Thermal Carbon Analysis Enabling Comprehensive Characterization

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Thermal carbon analysis enabling comprehensive characterization of lignin and its degradation products Keith Voeller, Honza Bilek, Jasmine Kreft, Alzbeta Dostalkova, Evguenii I. Kozliak, and Alena Kubatova ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02392 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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ACS Sustainable Chemistry & Engineering

Thermal carbon analysis enabling comprehensive characterization of lignin and its degradation products Keith Voeller,1,2 Honza Bílek,1 Jasmine Kreft,3 Alžběta Dostálková, 1,4 Evguenii Kozliak,1 Alena Kubátová1,* 1

Department of Chemistry, University of North Dakota, 151 Cornell St. Grand Forks, ND 58202,

USA 2

Present address: ARS USDA Human Nutrition Research Center, 2420 2nd Avenue North,

Grand Forks, ND 58203 3

Department of Chemical Engineering, University of North Dakota, 241 Centennial Dr., Grand

Forks, ND 58202, USA 4

Present address: Department of Biotechnology, University of Chemistry and Technology,

Prague, Technická 5, Praha 6 – Dejvice, 166 28, Czech Republic

* Corresponding Author: Phone: +1 701-777-0348, fax: +1 701-777-2331, Email: [email protected].

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Total 6733 (7000 max) Keywords: lignomics, thermal carbon analysis, desorption, pyrolysis, lignocellulose

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Thermal carbon analysis enabling comprehensive characterization of lignin and its degradation products Keith Voeller,1,2 Honza Bílek,1 Jasmine Kreft,3 Alžběta Dostálková, 1,4 Evguenii Kozliak,1 Alena Kubátová1,*

Abstract We have developed a novel thermal carbon analysis (TCA) method that provides both carbon mass balance and thermal fractionation profiles. Though not providing chemical structural information, this method enables a comprehensive characterization of both lignin and its degradation products, potential renewable and sustainable feedstocks. TCA is essential as a complement to a qualitative chemical speciation by thermal desorption-pyrolysis gas chromatography-mass spectrometry (TD-Py-GC-MS). Mono- and diaromatic oxygenated compounds were used as model compounds to optimize the method. The influence of various parameters such as solvents, amounts of sample loaded and temperature ramp configuration, were investigated. A multistep temperature program with TD and pyrolytic temperatures with and without oxygen was employed for analysis of untreated lignin, where up to 55 wt. % evolved in the presence of oxygen only, this fraction being unaccounted for by currently used methods. The TCA results were supported by thermogravimetric analysis with a matching heating ramp resulting in a similar mass distribution; however, TCA has the advantage of being selective for carbon. For lignin degradation products, the TD steps of TCA yielded similar recoveries as a solvent extraction followed by GC-MS. Thus, TCA may be used for screening significant product fractions to quantify the previously uncharacterized oligomer/polymer and char fractions.


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TOC/Abstract Graphic Synopsis

Towards sustainable generation of renewable aromatics, thermal carbon analysis enables comprehensive characterization of lignin degradation products and mass balance closure.



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Introduction Lignocellulosic biomass consists of three major components; cellulose, hemicellulose and lignin.1 Cellulose and hemicellulose are traditionally used for production of ethanol.1 While 50 million tons of lignin (accounting for as much as 15 to 40 wt. % of dry biomass) are produced annually, primarily as a byproduct from the ethanol and paper industries, its application is limited.2 Lignin has been targeted for its utilization as a potential source of value added materials, however, its primary current use is as a feed and fuel for heat and power generation.2-4 Chemical analysis of lignin and its degradation products is typically addressed with a suite of methods including spectroscopic, separation and thermal protocols. Spectroscopic methods such as nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR) and mass spectrometry (MS) (usually with matrix assisted laser desorption ionization) yield insights on lignin as a whole molecule, revealing aromatic units and inter-unit linkages, and provide details on different functionalities.5 However, these bulk methods cannot readily distinguish between the lignin feedstock and its degradation products as the same types of specific bonds and functionalities or fragments (in MS) may be present.6 The separation methods target individual constituents and include size-exclusion (SEC), gas and liquid chromatography (GC and LC), which are usually coupled with MS. SEC is often used for the determination of lignin’s molecular weight (MW) and mass distribution of its degradation products.7, 8 However, the frequently used polystyrene standards are not an adequate representation of lignin and its degradation products due to the heteropolymeric character of lignin.8 The additional functionalities add to stationary phase interactions (beyond the size exclusion effect) and ultimately lead to uncertainties in MW determination.9


