Quantitation of Sugar Content in Pyrolysis Liquids after Acid

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Quantitation of Sugar Content in Pyrolysis Liquids after Acid Hydrolysis Using High-Performance Liquid Chromatography without Neutralization Patrick A. Johnston† and Robert C. Brown*,†,‡ †

Bioeconomy Institute and ‡Department of Mechanical Engineering, Iowa State University, Ames, Iowa 50011, United States ABSTRACT: A rapid method for the quantitation of total sugars in pyrolysis liquids using high-performance liquid chromatography (HPLC) was developed. The method avoids the tedious and time-consuming sample preparation required by current analytical methods. It is possible to directly analyze hydrolyzed pyrolysis liquids, bypassing the neutralization step usually required in determination of total sugars. A comparison with traditional methods was used to determine the validity of the results. The calibration curve coefficient of determination on all standard compounds was >0.999 using a refractive index detector. The relative standard deviation for the new method was 1.13%. The spiked sugar recoveries on the pyrolysis liquid samples were between 104 and 105%. The research demonstrates that it is possible to obtain excellent accuracy and efficiency using HPLC to quantitate glucose after acid hydrolysis of polymeric and oligomeric sugars found in fast pyrolysis bio-oils without neutralization. KEYWORDS: HPLC, biomass, bio-oil, acid hydrolysis, levoglucosan, fast pyrolysis, carbohydrates

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and levoglucosan to glucose.8,9 However, these traditional HPLC methods can be cumbersome. Methods such as ASTM E1758 “Standard Method for the Determination of Carbohydrates by HPLC” and the National Renewable Energy Laboratory’s (NREL) “Determination of Structural Carbohydrates in Biomass” require a neutralization step after hydrolyzing polysaccharides and oligosaccharides to glucose.10,11 Researchers analyzing wood sugar using other liquid chromatography techniques have been successful in performing analysis without neutralization. Petterson et al.12 performed wood sugar analysis using anion-exchange chromatography with a pulsed amperometric detector (PAD) after hydrolysis without neutralization. The main problem with the conventional methods of analysis using neutralization is the loss of sugars if the pH is not carefully monitored. The NREL methodology states: “Samples should never be allowed to exceed a pH of 9, as this will result in a loss of sugars”.10 Miller13 showed spectrophotometer evidence that a significant reduction of glucose was observed with increasing concentrations of sodium hydroxide. Rover et al.14 determined that a 7% decrease of sugar was observed when the pH was increased to 11. A new HPLC method recently established at Iowa State University (ISU) enables researchers to avoid the neutralization step. Hence, total sugar in lignocellulosic fast pyrolysis oils can be analyzed more rapidly. The new method also helps to prevent degradation and loss of sugars that can sometimes result from neutralization if the pH is not carefully monitored.10

INTRODUCTION Direct liquefaction is the thermal decomposition of solid organic material into condensable vapors, liquids, or solutions of organic compounds. Examples include fast pyrolysis, solvent liquefaction, and hydrothermal processing. Under appropriate conditions, direct liquefaction can depolymerize lignocellulosic biomass into monosaccharides or disaccharides.1 Sugars have not received much attention because they have not traditionally been thought to be an important product of direct liquefaction and the difficulty of measuring them in the complex matrix of the liquid products of direct liquefaction. We are developing analytical methods for measuring sugars in the products of direct liquefaction with a particular focus on bio-oil, the liquid product from the fast pyrolysis of lignocellulosic biomass. Fast pyrolysis is the thermal decomposition of organic compounds in the absence of oxygen at temperatures in the range of 400−600 °C.2,3 Products include liquids (55−60 wt %), gases (12−22 wt %), and char (16−20 wt %).4−6 The liquids are a complex mixture of organic acids, alcohols, aldehydes, ketones, phenols, carbohydrates, esters, lignin oligomers, and water.3 The carbohydrates include monosaccharides and anhydrosugars, most prominently levoglucosan. Usually the yield of levoglucosan from the pyrolysis of lignocellulosic biomass is only a few percent, but Kuzhiyil et al.1 found that infusing biomass with a weak mineral acid solution prior to pyrolysis increased levoglucosan yields from the cellulose fraction to as much as 59 wt %. These sugars can be upgraded into transportation fuels and chemicals via fermentation to alcohols, catalytic synthesis to hydrocarbons, dehydration to heterocyclic aldehydes (furfurals) and aromatics (furans), and aqueous-phase re-forming to produce alkanes as building blocks of gasoline.3,7 Analytical techniques such as high-performance liquid chromatography (HPLC) are commonly used to identify and quantitate sugar concentrations by hydrolyzing oligosaccharides © XXXX American Chemical Society

