Progress 09/01/03 to 08/31/06
Outputs
Separation and identification of compounds: The method developed to identify organic acids in a corn stover hydrolysate applies an initial class separation of degradation products using traditional analytical sample preparation techniques. Class mixtures are then further separated using HPLC prior to tandem mass spectrometry analysis (MS/MS). Systematic investigation of ionization modes showed that negative atmospheric pressure chemical ionization is the best choice for investigating a wide variety of degradation products. The methodology for qualitative analysis of hydrolysates using HPLC-MS/MS involves collection of an initial full-scan chromatogram for class mixtures. The nominal mass data for parent ions then enables tentative assignment of a molecular formula to elutedcompounds. Once these data are obtained, MS/MS data are collected on each parent ion with the mass analyzer operated in selected ion mode. Fragmentation patterns for each parent ion are then used to
postulate a plausible structure for the degradation product. This approach has enabled tentative structural identification of ca. 40 organic-acid degradation products. Positive assignment of chromatographic peaks is based on comparison of the HPLC-MS/MS data with a reference standard. Quantification of compounds: An HPLC method has been developed and validated, affording simultaneous determination of 40 degradation products in a single chromatographic run, of which 20 have been quantified with a high degree of certainty. The concentrations of analytes determined using this method are consistent with previous reports and show the major components to be aliphatic acids, furfural, and 5-HMF, with lignin decomposition products (e.g., aromatic acids and aldehydes) present at much lower concentrations. Method accuracy has been assessed by spiking a duplicate hydrolysate sample with a precisely known concentration of each analyte prior to analysis. Average percent recoveries calculated for
spiked samples generally demonstrate reliable determination of analyte concentrations within relevant statistical ranges. Method precision, measured as the relative standard deviation (R.S.D.) among replicate determinations, ranged from 1% to 29% with an average R.S.D. in unspiked and spiked samples of 9.9% and 8.3%, respectively, well within the acceptable range for determination of the measured concentrations in such a complex matrix using HPLC (typically 10%-15%). Response to reaction severity: The severity function is often used to correlate pretreatment product concentrations with temperature, reaction time and pH. It was found that in general, the majority of degradation compounds accumulated higher concentrations as the reaction severity increased. Some compound concentrations showed an inverse response to severity. Although a general trend with severity was noted in most cases, it was also found that the traditional severity function did not adequately predict accumulation
trends for most of the compounds quantified. In particular, it was noted that the temperature effects on product accumulations were usually considerably greater than predicted by the severity function.
Impacts
To our knowledge, the quantitative HPLC method developed in our laboratory represents one of very few examples where simultaneous separation and quantitation of aliphatic acids, aromatic acids, phenols, and aldehydes is accomplished in a single chromatographic run. More significantly, to the best of our knowledge, this work represents the only example where this has been performed on a biomass hydrolysate. Prior to initiating this study HPLC-MS/MS had not been extensively explored for the analysis of hydrolysate components other than sugars. However, our work to date confirms this to be a powerful tool for qualitative interrogation of these complex matrices. Results from this study represent a major advance in analytical capability and are attracting the attention of other research groups investigating biomass pretreatment. The analytical methodology is also expected to significantly impact existing efforts to close mass balance relationships. Finally, this is the only
study known to us that has tracked the effect of pretreatment severity on a wide variety of degradation products. Results of the severity studies are contributing to the understanding of the mechanisms of biomass hydrolysis and should contribute to improved mechanistic understanding of the degradation chemistry occurring during biomass pretreatment.
Publications
- Shou-Feng Chen, R. A. Mowery, V. A. Castleberry, G. P. van Walsum, and C. K. Chambliss 2005. Simultaneous HPLC Determination of Aliphatic Acids, Aromatic Acids, and Neutral Degradation Products in Biomass Pretreatment Hydrolysates, accepted for publication in J. Chrom. A., October, 2005.
- Shou-Feng Chen, Richard A. Mowery, G. Peter van Walsum, and C. Kevin Chambliss. Identification and Quantitation of Organic Degradation Products in Dilute-Acid-Catalyzed Corn Stover Pretreatment Hydrolysates. Conference proceedings of the AIChE annual meeting, Cincinnati, OH, Oct 30 - Nov 4, 2005. Paper # 371e, 19 pages.