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GC-MS analysis following liquid-liquid extraction (LLE) is an excellent tool for identification of extractable and volatile lignin degradation products.6 While this fraction appears to be desirable as phenolic monomers are considered value-added chemicals, it often represents only a small portion of the overall carbon balance, as phenolic oligomers and polymers are not GC-elutable.10 The LC-MS with electrospray ionization is not well-suited for application to unknown products due to selective ionization and also fragmentation (loss of molecular ions) without commercially available standards, which are lacking particularly for large MW lignin species.11 Finally, thermal methods, e.g., thermogravimetric analysis (TGA) and pyrolysis (Py) GCMS6 are frequently employed to address lignin recalcitrance and limited solubility in common solvents. They are used either to provide an overall sample characterization, e.g., TGA, or to target specific constituents, e.g., Py-GC-MS.12 These thermal analysis methods are also applied in combination with other methods to achieve a more comprehensive sample characterization. TGA is often combined with differential scanning calorimetry or FTIR for analysis of reaction systems, to provide insights into the pyrolytic mechanisms and determine the thermal stability of isolated lignin.13 However, the main limitation is the lack of characterization of the nonvolatile portion of the sample, as up to 46 wt. % of lignin remains after analysis as biochar and ash.14 Py-GC-MS enables a detailed identification of individual compounds or pyrolytic breakdown product of lignin and its degradation products, although this method is typically set up as merely qualitative.15 The majority of Py-GC-MS studies are performed in an inert atmosphere within a range of 400–1000 °C (typically 600–800 °C).16 The most common method is a single step pyrolysis used to determine the syringol/guaiacol (S/G) ratio in lignin to classify the hardness of wood17 and to identify compounds from lignin degradation samples.16 However, 5 ACS Paragon Plus Environment


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this single pyrolysis step setup prevents differentiation of monomers from the oligomeric and polymeric products of higher MW. Only a few pyrolysis studies used calibration standards for product quantification, while most evaluated products of a single step pyrolysis based on normalized peak areas.18 To obtain insights into the structure of lignin and its degradation products, fractional PyGC-MS methods were implemented in which products are evolved at several sequential temperature steps (400–1050 °C). This was done to either investigate changes in S/G ratios of products or determine quantities of liquids and non-condensable gases.3 Pyrolytic, i.e., bondbreaking, conditions are considered to occur above 400 °C. Lignin, however, was shown to thermally degrade at temperatures as low as 230–260 °C.4 Therefore fractional lignin degradation were investigated at temperatures below pyrolytic conditions; i.e., conditions generally considered as thermal desorption (TD).19 The TD steps were integrated into fractional pyrolysis methods.20 Although an expansion of Py-GC-MS into lower, TD temperatures resulted in a better understanding of lignin degradation, quantification is still lacking and so normalized product yields are routinely reported.21 Looking for alternatives for lignin chemical analysis, we considered a different thermal method, which has been used in other fields of chemistry for characterization of complex matrices, such as atmospheric particulate matter (PM). Thermal optical analysis used in atmospheric studies is based on evolving carbon in the temperature range from 100 to 890 °C with and without oxygen while using a methanizer prior to a flame ionization detection (FID). A significant advantage of this method over TGA is the differentiation of organic and pyrolytic/coked black carbon (termed as elemental carbon in the atmospheric studies), which is distinguished using combination of thermal evolution with and without oxygen and by measuring 6 ACS Paragon Plus Environment