Received: May 14, 2014 Revised: July 28, 2014 Accepted: July 30, 2014

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dx.doi.org/10.1021/jf502250n | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Comparison of standard compounds using HPLC: (A) acidic hydrolysate (new method); (B) neutralized hydrolysate (traditional method).

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hydrolysis. These standards were hydrolyzed under the same conditions as the samples to establish complete hydrolysis to glucose and used as reference standards. The reference standards had purities of ≥99.0%. After hydrolysis, all samples were filtered with a 0.45 μm glass microfiber (GMF) syringe filter (Whatman, Pittsburgh, PA, USA) before HPLC analysis. With the traditional method an extra neutralization step was completed before the filtration. The neutralization step was completed as per ASTM E1748 by adding calcium carbonate slowly until a pH of between 5 and 6 was attained. The samples were filtered with a 0.45 μm GMF syringe filter before HPLC analysis. Instrumentation. The HPLC system used for the experiments was a Dionex Ultimate 3000 LC system (Sunnyvale, CA, USA) with a quaternary analytical pump and a Shodex refractive index (RI) detector (New York, NY, USA). Two different types of columns were used in the experiments. The analytical columns used were either a 300 mm × 7.7 mm i.d, 8 μm, HyperRez XP Carbohydrate (Thermo Fisher Scientific) or a 300 mm × 7.8 mm i.d., 9 μm, Aminex 87P (BioRad, Hercules, CA, USA). The guard column for the Aminex was a Micro-Guard de-ashing cartridge with a de-ashing holder (Bio-Rad). The guard column for the HyperRez was a Carbohydrate H+ cartridge with a guard holder (Thermo Fisher Scientific). The guard columns were used to eliminate possible interferences caused by anions and cations. The instrument parameters for the HyperRez were as follows: The mobile phase was ultrapure 18.2 MΩ deionized water with a flow rate of 0.2 mL/min, and the column temperature was set at 55 °C. The instrument parameters for the Aminex column were as follows: The mobile phase was ultrapure 18.2 MΩ deionized water with a flow rate of 0.6 mL/min, and the column temperature was set at 80 °C. The

MATERIALS AND METHODS

Reagents and Standards. Levoglucosan and cellobiosan were purchased from Carbosynth (Compton, Berkshire, UK) and had purities of ≥99.0%. Glucose and xylose were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and had purities ≥98.0%. Cellobiose and sorbitol were purchased from Acro̅s Organics (Thermo Fisher Scientific) and had purities of ≥99.0 and ≥98.5%, respectively. All samples and standards solutions were prepared using ultrapure 18.2 MΩ deionized water from a Barnstead E-Pure system (Thermo Fisher Scientific). The sulfuric acid used in all experiments was certified 10 N with an assay of 9.95−10.05 (Thermo Fisher Scientific). Pyrolysis Liquid Samples. The pyrolysis liquid used in this study was produced using a fast pyrolysis auger reactor and switchgrass feedstock.15,16 The reactor was a benchtop 2 kg/h screw (auger) type fast pyrolysis reactor. The feedstock is first mixed with the heat carrier media in the reactor. After the biomass has been thermochemically processed, the particulate matter (char) is removed from the gas by two cyclones. In the final stages the gases are condensed out at different temperatures to produce four fractions of bio-oil. The polysaccharide-rich portion was found mainly in the first two condensers and electrostatic precipitator (ESP) stage fractions. Acid Hydrolysis of Bio-Oil Samples. The anhydro and other polymeric/oligomeric sugars in the fast pyrolysis bio-oil were acid hydrolyzed with 400 mM H2SO4 at 125 °C for 44 min to glucose.17 Hydrolysis conditions were based on the previous work of Bennett et al.17 Aliquots of 6 mL of 400 mM H2SO4 and 60 mg of bio-oil were added to sealed glass vials. Pure compounds of levoglucosan and cellobiosan were used as check standards to determine complete B

