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

Capability to characterize lignin, lignocellulose, and their degradation products is essential for development of new renewable feedstocks. Electrospray ionization high-resolution time-offlight 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 intact 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 9,000 Da or higher, depending on the mass analyzer. The obtained Mn and Mw values of 1,500 and 2,500 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 matrixassisted laser desorption/ionization (MALDI) TOF MS, yet featuring a higher signal-to-noise ratio. The formation of multiply charged species was confirmed with ESI ion mobility HR Q-TOF MS

Fast and efficient size-based separations of polymers using ultra-high-pressure liquid chromatography

Ultra-high-pressure liquid chromatography (UHPLC) has great potential for the separations of both small molecules and polymers. However, the implementation of UHPLC for the analysis of macromolecules invokes several problems. First, to provide information on the molecular-weight distribution of a polymer, size-exclusion (SEC) columns with specific pore sizes are needed. Development of packing materials with large pore diameters and pore volumes which are mechanically stable at ultra-high-pressures is a technological challenge. Additionally, narrow-bore columns are typically used in UHPLC to minimize the problem of heat dissipation. Such columns pose stringent requirements on the extra-column dispersion, especially for large (slowly diffusing) molecules. Finally, UHPLC conditions generate high shear rates, which may affect polymer chains. The possibilities and limitations of UHPLC for size-based separations of polymers are addressed in the present study. We demonstrate the feasibility of conducting efficient and very fast size-based separations of polymers using conventional and wide-bore (4.6 mm I.D.) UHPLC columns. The wider columns allow minimization of the extra-column contribution to the observed peak widths down to an insignificant level. Reliable SEC separations of polymers with molecular weights up to ca. 50 kDa are achieved within less than 1 min at pressures of about 66 MPa. Due to the small particles used in UHPLC it is possible to separate high-molecular-weight polymers (50 kDa ≤ Mr ≤ 1-3 MDa, upper limit depends on the flow rate) in the hydrodynamic-chromatography (HDC) mode. Very fast and efficient HDC separations are presented. For very large polymer molecules (typically larger than several MDa, depending on the flow rate) two chromatographic peaks are observed. This is attributed to the onset of molecular deformation at high shear rates and the simultaneous actions of hydrodynamic and slalom chromatography

Challenges in polymer analysis by liquid chromatography

Synthetic polymers are very important in our daily life. Many valuable properties of polymers are determined by their molecular weight and chemical composition. Liquid chromatographic (LC) techniques are very commonly used for molecular characterisation of polymers. LC analysis of macromolecules is more challenging than analysis of low-molecular-weight compounds, because of polymer dispersity, chemical heterogeneity (several polymer distributions within one sample), poor solubility of many engineering plastics in common chromatographic solvents, and other factors. The present review focuses on difficulties associated with LC analysis of synthetic polymers. The approaches that allow bringing poorly soluble polymers within the scope of LC are discussed. Different LC modes used for polymer separations are reviewed and associated practical challenges are identified. Aspects of optimization of separations in terms of resolution (retention factors, selectivity and efficiency) and analysis time are discussed. Modern technologies (core-shell stationary phases, monolithic columns, and sub-2 μm particles) that may positively affect the trade-off between speed of analysis and efficiency are considered in this respect. Finally, the issue of detection in LC of polymers is addressed. The advantages and limitations of different detection techniques as well as hyphenated techniques are discusse

Comprehensive two-dimensional ultrahigh-pressure liquid chromatography for separations of polymers

Online comprehensive two-dimensional liquid chromatography (LC × LC) is a technique of great importance, because it offers much higher peak capacities than separations in a single dimension. When analyzing polymer samples, LC × LC can provide detailed information on two mutually dependent polymer distributions. Because both molecular-weight distributions and chemical-composition distributions are typically present in synthetic copolymers, combinations of interactive LC with size-exclusion chromatography (SEC) are especially useful for (co)polymer analyses. Commonly applied SEC separations in the second dimension take several minutes, so that a total LC × LC experiment typically requires several hours. This renders LC × LC unsuitable for routine analysis. In the present study we have explored possibilities to perform fast and efficient online comprehensive two-dimensional analysis of polymers using contemporary ultrahigh-pressure liquid chromatography in both dimensions (UHPLC × UHPLC). Gradient-elution UHPLC in the first dimension allowed efficient separations of polymers based on their chemical composition. SEC at ultrahigh-pressure conditions in the second dimension offered very fast, yet efficient separations based on molecular size. The demonstrated UHPLC × UHPLC separations of industrial polymers could be performed within 1 h and provided comprehensive information on two-dimensional distribution

Selective chromatographic separation of polycarbonate according to hydroxyl end-groups using a porous graphitic carbon column

Porous graphitic carbon (PGC) has shown unique separation efficiency in liquid chromatography for a wide range of substance classes. In the characterization of polymers PGC has particularly been used for analysis of polyolefins. Its retention mechanisms differ dramatically from those of silica-based stationary phases and therefore allow interesting applications. Due to its unprecedented retention mechanisms PGC does not only promise good separation performance for polyolefins but also for more polar polymers such as Polycarbonate (PC). In this study, we determined the critical conditions of PC on PGC usingCHCl3/dichlorobenzene (DCB) and CHCl3/trichlorobenzene (TCB) as eluents achieving separations according to hydroxyl end-groups, which was confirmed by MALDI-TOF-MS analysis. As the content of TCB at the critical point was lower compared to that of DCB, it was concluded that TCB is a stronger desorption promoting eluent than DCB for the present system. The temperature influence on the critical point was then investigated revealing that with increasing temperature the content of desorption promoting eluent has to be raised in order to achieve critical conditions. Furthermore, a peak shifting over time was observed using TCB as desorption promoting eluent, which was attributed to irreversibly adsorbed PC on the column material. However, when a flow cell-IR detector was applied monitoring the eluted samples, a recovery rate close to 100% was found