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laser transmittance as the sample evolves. Since all the carbon characterized in the biomass (e.g., lignin) is of organic nature, so the optical feature is not useful. Thus we postulate that thermal carbon analysis (TCA) desorption and pyrolysis profiles with and without oxygen may provide significant insights into the composition of lignin and its degradation products. In this study, we developed a quantitative TCA method based on the combination of TDPy and carbon analysis for characterization of lignin and its degradation products. This method aims to distinguish and quantify several fractions of organic carbon, i.e., volatile low-MW compounds, low-MW pyrolytic products, oligomeric fraction and the recalcitrant fraction ultimately yielding coke. We evaluated various TCA parameters including its temperature profile, sample loading, and the impact of solvent using standard lignin model low-MW compounds as well as untreated lignin with a goal to minimize both the volatility-based losses and coke formation caused by pyrolytic side reactions. The method was then applied to several different types of lignin and hydrotreated lignin samples and evaluated with respect to TGA, LLE with GC-MS, and TD-Py-GC-MS.

Experimental section Materials Solvents used included dichloromethane, DCM; and methanol, MeOH (VWR, Arlington Heights, IL, USA), which were GC and HPLC grade, respectively; as well as deionized water obtained from a Direct-Q 3 UV system purifier (Millipore, Billerica, MA, USA) with the total organic carbon content below 5 ppb. Glacial acetic acid (Fisher Scientific, Waltham, MA, USA) was HPLC grade. The standards used were purchased from Sigma-Aldrich (Milwaukee, WI,



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USA) see Supporting Figure S1 for structures. Bicreosol was synthesized using a published protocol.22 The details of this synthesis are provided under Supporting Information S2. Alkali lignin used for the hydrothermal treatment experiment was also purchased from Sigma-Aldrich with an elemental makeup of C (64.14 %), H (5.79 %), S (1.39 %) and N (0.46%). Hydrotreated samples were obtained upon an exposure of lignin to 300 °C (subcritical) water using a setup reported previously.23 Briefly, lignin (~0.25 g) and water (2.9 mL) were each added into stainless steel vessels with 0.71 cm i.d., being 6.325 cm long and having a total volume of 4.65 mL when closed, and pressure rated to 517 bar (Parker Hannifin Corp., Cleveland, OH, USA). The headspace, in the capped vessel, is essential to ensure that the internal pressure was governed by the steam/water equilibrium (i.e., pressure ~ 86 bar at 300 °C). [Safety note: It is imperative to have a sufficient headspace to allow formation of the steam/water equilibrium to avoid excessive pressures, which can occur within a completely filled cell].23 Vessels were heated in a GC oven, while being rotated at ~3 rpm. The reaction mixture was heated to a desired temperature monitored in a separate reaction vessel with a thermocouple. Then, the sample was kept at this temperature for 25 min. The reaction was quenched by flushing cold water over the vessel. Thermal carbon analysis A thermal optical analyzer from Sunset Laboratory Inc. (Portland, OR, USA) was employed to obtain quantitative thermal carbon profiles enabling a comprehensive carbon fractionation. The optical feature was not used, and hence, the term thermal carbon analysis. For TCA, the sample was introduced on a Pall Flex 2500QAT-UP tissue quartz filter (Pall Corp. East