dx.doi.org/10.1021/jf502250n | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Comparison of equivalent retention times of standard compounds (A) cellobiosan and (B) glucose with the traditional method. injection amount for the HyperRez column was 25 μL and the Aminex column was 20 μL.

duplicate, and triplicate injections were performed on each sample. The results displayed are based on total polymeric/ oligomeric sugars that are converted to glucose after acid hydrolysis. Figure 3 displays the sample chromatograms for both methods after hydrolysis. The sugar yield in milligrams per milliliter was calculated as percent (w/w) using the density of the solution used in the sample preparation. The result for the traditional method was 11.3% (w/w) and that for the new method was 11.2% (w/w). The two methods displayed equivalent results. Verification standards were tested before and after each set of tests. Reference standards of pure levoglucosan and cellobiosan were hydrolyzed with each set of samples to ensure complete hydrolysis conditions. All samples were tested in duplicate with triplicate injections. The percent RSD for the Aminex column was 1.03% and that for the HyperRez column was 1.13%. Levoglucosan was added to each bio-oil sample and hydrolyzed in duplicate and analyzed in triplicate. The average recovery yields of the two levoglucosan-spiked samples with a 95% confidence interval were 105 ± 0.20 and 104 ± 0.49%. Levoglucosan recovery yields were very representative of the total amount of sample spiked in the solution. Both columns had very good linearity and were able to resolve and separate the compounds of interest in the bio-oil samples. The new method produced total sugar results comparable to the traditional method. The HyperRez column,

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RESULTS AND DISCUSSION A comparison of glucose, cellobiosan, cellobiose, levoglucosan, sorbitol, and xylose standard compounds using both the Aminex 87P and the HyperRez columns is displayed in Figure 1. The concentration of the standard was approximately 10 mg/ mL. A complete set of calibration standards using glucose, cellobiosan, cellobiose, levoglucosan, sorbitol, and xylose was performed in duplicate on the HPLC using the HyperRez column. The same sugars, with the exception of cellobiosan, were measured on HPLC with the Aminex column. Cellobiosan has almost the exact same retention time as glucose (Figure 2). Because the acid hydrolysis reaction will convert the cellobiosan to glucose, this compound is not necessarily needed in the calibration standard. Its absence would, however, be an issue with the conventional column because incomplete hydrolysis of cellobiosan could give falsely high readings for glucose. The calibration curves had 12 points (same concentration in duplicate) in the range of approximately 0.5−10 mg/mL. The coefficient of determination for all standard compounds on both the HyperRez and Aminex columns was >0.999. Check standards were tested after each set of samples was complete. All samples were hydrolyzed in C

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Figure 3. Comparison of samples using HPLC: (A) acidic hydrolysate (new method); (B) neutralized hydrolysate (traditional method).

however, offers two distinct advantages. It enables researchers and refineries to quickly analyze hydrolyzed sugar samples, bypassing the time-consuming and tedious neutralization step. The HyperRez column also eliminates possible degradation of sugars that can occur during the conventional neutralization step if pH levels are not closely monitored. Our research demonstrated that it is possible to obtain excellent accuracy and efficiency using HPLC to quantitate glucose after acid hydrolysis of polymeric and oligomeric sugars found in fast pyrolysis bio-oils without neutralization.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Marjorie Rover for helpful discussions in the subject area, Dustin Dalluge for producing the pyrolysis liquids, and Nick Jaegers for assisting with laboratory experiments.

