Mass spectrometric studies of cellulose ethers

Cellulose based derivatives, such as cellulose ethers, today find many applications in a diverse range of fields. The numerous applications put high demands on the technological performance of the cellulose ethers. In order to be able to control the technological properties it is necessary to develop analytical methods by, which they can be correlated to the chemical structure. In some cases variations in the technological properties cannot be explained by the chemical characterisation methods available today. Thus, it is of high importance to investigate the suitability of new analytical techniques for chemical characterisation of cellulose ethers. In this thesis matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and nanoelectrospray ionisation (nano-ESI)-MS were studied as analytical techniques for cellulose ethers. Among the results, MALDI tandem mass spectrometry (MS/MS) and nano-ESI-MS/MS were demonstrated as exceptionally useful tools for the structure analysis of cellulose ether oligosaccharides. Nano-ESI-MS/MS was employed to characterise the selectivity of a cellulose depolymerising enzyme, endoglucanase, on methyl cellulose (MC). Novel dialkylamine derivatives of cellulose ether oligosaccharides were shown to increase the sensitivity in both MALDI- and nano-ESI-MS, and to facilitate structure analysis by MS/MS. Studies of the molar mass distribution of partially depolymerised MC were performed by size-exclusion chromatography semi-online to MALDI-MS. The sample preparation method for MALDI was found to have a significant influence on the outcome of the analysis. Furthermore, a simple experiment showed that, the combination of MALDI-MS and partial acidic depolymerisation could be used to distinguish between cellulose ethers of different degrees of substitution

Improved MALDI-TOF-MS analysis of partially depolymerised methyl cellulose by reducing end derivatisation

The depolymerized methyl cellulose was analyzed, using improved MALDI-TOF-MS by reducing the end derivatization. The methyl cellulose was depolymerized to give methylated oligo-(1->4)-β-D-anhydroglucoses (M-oligo-AG). To reduce the errors during measurement of degree of substitution (DS), M-oligo-AG was derivatized with Girard's T reagent performed on an equimolar mixture of maltooligosaccharides. The GT-derivatized M-oligo-AG (GT-M-oligo-AG) formed singly charged molecular ions with peaks at m/z values 91 higher than the corresponding peaks of M-oligo-AG. The results show that for GT-M-oligo-AG the relative signal intensities for DP=1 and 2 were increased significantly

Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and structural analysis of enzyme active sites

The properties of modified cellulose polymers, such as methylcellulose, are significantly influenced by the distribution of substituents along the polymer backbone. This distribution is difficult to determine due to the lack of suitable analytical methods. One approach is to use cellulose-degrading enzymes to gain information from the capability of the enzymes to cleave the bonds between glucose units. Endoglucanases are cellulase enzymes that can break internal glycosidic linkages and degrade low substituted regions of modified cellulose where the substituents do not interfere with the enzyme active site. In this work methyl cellulose was degraded using five endoglucanases from glycosyl hydrolase families 5 and 7 from three different species. The products were analyzed with reducing end analysis, chromatography (SEC-MALS-RI), and MALDI-TOFMS. The results were correlated with available determined enzyme structures and using structural alignment for unknown enzyme structures. This was performed in order to elucidate the relationship between active site structures and sensitivity for substituents on derivatized cellulose. The evaluation of endoglucanase hydrolysis of methyl cellulose showed that differences in sensitivity could be related to differences in steric hindrance of substituents in the active site, which could explain differences within family 5 and 7 enzymes, as well as the generally higher substituent tolerance for family 5 enzymes. This information is important for use of endoglucanases as tools for characterization of substituent distribution. The results are also valuable since soluble cellulose derivatives are generally used as substrates during enzyme characterization and in endoglucanase activity assays

Size and Structure Characterization of Ethylhydroxyethyl Cellulose by the Combination of Field-Flow Fractionation with Other Techniques. Investigation of Ultralarge Components

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 104 up to 109 g·mol-1. The highest viscosity class EHEC III was less size-dispersed covering molar masses from 5 × 105 to 5 × 107 g·mol-1. Filtering of the EHEC II solution removed small amounts of compact UHM material. Enzyme treatments were performed on EHEC II to further characterize it. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and anion ion-exchange chromatography coupled to pulsed amperometric detection showed that the UHM component contained EHEC

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of partially depolymerised carboxymethyl cellulose

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) of partially depolymerised carboxymethyl cellulose (CMC) have been investigated. The depolymerisation was either enzymatic or acidic. Fractions of enzymatically depolymerised CMC were collected from size-exclusion chromatography (SEC) and further investigated by MALDI-TOFMS. 2,5-Dihydroxybenzoic acid was used as matrix, dissolved in H2O due to the poor solubility of CMC in suitable organic solvents. The samples were dried by two methods, in ambient atmosphere and at reduced pressure. Under reduced pressure the sample spot homogeneity increased. This drying method, however, produced additional adduct peaks in the mass spectra originating from ion exchange on the CMC oligomers. Analysis of CMC could be performed in both negative and positive ion modes. Mass discrimination and variation in ionisation efficiency were demonstrated by comparing mass spectra with SEC data. Measurements of the degree of substitution (DS) were performed on three CMCs with different DS values, which were depolymerised in trifluoroacetic acid. The three CMCs were easily distinguished from one another, but the obtained DS values deviated from the values supplied by the manufacturer. Copyright © 2003 John Wiley & Sons, Ltd

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of partially depolymerised methyl cellulose

Methyl cellulose (MC) was partially depolymerised and the oligomers thus obtained were studied by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). The depolymerisation was either enzymatic or acidic. Fractions of enzymatically depolymerised MC were collected from size-exclusion chromatography and subjected to a sample preparation investigation. Several MALDI matrices and solvents were evaluated. The results showed that the solvent choice had a significant effect on the measured degree of substitution (DS). Aprotic solvents produced higher DS values, which was most likely due to poor solubility of species with low DS. The obtained signal intensity, however, did not correlate with the solubility but seemed to be more dependent on certain matrix/solvent combinations. All the matrices attempted produced mass spectra with sufficient signal intensity for accurate peak area calculation. The choice of matrix did not have any significant effect on the measured DS. Sample spots obtained from organic solvents had a more homogeneous distribution of the analyte and smaller crystals than those obtained from water. This increased both the reproducibility and peak resolution and in addition the analysis time was shorter. DS measurements were performed on two acidically depolymerised MCs with different nominal DS values. It was easy to distinguish between the two MCs, and the measured DS values agreed well with the values supplied by the manufacturers. Copyright © 2003 John Wiley & Sons, Ltd

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated