue [9,25]. ‘A modification of the standard Klason lignin procedure requires less than 300 mg ofthe sample and uses an autoclave at 121°C for Ih for secondary hydrolysis. ‘The results from this acid-insoluble lignin procedure correlate well with those from ‘he stndard method (26). fractionation method, known as the Van Soest method, has been widely used with herbaceous materials (27) 18.4.2 Determination of Acid-Soluble Lignin ‘When using a Klason-type procedure to determine the lignin content of a lignocellue losic material, a portion ofthe lignin is solubilized. This acid-soluble lignin (ASL) amounts to 0.2%~0.5% of softwood and 3%-5% of hardwoods (28). The method ‘most commonly used to analyze acid-soluble lignin is ultraviolet (UV) spectroscopy, Which relies on the ASL's adherence to Becr' law {29}. AC first the UV measurements, ‘were made at 280 nm (30,31), but later the recommended measurement wavelength METHODS FOR THE CHEMICAL ANALYSIS OF BIOMASS PROCESS STREAMS 29 was changed to 205 nm to avoid interference from 5-(hydroxymethyl)-2-furaldehyde and furfural, common acidic degradation products of carbohydrates which have absor- ‘bance maximums near 280 nm [32,33,34]. Some researchers have also used 240 nm for measuring ASL (35] , but a UV scan of acid-soluble lignin from 200 to 300 nm reveals that measurement at 205 nm is the better choice. To determine ASL spectropbotometrcally, the absorptivity of the ASL from ‘that particular type of biomass must be known. This value can be empirically deter- ‘mined by preparing some type of standard ASL solution. Preparative liquid chroma- tography [31] and chemical isolation techniques [36] have been used to obtain an ASL. By analyzing these solutions with UV spectroscopy at 205 nm, an ASL's ab- sorptivity can be calculated using Beer's law and the value used to determine the concentration of that ASL in hydrolyzate. TAPPI Useful Method 250 [37] recom- ‘mends the use ofan average absorptivity valve of 110 L/g-cm, obtained from a variety ‘of woods, to calculate the ASL content in wood and pulp. To avoid the use of model lignin compounds to determine absorptivity, an approach has been suggested that in- corporates a correction for any degradation products formed during a "reKlasonatio procedure in conjunction withthe absorbance at 205 nm of the resulting ASL solution tocaleulate the ASL absorptivity [38]. 18.43 Determination of Total Lignin ‘Total lignin ina variety of lignocellulosic samples has been determined by measuring the UV absorbance at 280 nm of a finely ground sample that had been dissolved in a solution of cetyl bromide in acetic acid (39]. As withthe ASL. calculating total ignin inthe sample requires an absorptivity value obtained by using the same UV procedure that corresponds tothe isolated lignin preparation or to the stating biomass sample ‘whose lignin content had already been measured by the Klason method, Browning provides information on additional wet-chemical methods for determining total lignin 228) Infrared (IR) spectroscopy has been used successfully to characterize isolated lignin preparations and to document changes produced in lignin by various treatments [40], The IR spectrum of lignin in wood has been obtained with a double-beam spec- trophotometer by preparing potassium bromide pellets with a ground sample, which is then placed inthe sample beam, and withthe holocellulose from the same material, placed in the reference beam [41], This approach has been greatly simplified with the advent of Fourier transform infrared (FTIR) spectroscopy. FTIR has also been used to obtain spectra from the surface of ground or powdered samples by diffuse reflee- tance. A series of equations was developed to semi-quantitatively predict the lignin, slucose, and xylose content of the wood solid by diffuse reflectance infrared Fouriet transform (DRIFT) [42,43). “Molecular beam mass spectrometry (MBMS) has been use to characterize herbaceous biomass [44]. The MBMS pyrolyzed materials at 600°C and analyzed the volatile pyrolysis products inreal time. If suitable calibration standards are availabe, Pytoisis mass-specia multivariate statistical analysis can be used for semiquantitative 40 HANDBOOK ON BIOETHANOL: PRODUCTION AND UTILIZATION analysis. However, in the cas of lignin in herbaceous biomass materials, a