|
Progress 10/01/03 to 09/30/04
Outputs
Separation and identification of compounds: The method developed to identify organic acids in a corn stover hydrolysate applies an initial class separation of degradation products using traditional analytical sample preparation techniques. Class mixtures are then further separated using HPLC prior to tandem mass spectrometry analysis (MS/MS). Systematic investigation of ionization modes showed that negative atmospheric pressure chemical ionization is the best choice for investigating a wide variety of degradation products. The methodology for qualitative analysis of hydrolysates using HPLC-MS/MS involves collection of an initial full-scan chromatogram for class mixtures. The nominal mass data for parent ions then enables tentative assignment of a molecular formula to elutedcompounds. Once these data are obtained, MS/MS data are collected on each parent ion with the mass analyzer operated in selected ion mode. Fragmentation patterns for each parent ion are then used to
postulate a plausible structure for the degradation product. This approach has enabled tentative structural identification of ca. 40 organic-acid degradation products. Positive assignment of chromatographic peaks is based on comparison of the HPLC-MS/MS data with a reference standard. Quantification of compounds: An HPLC method has been developed and validated, affording simultaneous determination of 40 degradation products in a single chromatographic run, of which 20 have been quantified with a high degree of certainty. The concentrations of analytes determined using this method are consistent with previous reports and show the major components to be aliphatic acids, furfural, and 5-HMF, with lignin decomposition products (e.g., aromatic acids and aldehydes) present at much lower concentrations. Method accuracy has been assessed by spiking a duplicate hydrolysate sample with a precisely known concentration of each analyte prior to analysis. Average percent recoveries calculated for
spiked samples generally demonstrate reliable determination of analyte concentrations within relevant statistical ranges. Method precision, measured as the relative standard deviation (R.S.D.) among replicate determinations, ranged from 1% to 29% with an average R.S.D. in unspiked and spiked samples of 9.9% and 8.3%, respectively, well within the acceptable range for determination of the measured concentrations in such a complex matrix using HPLC (typically 10%-15%). Response to reaction severity: The severity function is often used to correlate pretreatment product concentrations with temperature, reaction time and pH. It was found that in general, the majority of degradation compounds accumulated higher concentrations as the reaction severity increased. Some compound concentrations showed an inverse response to severity. Although a general trend with severity was noted in most cases, it was also found that the traditional severity function did not adequately predict accumulation
trends for most of the compounds quantified. In particular, it was noted that the temperature effects on product accumulations were usually considerably greater than predicted by the severity function.
Impacts
To our knowledge, the quantitative HPLC method developed in our laboratory represents one of very few examples where simultaneous separation and quantitation of aliphatic acids, aromatic acids, phenols, and aldehydes is accomplished in a single chromatographic run. More significantly, to the best of our knowledge, this work represents the only example where this has been performed on a biomass hydrolysate. Prior to initiating this study HPLC-MS/MS had not been extensively explored for the analysis of hydrolysate components other than sugars. However, our work to date confirms this to be a powerful tool for qualitative interrogation of these complex matrices. Results from this study represent a major advance in analytical capability and are attracting the attention of other research groups investigating biomass pretreatment. The analytical methodology is also expected to significantly impact existing efforts to close mass balance relationships. Finally, this is the
only study known to us that has tracked the effect of pretreatment severity on a wide variety of degradation products. Results of the severity studies are contributing to the understanding of the mechanisms of biomass hydrolysis and should contribute to improved mechanistic understanding of the degradation chemistry occurring during biomass pretreatment.
Publications
- Shou-Feng Chen, Richard A. Mowery, G. Peter van Walsum, and C. Kevin Chambliss. 2005. Identification and Quantitation of Organic Degradation Products in Dilute-Acid-Catalyzed Corn Stover Pretreatment Hydrolysates. Presented at the AIChE annual meeting, Cincinnati, OH, Oct. 30-Nov 4, 2005.
- Shou-Feng Chen, Richard A. Mowery, Melinka Arispe-Angulo, Vanessa A. Castleberry, G. Peter van Walsum, and C. Kevin Chambliss. 2005. Identification and Quantitation of Organic Degradation Products in Dilute-Acid-Catalyzed Corn Stover Pretreatment Hydrolysates. Presented at the 27th Symposium on Biotechnology for Fuels and Chemicals, Denver, CO. May 1-4, 2005.
- G. Peter van Walsum. 2005. Towards a Diagnostic Assessment of Microbial Inhibition in Bioconversion of Lignocellulose to Fuels and Chemicals. Presented at the University of Minnesota Research Colloquium. April 14, 2005.
- S.-F. Chen, R. A. Mowery, V. A. Castleberry, G. P. van Walsum, and C. K. Chambliss, 2004. HPLC-MS Analysis of Organic Acids in Biomass Pretreatment Hydrolysates, Presented at the 26th Symposium on Biotechnology for Fuels and Chemicals, Chattanooga, TN, May 9-12, 2004.
- S.-F. Chen, R. A. Mowery, V. A. Castleberry, G. P. van Walsum, and C. K. Chambliss 2005. Simultaneous HPLC Determination of Aliphatic Acids, Aromatic Acids, and Neutral Degradation Products in Biomass Pretreatment Hydrolysates, accepted for publication in J. Chrom. A., October, 2005.
- Shou-Feng Chen, Richard A. Mowery, G. Peter van Walsum, and C. Kevin Chambliss. 2005. Identification and Quantitation of Organic Degradation Products in Dilute-Acid-Catalyzed Corn Stover Pretreatment Hydrolysates. Conference proceedings of the AIChE annual meeting, Cincinnati, OH, Oct. 30-Nov. 4, 2005. Paper # 371e, 19 pages.
- Mowery, R. A.; Chen, S.-F.; Castleberry, V. A.; van Walsum, G. P.; Chambliss, C. K. 2006. Qualitative analysis of organic acids in a corn stover hydrolysate using high-performance liquid chromatography with tandem mass spectrometry, manuscript in preparation.
|