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Hills, NY, USA) into the oven. The sample was thermally desorbed and pyrolyzed at tested temperatures in helium atmosphere followed by the addition of oxygen to evolve the coked fraction. The instrument was initially set up to purge ambient carbon dioxide for 2.4 min at 30 °C prior to the data acquisition. This step was later included as part of an acquisition allowing to quantify evolved CO2 and volatile components. The final temperature program consisted of a series of 6-min steps: TD steps at the ambient temperature, 200 and 300 °C, and Py temperatures, 400, 500 and 890 °C in the helium atmosphere, after that the oven was cooled to 550 °C and the gas mixture of He with 10% of O2 to evolve the remainder of the sample starting at 550 °C for 45 s followed by 625 °C for 45 s, 700 °C for 45 s, 775 °C for 45 s and 890 °C for 120 s. Once all sample carbon evolved, helium with 5% methane was introduced for internal calibration. The carbon that evolved during all temperature steps was converted to CO2 by flowing over a heated MnO2 catalyst at 870 °C followed by a conversion to CH4 in a methanizer and then detected with a FID. The external calibration (1.0–80 µg of C loaded) was performed using sucrose (ACS grade, Alfa Aesar, Ward Hill, MA, USA) dissolved in water. All experiments were performed in triplicate and reported as means with one standard deviation. The peak integrations were performed using Origin Pro 9.1 (Northampton, MA, USA). TGA A SDT Q600 TGA (TA Instruments, New Castle, DE, USA) was operated under N2 at a flow rate of 20 mL/min. Alkali lignin (~20 mg) was analyzed with a temperature ramp of 25 °C/min (to ensure controlled heating) between sequential steps of 200, 300, 400, 500 and 850 °C, each held for 5 min. The TGA lignin analysis was performed in triplicate.



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LLE-GC-MS For LLE, a 1.00 mL aliquot of an aqueous sample was spiked with 50 µL of 4chloroacetophenone (10.0 mg/mL) as a recovery standard, and then acidified to pH 4 with glacial acetic acid and extracted by vortexing three times with DCM (1.00 mL). The combined DCM phase was spiked with an internal standard, 75 µL o-terphenyl (10.0 mg/mL). The GC-MS (Agilent GC 7890 with 5975C MS) was equipped with a 51-m HP-5MS column (0.25 µm film thickness and 0.25 mm i.d.). The temperature program started at 40 °C for 1 min, followed by a gradient of 40 °C/min to 80 °C, and then 25 °C/min to 320 °C, and held for 5 min. The extract (0.2 µL) was injected in a splitless mode (20 s) at 300 °C at a helium flow rate of 1.5 mL/min. The MS transfer line was heated at 280 °C and solvent delay was set to 4.5 min. The MS with electron ionization was employed in the mass range of 33–550 m/z. The quantification was based on the calibration using characteristic ions for individual analytes (see Supporting Information S3). TD-Py-GC-MS TD-Py-GC-MS was performed using a CDS Analytical, Inc. (Oxford, PA, USA) 5200 pyroprobe (run in a direct pyrolysis mode) connected to the same GC-MS described above, but no solvent delay was used and the GC inlet was kept at 300 °C in a split mode 10:1. Prior to the analysis, the quartz tube with quartz wool was cleaned outside of the probe at 1200 °C for 5 s. The sample (2.0–5.0 µL) was then introduced onto the quartz wool and the inserted probe was heated at 10 °C/s to sequential fractional heating of 200, 300, 400, 500 and 870 °C. The probe was held at each temperature for 30 s to transfer on GC completing analysis after each heating step. The transfer line (leading to the GC inlet) and valve oven were kept at


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300 and 320 °C, respectively. The pyroprobe assembly (surrounding the heated probe) was held at 300 °C. Results and Discussion TCA applicability to lignin characterization The TCA characterization of alkali lignin (Figure 1) demonstrates the whole (bulk) sample thermal carbon fractionation allowing for mass balance closure. Fig. 1A shows the instrument readings while Fig. 1B presents the thermal fractionation data. First, the quantitative data comparable to those known for TGA were obtained at 100–850 °C in the He atmosphere. The subsequent heating with a mixture of He/O2 at 550–890 °C provided concentrations of the most recalcitrant, soot-like carbon fraction, which accounts for nearly 50% of the introduced carbon. The obtained TCA profile shows that a significant portion of lignin evolved at TD temperatures of

Thermal Carbon Analysis Enabling Comprehensive Characterization

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