Biofuels Program Biomass Analysis Technology Team Laboratory Analytical Procedure LAP-019CS Procedure Title: Hydrolysis of Corn Stover for Compositional Analysis Author(s): Bonnie Hames, Fannie Posey-Eddy, Chris Roth, Ray Ruiz, Amie Sluiter, David Templeton Date: 04-01-2002 ISSUE DATE: 04-01-2002 SUPERSEDES:
Hydrolysis of Corn Stover Laboratory Analytical Procedure #019 1. Introduction 1.1 Corn Stover is composed largely of three biopolymers; cellulose, a polymer of glucose; hemicellulose, an acetylated arabinoxylan with minor amounts of galactose and mannose; and lignin, a complex phenolic polymer. This procedure uses a two-stage hydrolysis process to separate the complex, polymeric biomass matrix into forms that can be more easily measured and quantified. During hydrolysis, the polysaccharides present in a corn stover sample are hydrolyzed to their component sugars. The monomeric sugars and associated by-products can then be quantified by ion-moderated partition HPLC. After hydrolysis, a large portion of the lignin remains insoluble in acid and can be analyzed gravimetrically. The acid soluble lignin can be measured spectroscopically. 1.2 The sample is taken through a primary 72% sulfuric acid hydrolysis, followed by a secondary 4% sulfuric acid hydrolysis. 1.2 This procedure is similar to portions of ASTM E1758, Standard Test Method for the Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography. 2. Scope 2.1 This procedure describes a two-stage hydrolysis process where the sample is taken through a primary 72% sulfuric acid hydrolysis, followed by a secondary 4% sulfuric acid hydrolysis. 2.2 This procedure is performed on extractives-free corn stover, following LAP 010CS Determination of Extractives in Corn Stover. 2.3 This procedure is performed prior to and is required for LAP 002CS Determination of Structural Carbohydrate Content in Corn Stover Feedstocks by High Performance Liquid Chromatography, LAP 003CS Determination of Acid-Insoluble Lignin in Corn Stover, LAP 003CS Determination of Acid-Soluble Lignin in Corn Stover, LAP 017 Determination of O-Acyl Groups in Biomass by High Performance Liquid Chromatography. 2.4 This procedure has been optimized for the analysis of carbohydrate content in extractivesfree corn stover. 2.5 All analyses shall be performed according to the guidelines established in the Biofuels Program Experimental Data Quality Assurance Plan (QAP).
3. Terminology 3.1 *** 4. Significance and Use 4.1 This procedure uses a two-stage hydrolysis process to separate the complex, polymeric biomass matrix into forms that can be more easily measured and quantified. 2.4 This procedure is performed prior to and is required for LAP 002CS Determination of Structural Carbohydrate Content in Corn Stover Feedstocks by High Performance Liquid Chromatography, LAP 003CS Determination of Acid-Insoluble Lignin in Corn Stover, and LAP 003CS Determination of Acid-Soluble Lignin in Corn Stover 5. Interferences 5.1 This procedure has been optimized for the particle size range specified in LAP 021 Preparation of Corn Stover for Compositional Analysis. 5.1.1 The application of this procedure to smaller particle sizes may result in a low bias in carbohydrate content measurements due to excessive degradation of monomeric sugars. 5.1.1.1 Excessive degradation may result in a high bias in the lignin content measurement due to the insolubility of many carbohydrate degradation products. 5.1.2 The application of this procedure to larger particle sizes may result in a low bias in carbohydrate content measurements due to incomplete hydrolysis to monomeric sugars. 5.1.2.1 Incomplete hydrolysis may result in a high bias in the lignin content measurement due to the insolubility of polymeric carbohydrates. 5.2 Test specimens not suitable for analysis by this procedure include acid- and alkalinepretreated biomass samples that have not been washed. Unwashed pretreated biomass samples containing free acid or alkali may change visibly on heating. 5.3 Samples with ash contents above 10% may contain soil, which may result in a low bias in structural sugar content measurement due to side reactions to products not measured in that procedure. 5.4 Failure to remove extractable materials such as starch and non-structural sugars may result in a high bias in structural sugar content measurements.
