Atmospheric pressure ionization mass spectrometry as a tool for structural characterization of lignin.

RATIONALE: Lignin occurs in a broad range of forms, e.g., native as the main support for plant walls, and processed, for which its structure depends on the nature of the industrial isolation method, such as in paper production or in biorefineries. Due to the variety of lignin sources, there is no unified agreement on the structure of lignin or even its molecular weight (MW). METHODS: The focus of this review is on the application of atmospheric pressure ionization methods to lignin analysis by mass spectrometry (MS), namely electrospray ionization (ESI) or direct analysis in real-time (DART). Specific parameters affecting ionization including electrolytes and solvents are discussed. RESULTS: The main challenge for MW determination of lignin is its heteropolymer character as well as the mass range limitations of MS instrumentation. To date, only a few studies have successfully used the mass range above m/z 1500. We present the advantage of ESI in generating multiply charged ions, allowing for a further increase in the mass range of deconvoluted mass spectra. While some methods such as DART do not address the mass range problem, they may serve as excellent imaging tools suitable for structural characterization of lignin. CONCLUSIONS: A literature review presents the recent accomplishments in lignin MS analysis by atmospheric pressure ionization techniques. Although significant breakthroughs have been made, it is essential to further improve the operating conditions and validate the methods for a broader range of feedstocks with the results being confirmed using other methods.

Influence of early stages of triglyceride pyrolysis on the formation of PAHs as coke precursors.

Molecular beam (MB) time-of-flight mass spectrometry has been used to investigate thermal decomposition of triolein, to reveal the mechanisms of low temperature soot/coke formation characteristic for triglycerides (TGs). Mass detected pyrolysis products were observed at incremented temperatures using both VUV single photon ionization (general product detection) and REMPI based selective detection of aromatic products. To augment the simple mass characterizations, we have employed stoichiometric considerations; we have supplemented the analysis further by using the detailed information available from product analysis of batch reactor TG cracking. Both the VUV photoionization and batch reactor studies indicated that formation of C7-sized stable products is a marker of significant triolein decomposition that is coupled with PAH formation. A significant fraction of the C7 species observed likely formed as a result of a C-C bond scission at the allylic position to the ω-9 double bond of oleic acid. REMPI detection indicated a high specificity for PAH formation at three distinct molecular weight values, 276, 352 and 444 amu (the latter being a fullerene precursor). The stoichiometric analysis has shown that these PAHs likely arise from condensation reactions of either C7- or C8-sized fragments (three, four and five, respectively). The C8-sized intermediate would become essential whenever the PAH product of C7 fragment condensation contained an odd number of carbon atoms, resulting in a less stable aromatic structure with an incomplete double bond conjugation. MB experiments involving either addition or in situ generation of hydrogen resulted in an enhancement of lower molecular weight PAH formation, i.e., a decrease in the effective number of condensing fragments. In contrast, an increase in temperature yielded the opposite effect.

Unfolding of Lignin Structure Using Size-Exclusion Fractionation.

The heterogeneous and recalcitrant structure of lignin hinders its practical application. Here, we describe how new approaches to lignin characterization can reveal structural details that could ultimately lead to its more efficient utilization. A suite of methods, which enabled mass balance closure, the evaluation of structural features, and an accurate molecular weight (MW) determination, were employed and revealed unexpected structural features of the five alkali lignin fractions obtained with preparative size-exclusion chromatography (SEC). A thermal carbon analysis (TCA) provided quantitative temperature profiles based on sequential carbon evolution, including the final oxidation of char. The TCA results, supported with thermal desorption/pyrolysis gas chromatography-mass spectrometry (TD-Py-GC-MS) and 31P NMR spectroscopy, revealed the unfolding of the lignin structure as a result of the SEC fractionation, due to the disruption of the interactions between the high- and low-MW components. The "unraveled" lignin revealed poorly accessible hydroxyl groups and showed an altered thermal behavior. The fractionated lignin produced significantly less char upon pyrolysis, 2 vs. 47%. It also featured a higher occurrence of low-MW thermal evolution products, particularly guaiacol carbonyls, and more than double the number of OH groups accessible for phosphitylation. These observations indicate pronounced alterations in the lignin intermolecular association following size-exclusion fractionation, which may be used for more efficient lignin processing in biorefineries.