strong cor- relation between MBMS results and the conventional analysis for Klason lignin was ‘not found {45}. Differences in the two methods may arise from the ability of MBMS to separate the lignin from the protein; also, the MBMS lignin determination includes phenolic acid groups that are primarily constituents ofthe hemicellulosic fraction, Proton nuclear magnetic resonance (PMR) spectroscopy has been used ex- tensively inthe study of lignin and lignin model compounds despite its limitations with complex, high molecular weight materials [46~48].. Semiquantitative lignin values can be calculated from the integrated arcas ofthe spectral ranges corresponding 10 specific types of protons per C, unt in the prepared sample. ‘The speciza of lignin, as well as of purified cellulose and papers, have been obtained by electron spectroscopy for chemical analysis (ESCA) [47,49]. ESCA has also been used to semiquanttatvely determine the relative amounts of lignin and poly- saccharides onthe surfaces of wood fibers [50}. 18.5 DETERMINATION OF CARBOHYDRATES Inrecent years, techniques for analyzing carbohydrates in lignocellulosics have been rapidly developed and refined. Acid hydrolysis, coupled with chromatographic tech- niques, has quickly replaced more tedious fractionation and gravimetric techniques, such a the Van Soest method for forage fibers [27]. The cellulose and hemicellulose content ofa biomass sample can be estimated by hydrolyzing the sample and quanti- fying the resulting monosaccharides using a chromatographic technique. Typically cellulose is assumed to be equal to the total glucan, even though any glucomannan and galactoglucomannan in the hemicellulose will contribute to this total glucan value [51]. Hemiceliulose is assumed to be the total polysaccharide fraction of extractive- free biomass, less the cellulose, 185.1 Hydrolysis of Polysaccharides before Quantification To analyze the carbohydrates ofa lignocellulosic sample, the material must frst be digested chemically, In biomass analysis, the most commonly used method involves ‘two-stage sulfuric acid hydrolysis in which the biomass sample is first reated with concentrated acid, usually 720.1% w/w sulfuric acid, at 30°C for I h (9} or 2h [52 This hyérolysis step is followed by dilution to 4% acid and further hydrolysis in an autoclave at 121°C or under reflux for an additional hour. In the hydrolysis reaction, the glycosidic linkages of the polysaccharides in a biomass sample are cleaved by adding water, which produces monomeric compounds such as glucose and xylose. ‘These can then be determined quantitatively by chromatographic techniques. Degra- dation products such as furfural and 5-(hydroxymethyi)-2-furaldehyde, are also formed. To quantify the volatile degradation products, sealed vessels must be used uring the high-temperature acid hydrolysis step to retain volatile constiruents that ‘would otherwise be lost [52.53] METHODS FOR THE CHEMICAL ANALYSIS OF BIOMASS PROCESSSTREAMS 401 18.5.2 Determination of Sugars by Paper Chromatography Biomass hydrolysis solutions contain a complex array of sugars, phenolics, organic acids, furfurals, and other degradation products. All species mast somehow be an alyzed. The original procedure to separate these compounds used paper chromatog raphy, where the sugars ae eluted from the paper and quantified separately by pho- tometrc detection ofthe sugar-educing power [928,54] 185.3 Determination of Sugars by Gas Chromatography Awaditional means of carbohydrate analysis has been by gas chromatography (GC). Following acid hydrolysis of a biomass sample, the sugars must be converted into volatile derivatives before the GC separates them. In one commonly used method, the ‘monosaccharides ina hydrolycate are first reduced to alditols with sodium borohydride and are then acetylated by adding sulfuric acid and acetic anhydride (55-58). A scaled-down version of this method, using 10-mg or smaller samples, has also been suggested {59]. In another modification of the TAPPI procedure, I-methyl imidazole {is added to catalyze the reaction (60]. The derivatization step in this modified proced- ure takes only 10 min without special heating, compared to 1 h at 60°C required in the ‘TAPPI method. In 2 simplified derivatization procedure, the acetylation of the nonreduced aldoses occurs