6. Apparatus 6.1 Analytical balance readable to 0.1 mg. 6.3 Convection ovens with temperature control to 45 ± 3 C and 105 ± 3 C. 6.4 Autoclave capable of maintaining 121 ± 3 C. 6.5 Water bath set at 30 ± 3 C. 6.6 Desiccator containing anhydrous calcium sulfate. 7. Reagents and Materials 7.1 Reagents 7.1.1 72% w/w H 2 SO 4 (12.00 ± 0.02 M or specific gravity 1.64 ± 0.01 at 15.6 C. See appendix for preparation procedure. 7.1.2 High purity sugars for standards and CVS (98%+) - D (+) cellobiose, glucose, xylose, galactose, arabinose, and mannose. Sugar samples used for standards should be from a different lot or manufacturer than those used for CVS. Refer to LAP 02 Determination of Carbohydrate content in Corn Stover for the use of these sugar standards. 7.1.3 An appropriate QA standard such as NIST Standard Reference Material 8491 Bagasse or a well-characterized corn stover sample. 7.6.1 Calcium carbonate, ACS reagent grade. 7.6.2 Purified water, (Nanopure or HPLC grade) 7.2 Materials 7.2.1 Glass test tubes, 16x100 mm OR 16x150 mm. 7.2.2 Glass stir rods approximately 5 cm longer than test tubes in 8.2.1 7.2.3 125 ml glass serum bottles, crimp top style, with rubber stoppers and aluminum seals to Procedure #002 Issue Date: 8/12/96 Supersedes: 1/30/95 Page 4 of 11
fit. 7.2.4 Calibrated pipette to dispense 3.0 ± 0.01, and 84.0 ± 0.04 ml 8. ES&H Considerations and Hazards 8.1 Follow all applicable NREL Laboratory Specific Hygiene Plan guidelines. 8.2 Operate all equipment in accordance with NREL Safe Operating Procedures. 8.3 H 2 SO 4 is very corrosive and must be handled carefully 8.4 Use caution when handling hot glass bottles after the autoclave step, as they may have become pressurized, creating an explosion hazard. 9. Sample Preparation and Pre-Analysis Requirements 9.1 Prior to analysis using this procedure, corn stover samples should be processed according to LAP-021 Preparation of Corn Stover for Compositional Analysis and LAP-010 Determination of Extractives in Corn Stover. 9.2 This procedure is suitable for air-dried, lyophilized, and extracted biomass samples, as well as for samples that have been oven dried at a temperature of 45 C or less. It is not suitable for samples that have been dried at a temperature exceeding 45 C. 9.3 Material with a total solids content less than 85%, on a 105 C dry weight basis, will require oven drying, or air drying prior to milling or analysis. The prepared sample should be stored in a manner to ensure its moisture content does not change prior to analysis. Note: Preparing samples for analysis by oven drying can produce hard chunks of material. This material must then be milled to reduce the size of the large pieces to less then 2 mm in diameter. The sample is then re-dried prior to testing. 9.4 Recommended batch size is 3-5 samples. These samples will be run in duplicate with QA standards and sugar recovery samples for a total of 10-14 samples. Allow a minimum of eight hours for the preparation of HPLC samples. Acceptable overnight stopping points will be noted. 9.5 The test specimen shall consist of approximately a minimum of 3 g of prepared extractives- Procedure #002 Issue Date: 8/12/96 Supersedes: 1/30/95 Page 5 of 11
free stover. A minimum of 5 g will be required for summative analysis. The test specimen shall be obtained in such a manner to ensure that it is representative of the entire lot of material being tested. 10. Procedure 10.1 Note: The total solids content of the original sample, %T as received, must be determined using LAP-001, prior to any preparatory steps. The total solids content of the sample based on its preparation, %T C-L, must also be known. 