Quantitative insights on de/repolymerization and deoxygenation of lignin in subcritical water.

The objective of the study was to investigate alkali lignin polymerization/depolymerization pathways in subcritical water (SW) without additives. Following a SW treatment at 200, 250, 275 and 300 °C, the products were subjected to a comprehensive suite of analyses addressing the product speciation and molecular weight (MW) distribution. The MW reduction (1.4 times) in the solid products following the SW treatment indicated a surprisingly reduced impact of cross-linking/repolymerization at 300 °C and lower temperatures. This was further confirmed by thermal carbon analysis (TCA) showing a reduction in pyrolytic charring after the SW treatment. The TD-Py gas chromatography analysis of the SW treated lignin indicated that the solid residue is less oxygenated than the initial lignin (23 vs. 29% as confirmed by elemental analysis). Thus, deoxygenation rather than re-polymerization appears to be the main process route in the absence of catalysts within the temperature range considered.

Metformin Uptake and Translocation in Chickpeas: Determination Using Liquid Chromatography-Mass Spectrometry.

Multiple chronic conditions (MCCs) such as diabetes, hypertension, heart disease, arthritis, asthma, and common respiratory problems are prevalent in over one-fourth of Americans, and separate drugs are prescribed to manage each of the diseases. The nutritive crop seeds loaded with multiple drugs could be a cheap and sustainable alternative to drugs produced by pharmaceutical companies. Our long-term goal is to produce chickpea seeds containing comparable dosages of multiple drugs regularly prescribed for managing MCC. In this work, we conducted experiments to understand the uptake and translocation of metformin into the tissues of chickpea to demonstrate the applicability of LC-HR-ToF-MS in determining metformin concentration, and to investigate responses of increased dosage of metformin and it's accumulation into the chickpea seed. We treated the chickpea plants with 100 and 500 mg/L metformin chloride and analyzed its concentration in the leaf, stem, and seeds. We observed that metformin was successfully uptaken by chickpeas plant and translocated to stem, leaf, and seeds in both treatments. We also observed that the metformin concentration is responsive and as high as 349 times increase in seed when the dosage was increased from 100 to 500 mg/L.

Simultaneous high-temperature gas chromatography with flame ionization and mass spectrometric analysis of monocarboxylic acids and acylglycerols in biofuels and biofuel intermediate products.

Triacyl-, diacyl- and monoacylglycerols (TAGs, DAGs, MAGs) along with monocarboxylic acids (MCAs) are intermediate products in many triacylglycerol oil-to-biofuel conversion pathways. Accumulation of these compounds leads to poor biofuel characteristics and may result in fuel system damage. We developed a method for simultaneous identification and quantification of a wide range of MCAs (C4-C18), MAGs, DAGs, and TAGs. The method is based on trimethylsilylation followed by high temperature GC with programmed temperature vaporizer (PTV) injection coupled to parallel FID and MS detectors (HTGC-FID/MS). To minimize the discrimination of both low and high molecular weight species typically occurring on the injector, we optimized injection conditions using a central composite design. The critical variables were the time at initial temperature (40 °C), splitless time, and the interaction between these two parameters. Among three tested electron ionization source/quadrupole analyzer temperatures, a 350/200 °C setting provided the highest response and signal-to-noise ratio for TAGs and did not have an effect on MAGs and DAGs. Similar results were obtained when quantifying target analytes in intermediate products of soybean oil cracking with FID and MS (using specific acylglycerol fragmentation ions). The instrumental FID limits of detection (LODs) were 0.07-0.27 ng for most of the target analytes. Selected ion monitoring (SIM) LODs were 0.01-0.05 ng for MCAs and 0.03-0.14 ng for acylglycerols. For the total ion current (TIC), LODs observed increased with acyl chain length and degree of unsaturation, resulting in an increase from 0.05 to 0.18 ng for MCAs (C5 to C18) and from 0.03 to 1.8 ng for acylglycerols (TAGs C8 to C22). Deviations in the repeatability of sample preparation, intra- and inter-day analyses, including sample stability over an eight-day time period, did not exceed 10% variance. These results demonstrate that the developed method is accurate and robust for the determination of acylglycerols and MCAs produced during the processing of TAGs into biofuels.