directly in the sulfuric acid hydrolysate (61). In this method, each sugar will give more than one peak. The quantification can be based either on single iso- ‘meric peaks by conventional multipoint calibration, or on several peaks by partial least-square calibration, Easty and Thompson (1] report that sugars in wood hydrolyzates can also be determined as acetates of aldononitrles. The sample is hydrolyzed, neutralized, and ‘evaporated to dryness. The residue is then heated with pyridine and hydroxylamine hydrochloride followed by acetylation with acetic anhydride. Another approach in- volves preparing volatile trimethysill derivatives by adding anhydrous pyridine to the hydrolysis residue, and then treating that mixture with hexamethyldisilazane and tri ‘methylchlorosilane (62]. Laver etal. [63] showed that a separate derivative is formed from each anomeric form of each sugar in the mixture. ‘Sugars are normally identified by their retention times in routine GC sugar ‘determinations, but a mass spectrometer used in conjunction with the GC will provide conclusive idemification of each peak. Kochetkov and Chizhov [64] and Lonngren ‘and Svensson [65] reviewed the principles of mass spectrometry (MS) of carbohydrate Gerivatives. GCIMS is used not only for simple sugar derivatives, but also to identify the volatile peralkylated derivatives of disaccharides, wisaccharides, and selected tetrasaccharides [66]. 4€2 HANDBOOK ON BIOFTHANOL: PRODUCTION AND UTILIZATION 18.5.4 Determination of Sugars by High-Performance Liquid Chromatography Gas chromatography has traditionally been used to analyze carbohydrates, but the introduction and continued refinement of high-performance liquid chromatography (HPLC) has resulted in instrumental approaches well-suited for analyzing carbohyd- ‘ate mixtures. The two major advantages of HPLC over GC are (1) the elution times ae typically shorter; and (2) sample preparation is simpler because there is no need to derivatize ‘The first sugar separations by HPLC used strongly basic anion-exchange resin, but later efforts showed that carbohydrate mixtures could also be separated by ‘pation chromatography on cation-exchange resins {67~70]. The sugars are thought to form complexes based on the configuration of their hydroxyl groups with the metal ionically bound to a sulfonated polystyrene-divinylbenzene copolymer, and the separa- tion is governed by the stereochemistry of the carbohydrate [71]. Researchers gener- ally use degassed HPLC-grade water a the eluent and refractive index as the means of detection for ion-moderated partition carbohydrate chromatography (72 By changing the type of metal ionially bound to the resin the behavior of the column toward the sugar molecules can be altered (73], thereby allowing the ion- moderated partion chromatography column packings to be optimized for separating specific monosaccharides (74). A cation-exchange resin in the calcium form has been shown to be suitable for the anomeric analysis of disaccharides liberated from biomass polysaccharides (75]. Resins in the calcium and silver forms have been used for oli gosaccharide separations [76]. Monosaccharides have been completely resolved in biomass hydrolyzates on a resin in the lead and strontium forms (6,52.73). Biomass hycrolyzates are complex mixtures that typically contain dispropor- tionate amounts of carbohydrates. The large quantity of one carbohydrate can mask the presence of a smaller quantity of another ifthe two components have similar re- tention times, Cation-exchange resins inthe lead form are particularly well-suited to analyze the monosaccharides typically derived from lignocellulosic materials, despite disproportionate quantities of carbohydrates, Many types of lignocellulosic, including ‘hardwoods (6, hardwood bark (77), pretreated biomass {78], herbaceous crops [79], ‘corn residues [80], and fermentation residues (7] have been successfully analyzed using the lead-form column. This column can also quantify rhamnose and fucose, e- ported to be moieties in the hemicellulose of some woods (81). HPLC has also separated carbohydrates on bonded-phase silica packings ‘where polar or nonpolar functional groups are covalently bound to the surface of mic- roparticulate silica gel. A bonded phase containing the polar aminoalky| functional _oup