10.1 Determine the total solids content of each extractives-free stover sample by LAP-001 and record this value as %T final. Note 1: Samples for total solids determination (LAP-001) must be weighed out at the same time as the samples for the carbohydrate- lignin determination. If this is done later, it can introduce an error in the calculation because ground biomass can rapidly gain or lose moisture when exposed to the atmosphere. Note 2: Material with a total solids content less than 85%, on a 105 C dry weight basis, will require oven drying, or air drying prior to this analysis 10.7 Into a tared 16x100 mm OR 16x150 mm test tube, weigh 300.0 ± 5 mg of the (extractivesfree stover and record to the nearest 0.1 mg. Record as W 1, the initial sample weight in miligrams. Each sample must be run in duplicate, at minimum 10.4 Into a tared 16x100 mm OR 16x150 mm test tube weigh 300.0 ± 5 mg of the sugar recovery standards and record to the nearest 0.1 mg. Record as W 1, the initial sample weight in miligrams. Each sample must be run in duplicate, at minimum Note 1. The sugar recovery standards (SRS) will be taken through the remaining steps in the procedure in parallel with the samples. The calculated recovery of the SRSs will be used to correct for losses due to the destruction of sugars during the hydrolysis process. It may be useful to run selected SRSs in duplicate if a more accurate analysis is desired for the selected sugars. 10.5 Into a tared 16x100 mm OR 16x150 mm test tube, weigh 300.0 ± 5 mg of the QA standard and record to the nearest 0.1 mg. Record as W 1, the initial sample weight in miligrams. Each sample must be run in duplicate, at minimum Procedure #002 Issue Date: 8/12/96 Supersedes: 1/30/95 Page 6 of 11
Note 2. The QA standard will be taken through the remaining steps in the procedure in parallel with the samples. The QA standard is a well-characterized material used as a quality control check to test the accuracy of the analysis. Suitable quality assurance standards should have a composition similar to the test samples. Standard Reference Materials of some biomass types may be obtained from NIST. 10.6 Add 3.00 ± 0.01 ml (4.92 ± 0.01 g) of 72% H 2 SO 4 and use a glass stirring rod to mix for 1 minute, or until the sample is thoroughly wetted. 10.7 Place the test tube in the water bath set at 30 ± 1 C and hydrolyze for 60 ± 5 minutes. Note 1: Each sample must be hydrolyzed for 60 ± 5 minutes. For large batches or inexperienced users it may be advisable to stagger hydrolysis start time by five minutes to account for the time required to perform transfer step 11.9. 10.9 Stir the sample every 5-10 minutes to assure complete mixing and wetting. Note 1: This step is critical to assure even acid particle contact and uniform hydrolysis. 10.10 Upon completion of the 60 minute hydrolysis step, quantitatively transfer the contents of each tube to its own serum bottle and dilute to a 4% acid concentration by adding 84.00 ± 0.04 ml purified water. Note 1: The dilution and transfer step can be performed in different ways: by volume using volumetric pipettes or an automatic burette or by weight using a balance accurate to.01 grams. The total weight added to the tared bottle is 89.22 g (0.3 g sample, 4.92 g 72% H 2 SO 4, and 84.00 g deionized water). Since the specific gravity of the 4% acid solution is 1.0250 g/ml, the total volume of solution, V F, is 87.0 ml. Note 2: The quantitative transfer is best accomplished by rinsing with a series of aliquots of 10 ml or less. 10.11 Stopper each of the bottles, cover with a crimped aluminum seal. Place bottles in an autoclave-safe tray. 10.12 Set the autoclave to a liquid cycle. Autoclave the samples in their sealed bottles for 1 hour at 121 ± 3 C. Procedure #002 Issue Date: 8/12/96 Supersedes: 1/30/95 Page 7 of 11
10.13 After completion of the autoclave cycle, remove the tray of samples and allow to cool for about 20 minutes,until the bottles can be safely handled. Remove the seals and stoppers. 10.14 These autoclaved solutions will be used for the determination of, acid-insoluble residue, acidsoluble lignin, carbohydrates, uronic acids, and O-acyl group content. The procedure for determination of each of these components is described in the following LAPS. LAP 02CS Determination of Carbohydrates in Corn Stover by HPLC LAP 03CS Determination of Acid-Insoluble lignin in Corn Stover LAP 04CS Determination Acid-Soluble Lignin in Corn Stover LAP 17CS Determination of O-Acyl Groups in Biomass by HPLC LAP TBD Determination of Uronic Acids in Corn Stover (in development) 11. Calculations None 12. Report 12.7 Record the following values for use in the LAPS listed in section 10.14. 