Electrospray Ionization with High-Resolution Mass Spectrometry as a Tool for Lignomics: Lignin Mass Spectrum Deconvolution.

The capability to characterize lignin, lignocellulose, and their degradation products is essential for the development of new renewable feedstocks. Electrospray ionization high-resolution time-of-flight mass spectrometry (ESI-HR TOF-MS) method was developed expanding the lignomics toolkit while targeting the simultaneous detection of low and high molecular weight (MW) lignin species. The effect of a broad range of electrolytes and various ionization conditions on ion formation and ionization effectiveness was studied using a suite of mono-, di-, and triarene lignin model compounds as well as kraft alkali lignin. Contrary to the previous studies, the positive ionization mode was found to be more effective for methoxy-substituted arenes and polyphenols, i.e., species of a broadly varied MW structurally similar to the native lignin. For the first time, we report an effective formation of multiply charged species of lignin with the subsequent mass spectrum deconvolution in the presence of 100 mmol L-1 formic acid in the positive ESI mode. The developed method enabled the detection of lignin species with an MW between 150 and 9000 Da or higher, depending on the mass analyzer. The obtained M n and Mw values of 1500 and 2500 Da, respectively, were in good agreement with those determined by gel permeation chromatography. Furthermore, the deconvoluted ESI mass spectrum was similar to that obtained with matrix-assisted laser desorption/ionization (MALDI)-HR TOF-MS, yet featuring a higher signal-to-noise ratio. The formation of multiply charged species was confirmed with ion mobility ESI-HR Q-TOF-MS. Graphical Abstract ᅟ.

Size exclusion chromatography of lignin: The mechanistic aspects and elimination of undesired secondary interactions.

Characterization of lignin and its degradation products, more specifically determination of their molecular weight (MW) distribution, is essential for assessment and applications of these potentially renewable phenolics. Several representative gel filtration and gel permeation systems were evaluated in this work focusing on understanding of undesired secondary non-SEC interactions while utilizing four sets of commercially available polymeric standards as well as low-MW lignin model compounds including diarene standards synthesized in-house. The gel permeation column with a nonpolar highly cross-linked porous polystyrene/divinylbenzene-based stationary phase provided the most effective separation by MW for both low and high MW model compounds. Notably, the column with a higher pore and lower particle size provided a better resolution towards polymeric standards, even though the particle size effect was downplayed in the earlier SEC studies of lignin. For two other evaluated gel filtration and gel permeation columns, the separation was strongly affected by functionalities of the analytes and correlated with the compounds' pKa rather than MW. We showed that the separation on the stationary phases featuring polar hydroxyl groups led to specific column-analyte secondary interactions, perhaps based on their hydrogen bonding with lignin. Further, the SEC column evaluation yielded similar results with two sets of chemically different standards. This setup may be used as a general approach to selecting an applicable column for lignin SEC analysis. We confirmed the obtained results with a different independent method implementing a novel approach for lignin number-average MW (Mn) calculation based on laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) data. The determined Mn corroborated the SEC results.

Production of lignin based insoluble polymers (anionic hydrogels) by C. versicolor.

Unlike previous lignin biodegradation studies, white rot fungi were used to produce functional biopolymers from Kraft lignin. Lignin-based polymers (hydrogel precursors) partially soluble in both aqueous and organic solvents were produced employing a relatively fast (6 days) enzymation of Kraft lignin with basidiomycetes, primarily Coriolus versicolor, pre-grown on kenaf/lignin agar followed by either vacuum evaporation or acid precipitation. After drying followed by a treatment with alkaline water, this intermediate polymer became a pH-sensitive anionic hydrogel insoluble in either aqueous or organic solvents. The yield of this polymer increased from 20 to 72 wt% with the addition of 2% dimethylsulfoxide to distilled water used as a medium. The mechanical stability and buffering capacity of this hydrogel can be adjusted by washing the intermediate polymer/hydrogel precursor prior to drying with solvents of different polarity (water, methanol or ethanol). Any of these polymers featured a significant thermal resilience assessed as a high thermostable "coked" fraction in thermal carbon analysis, apparently resulting from significant covalent cross-linking that occurs during the treatment of their intermediate precursors.