(1) Kuzhiyil, N.; Dalluge, D.; Bai, X.; Kim, K. H.; Brown, R. C. Pyrolytic sugars from cellulosic biomass. ChemSusChem 2012, 5, 2228−2236. (2) Bridgwater, A. V. An introduction to fast pyrolysis of biomass for fuels and chemicals. In Fast Pyrolysis of Biomass: A Handbook; CPL Press: Newbury, UK, 1999; pp 1−15. (3) Brown, R. C. Introduction to thermochemical processing of biomass into fuels, chemicals, and power. In Thermochemical Processing of Biomass Conversion into Fuels, Chemicals, and Power; Brown, R. C., Ed.; Wiley: Chichester, UK, 2011; p 330. (4) Mullen, C. A.; Boateng, A. A.; Goldberg, N. M.; Lima, I. M.; Laird, D. A.; Hicks, K. B. Bio-oil and bio-char production from corn cobs and stover by fast pyrolysis. Biomass Bioener. 2010, 34, 67−74. (5) Agblevor, F. A.; Besler, S.; Wiselogel, A. E. Fast pyrolysis of stored biomass feedstocks. Energy Fuels 1995, 9, 635−640. (6) Wright, M. M.; Daugaard, D. E.; Satrio, J. A.; Brown, R. C. Techno-economic analysis of biomass fast pyrolysis to transportation fuels. Fuel 2010, 89, S2−S10.

AUTHOR INFORMATION

Corresponding Author

*(R.C.B.) Phone: (515) 294-7934. Fax: (515) 294-3091. Email: [email protected]. Funding

We acknowledge Phillips 66 Co. for financial support of this project. Notes

The authors declare no competing financial interest. D

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(7) Lewkowski, J. Synthesis, chemistry and applications of 5hydroxymethylfurfural and its derivatives. ARKIVOC 2001, 17−54. (8) Yu, Z.; Zhang, H. Pretreatments of cellulose pyrolysate for ethanol production by Saccharomyces cerevisiae, Pichia sp. YZ-1 and Zymomonas mobilis. Biomass Bioenergy 2003, 24, 257−262. (9) Helle, S.; Bennett, N. M.; Lau, K.; Matsui, J. H.; Duff, S. J. A kinetic model for production of glucose by hydrolysis of levoglucosan and cellobiosan from pyrolysis oil. Carbohydr. Res. 2007, 342, 2365− 2370. (10) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. Determination of Structural Carbohydrates and Lignin in Biomass; National Renewable Energy Laboratory: Golden, CO, USA, 2008 (revised 2012); http://www.nrel.gov/docs/ gen/fy13/42618.pdf. (11) ASTM. Standard test method for determination of carbohydrates in biomass by high performance liquid chromatography. In Book of Standards; ASTM: Philadelphia, PA, USA, 2007; Vol. 11.06, ASTM E1859. (12) Pettersen, R. C.; Schwandt, V. H. Wood sugar analysis by anion chromatography. J. Wood Chem. Technol. 1991, 11, 495−501. (13) Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426−428. (14) Rover, M. R.; Johnston, P. A.; Jin, T.; Smith, R. G.; Brown, R. C.; Jarboe, L. Production of clean pyrolytic sugars for fermentation. ChemSusChem 2014, 7, 1662−1668. (15) Brown, J. N.; Brown, R. C. Process optimization of an auger pyrolyzer with heat carrier using response surface methodology. Bioresour. Technol. 2012, 103, 405−414. (16) Brown, J. N. Development of a Lab-Scale Auger Reactor for Biomass Fast Pyrolysis and Process Optimization Using Response Surface Methodology; Iowa State University: Ames, IA, USA, 2009. (17) Bennett, N. M.; Helle, S. S.; Duff, S. J. Extraction and hydrolysis of levoglucosan from pyrolysis oil. Bioresour. Technol. 2009, 100, 6059−6063.

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