13. Precision and Bias W 1 = Initial air-dried weight of the extractives-free biomass samples, QA standard and sugar standards %T final = % Solids in the extractives-free biomass samples andqa standard. 13.7 The precision and bias determinations are part of the LAPS listed in section 10.14 14. Quality Control 14.1 Reported significant figures: not applicable 14.2 Replicates: At minimum, all samples and the method verification standard are to be analyzed in duplicate. 14.3 Blank: The only requirement is a reagent blank, which starts out as an empty 16x100 mm test tube (ie, no sample) which is taken through all the procedural steps. Procedure #002 Issue Date: 8/12/96 Supersedes: 1/30/95 Page 8 of 11
14.4 Relative percent difference criteria :These values are reported for the determinations made in the LAPS listed in section 10.14 14.5 Method verification standard: A method verification or QA standard must be run in duplicate with every batch. This method utilizes a well characterized standard material suitable for analysis. For example, NIST 8491 (baggasse) may be used as the MVS in carbohydrate analysis of grasses. 14.5 Calibration verification standard: Calibration verification standards shall be independently prepared and analyzed as described in section 11.19 of this procedure. 14.6 Sample size: The test specimen shall consist of approximately a minimum of 3 g of prepared extractives-free stover. A minimum of 5 g will be required for summative analysis. If there is insufficient sample, the result will be flagged and the lack of precision data should be noted. 14.7 Sample storage: Samples should be stored in an airtight container and refrigerated. 14.8 Standard storage: Standards should be kept frozen in airtight vials or test tubes. Vortex mix the standards vigorously upon thawing to ensure thorough mixing. 14.9 Standard preparation: Standards are prepared according to section 11.18 of this procedure. 14.10 Definition of a batch: Any number of samples that are analyzed and recorded together. The maximum size of a batch is limited by the equipment constraints. A batch cannot be larger than what is practical for the equipment used. 14.11 Control charts: The result of each replicate analysis of the method verification standard is recorded along with the average, RPD, and a laboratory book/page reference. The average value obtained for each analysis of the method verification standards is to be control charted. These values are reported for the determinations made in the LAPS listed in section 10.14. 15. Appendixes A Preparation of 72% w/w H 2 SO 4. 16. References 16.1 Moore, W.E., and D.B. Johnson. 1967. Procedures for the Chemical Analysis of Wood and Wood Products. Madison, WI: U.S. Forest Products Laboratory, U.S. Department of Procedure #xxx Issue Date: Supersedes: Page 9 of **
Agriculture. 16.2 NREL Biofuels Program Laboratory Analytical Procedure #001, "Standard Method for the Determination of Total Solids in Biomass". 16.3 NREL Biofuels Program Laboratory Analytical Procedure #003CS, "Determination of Acid- Insoluble Residue in Corn Stover". 16.4 NREL Biofuels Program Laboratory Analytical Procedure #004CS, "Determination of Acid- Soluble Lignin in Corn Stover". 16.5 NREL Biofuels Program Laboratory Analytical Procedure #010CS, "Standard Method for the Determination of Extractives in Corn Stover". 16.6 TAPPI Test Method T264 om-88, "Preparation of Wood For Chemical Analysis." In Tappi Test Methods. Atlanta, GA: Technical Association of the Pulp and Paper Industry. 16.7 Vinzant, T.B., L. Ponfick, N.J. Nagle, C.I. Ehrman, J.B. Reynolds, and M.E. Himmel. 1994. "SSF Comparison of Selected Woods From Southern Sawmills." Appl. Biochem. Biotechnol., 45/46:611-626. 16.8 NREL Biofuels Program Laboratory Analytical Procedure #015CS, "HPLC Analysis of the Liquid Fractions of Process Samples for Organic Acids, Glycerol, HMF, and Furfural". 16.9 T. Vinzant, L. Ponfick, N. Nagle, C. Ehrman, J. Reynolds, and M. Himmel, "SSF Comparison of Selected Woods from Southern Sawmills." 1994. Applied Biochemistry and Biotechnology Vol. 45/46 pp 611-626. Procedure #xxx Issue Date: Supersedes: Page 10 of **