and gradient elution with water in acetonitrile can be used to separate mono- saccharides, disaccharides, and trisaccharides; however, glucose and galactose will not completely resolve [82]. ‘Development of efficient anion-exchange resins in pellicular form played a ‘major role in the rapid advances in high-performance anion exchange chromatography (HPAEC). Reducing monosaccharides and oligosaccharides can now be separated “METHODS FOR THE CHEMICAL ANALYSIS OF BIOMASS PROCESS STREAMS <3, under alkaline conditions rapidly enough that isomerization and degradation are usu- ally negligible (83). Under alkaline conditions the hydroxyl carbohydrate groups are transformed into oxyanions, allowing carbohydrate to be chromatographed as anions without such additives as borates. HPAEC soon developed into a powerful tool for carbohyerate research and, when coupled with pulsed amperometric detection (PAD), can be used to measure carbohydrates with high sensitivity without needing pre- ot post-colurnn derivatization. HPAEC-PAD has been used to quantify carbohydrates, including sugar alcohols, aldoses, Ketoses, polysaccharides, and aminosaccharides (84). HPAEC-PAD is a valuable tool in biomass sample analysis. The anion- exchange results agree very closely with those obtained from traditional lead-form cation-exchange chromatography [85]. A lignocellulosic sample is hydrolyzed with sulfuric acid as described earlier, but i is not necessary to neutralize or concentrate the hydrolyzate before column injection [86]. Monosaccharides in biomass hydroly- zates can be completely separate under isocratic conditions using 5 to 20 mM sodium hydroxide, bu post-column alkalinization is required when an clucnt of such low pH is used [68,87]. Sodium hydroxide does not need to be added post-column, and mono- saccharides in a hytroyzatecan remain separated by using two columns in series and a gradient elution profile that incorporates a column regeneration step (88). MBMS can semiquanttatively analyze cellulose in herbaceous biomass mat- cals if suitable calibration standards are available (44). Good correlation was ob- tained between the MBMS results and the conventional carbobydrate analysis for the carbohydrates. Diffuse-rflectance FTIR spectroscopy can aso be used to semi- ‘quantitatively predict glucose and xylose content of the wood solids [43]. Both tech- niques were discussed in Subsection 15.4.3. 18.5.5 Determination of Uronic Acids (One approach for determining total uronic acids in a biomass sample is based on de- ccarboxylating the uronic acid moieties with strong mineral acid. The carbon dioxide (CO,) released during decarboxylation is trapped and chemically determined by a variety of techniques [9,89]. A correction must be made for the CO, evolution from the decomposition of nonuronic acid carbohydrates. ‘The carbazole-sulfuric acid colorimetric method has also been widely used todetermine hexuronic acids (28]. A related method uses harmine [90]. An alterna- tive approach reacts the hydrolyzate with 3,5-dimethyl phenol followed by colorimet- ric determination of the complex formed with glucuronic acid 91). ‘The colorimetric and decarboxylation methods cannot differentiate between uronic acids, and are insensitive to monosaccharides bonded to uronic acid moieties, HPLC does not suffer from these limitations, and methods for analyzing organic acids (Gneluding uronic acids) from biomass have been developed. In one method, a strong anion-exchange column in the borate form was eluted with a series of borate buffers to separate individual monosaccharides as well as uronic acids (92). Ion-moderated ‘chromatography has also been used, but the accurate determination ofthe uronic acids 401 HANDBOOK OW BIOETHANOL: PRODUCTION AND UTILIZATION in a hydrolyzate sample can be compromised by the presence of glucose, which has retention time similar to that of the uronic acids. Removing monosaccharides by first adsorbing the uronic acids in the hydrolyzate to a mixed-bed ion-exchange column, then eluting the adsorbed acids after the sugars have been rinsed away, enables the sample to be analyzed by HPLC without interference by the sugars [53]. 18.5.6 Determination of Degradation Products During the acid hydrolysis of lignocellulosics materials, carbohydrate degradation products are formed from hexose sugars (primarily 5-(hydroxymethyl)-2-furaldehyde ‘and levulinc acid) and from pentose sugars (primarily furfural). Other biomass acid degradation products, such as acetic acid and methanol, may also be formed. To quan- tify the volatile degradation products, sealed vessels must be used during the high- temperature acid hydrolysis ofthe materials to retain volatile constituents that would otherwise be lost [52,53] HPLC provides an ideal tool to quantify votatile and nonvolatile acid degra- dation products (53,67). Volatiles such as acetic acid and furfural in an aqueous solution can also be determined by GC [78,93]. Methanol, formed from cleaved meth- oxyl groups, can be formed under acidic conditions from demethoxylized 4-O-Me- slucuronic acid and lignin. It is most frequently analyzed using ASTM Test Method 11166 {94}, although HPLC and GC techniques can also be used, 18.6 DETERMINATION OF EXTRACTIVES After a lignocellulosic sample, the extraction solvent can be analyzed further for low ‘molecular weight sugars, amino acids, phenolic compounds, resin acids, and other classes of compounds. Measuring and characterizing the amount of extractives in a sample can be complex procedures. For example, a multistep procedure (which in- cludes solvent extraction with acetone and measurement of total extractives; fraction- ation of the extracts into strong acids, weak acids, and neutrals by ion-exchange chromatograpy; and finally, using GC to identify and quantify te fraction compon- ents) was developed for wood and bark (95}. ‘Significant quantities of volatile components may be present in many soft- ‘woods [96], but are negligible in hardwoods [28]. Typically, determining volatile oils. in wood involves a distillation step followed by condensing or adsorbing the vapors conto a suitable material, such as charcoal [97]. GC headspace analysis followed by MS identification has been used successfully to analyze volatile components released into the headspace ofa glass vessel filled with wood chips (98). Soxhlet extraction is normaly used to isolate the nonvolatile extractives, ‘employing a solvent selected based on the purpose of the analysis (8]. Quantifying ‘nonvolatile extractives is straightforward, but identifying and quantifying individual [METHODS FOR THE CHEMICAL ANALYSIS OF BIOMASS PROCESS STREAMS 405 components is much more complex. Several strategies have been proposed for separ- ating nonvolatile extractive into groups of components having similar properties 11,28,99), ‘To analyze phenolics in extractives, the sample is first acidified, then the phenolic acids are extracted by ethyl acetate and analyzed by HPLC [100], Polar extractives (including monosaccharides, sucrose, arabinogalactans, pectin, cycitols, and low molecular weight carboxylic acids) tend to be more concen- ‘rated in bark and foliage than inthe wood {1}. Isolating and quantifying many ofthe polar extractives involves an inital extraction with a polar solvent followed by deriva- tizing and GC analysis on a capillary column [101]. An enzymatic approach is used todetermine starch. For feedstocks such as agricultural residues and some hardwoods that contain significant amounts of starch, the extractives are analyzed using 2 thermo- stable a-amylase in combination with an amyloglucosidase (4]. The starch contents are often negligible in woody and other highly lignified feedstocks. 18.7 DETERMINATION OF ASH Ash isthe percentage of residue that remains after a lignocellulosic material is dry oxidized, and is an approximate measure of the mineral content and other inorganic matter inthe sample. In the standard procedure for determining ash in lignocellu- losics, the sample, ina tared crucible, is heated gradually to 575225°C in a muffle famace to carbonze the sample without aming (102-104). This temperature is main- tained unt ll the carbon is bumed away. The crucible is then cooled ina desiceator and weighed. Results are normally reported on the moisture-free basis. 18.8 DETERMINATION OF METALS ‘The metals present in a lignocellulosic sample may be analyzed by a variety of atomic spectrometric techniques once the organic matter in the sample has been destroyed. Of the two general procedures for destroying organic matter (dry ashing and wet

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