LAP-003CS. Procedure Title: Author(s): Bonnie Hames, Fannie Posey-Eddy, Chris Roth, Ray Ruiz, Amie Sluiter, David Templeton.
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1 Biofuels Program Biomass Analysis Technology Team Laboratory Analytical Procedure LAP-003CS Procedure Title: Determination of Acid-Insoluble Lignin in Corn Stover Author(s): Bonnie Hames, Fannie Posey-Eddy, Chris Roth, Ray Ruiz, Amie Sluiter, David Templeton Date: ISSUE DATE: SUPERSEDES:
2 Determination of Acid-Insoluble Lignin in Corn Stover Laboratory Analytical Procedure #003CS 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. 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 This procedure is similar to the ASTM Standard Test Method E1721 for the Determination of Acid Insoluble Residue in Biomass 1.3 A determination of total lignin includes both acid-insoluble lignin and acid-soluble lignin. 2. Scope 2.1 This test method has been optimized for the determination of acid-insoluble lignin in corn stover agricultural residue All results are reported relative to the 105 o C oven-dried weight of the sample. Since the measurements are made on the extractives-free material, the results may also be reported on an extractives-free basis. 2.2 This method should be performed in conjunction with LAP 04 Determination of Acidsoluble Lignin in Corn Stover for a determination of total lignin content. 2.2 The acid-insoluble residue collected contains somecondensed proteins from the original sample. An independent nitrogen analysis is required to estimate the protein content of this residue. The protein estimate is subtracted from the weight of the acid insoluble residue to provide a measure of the acid-insoluble lignin content. Physical separation of the acid insoluble lignin from the condensed protein fraction is outside the scope of this procedure. 2.3 All analyses shall be performed according to the guidelines established in the Biofuels Program Experimental Data Quality Assurance Plan (QAP). 3. Terminology 3.1 Acid-insoluble lignin is defined to be the residue retained on a medium porosity filter crucible after the primary 72% and secondary 4% H 2 SO 4 hydrolysis steps described in Lap 19-
3 Hydrolysis of Biomass for Compositional Analysis, with correction for acid insoluble ash and protein. 4. Significance and Use 4.1 The acid-insoluble lignin content is used in conjunction with other assays to determine the total composition of biomass samples. 5. Interferences 5.1 The results of acid-insoluble lignin analysis are affected by incomplete hydrolysis of biomass. Unless the carbohydrates are hydrolyzed completely, the lignin analysis will be biased high and the carbohydrate analysis will be biased low. Take care to mix the acid/biomass slurry thoroughly at the beginning and periodically throughout the concentrated acid hydrolysis. The presence of cellobiose in a hydrolysis liquor is an indicator of incomplete hydrolysis. Incomplete hydrolysis may be a result of too large a particle size. 5.2 The partitioning of the lignin between acid-insoluble lignin and acid soluble lignin is a function of severity. The severity is dependent on acid concentration, temperature, hydrolysis time and acid-particle contact. Specifications within this procedure must be followed closely to achieve reproducible results. Because of partitioning, complete lignin analysis includes both acid-soluble lignin LAP XX and acid-insoluble lignin. 5.3 Some protein-derived materials can also form acid-insoluble substances that are collected with acid-insoluble lignin. A protein correction is required for an accurate lignin value. 6. Apparatus 6.1 Analytical balance readable to 0.1 mg. 6.2 Convection oven with temperature control of 105 ± 3 o C. 6.3 Muffle furnace. An electric furnace is recommended for igniting the sample. The furnace should be fitted with an indicating pyrometer or thermocouple, so that the required temperature of 575 ± 25 o C can be maintained Filtration set-up including vacuum source and vacuum adapters for crucibles Desiccator containing anhydrous calcium sulfate. 7. Reagents and Materials 7.1 Test samples of biomass after processing through LAP 19 Hydrolysis of Corn Stover for
4 Compositional Analysis 7.2 Test samples of the QA standard after processing through LAP 19 Hydrolysis of Corn Stover for Compositional Analysis 7.3 Filtration flasks, 250 ml or larger ml ceramic filtering crucible, medium porosity Coors # or equivalent. 7.5 Hi temperature glass markers. 7.6 Bottles, wide-mouth 100 ml for sample storage 8. ES&H Considerations and Hazards 8.1 Follow all applicable NREL Laboratory Specific Hygiene Plan guidelines 9. Sample Preparation and Pre-Analysis Requirements. 9.1 Prior to analysis using this procedure, corn stover samples should be processed according to NREL LAP 21 Preparation of Biomass for Compositional Analysis, The total solids content of the "as received" test specimen (prior to any drying or extraction steps) must be known. Record this value as T as received. 9.2 Prior to analysis using this procedure, corn stover samples should be processed according to NREL LAP 10 "Standard Method for the Determination of Extractives in Biomass". As part of that procedure, the percent extractives in the prepared sample, on a 105 o C dry weight basis, is determined. This value, recorded as % extractives, is used to convert the % lignin reported on an extractives-free basis to an as received (whole sample) basis. 9.3 Prior to analysis using this procedure, corn stover samples should be processed according to NREL Lap 19 Hydrolysis of Biomass for Compositional Analysis. The weight, W 1, and total solids content, %T final, of the "extractives-free" test specimen as recorded in LAP 19 will be used to determine weight percent composition This procedure is performed after LAP 19 Hydrolysis of Biomass for Compositional Analysis and begins with the autoclaved solutions of hydrolyzed biomass This procedure uses the same bulk test materials as the following LAPS. If these tests will be performed, care must be taken not to consume the entire sample in this analysis.
5 10. Procedure LAP 02CS Determination of Carbohydrates in Corn Stover by HPLC 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) 10.1 Using the high temperature markers, individually label the crucibles needed for analysis and ignite them in the muffle furnace at 575 ±25 o C to achieve a constant weight of ±0.3 mg. Store the ignited crucibles in a desiccator until needed. Record the weight of the ignited crucible, W 2 to the nearest 0.1 mg. To improve tare accuracy, avoid direct contact with skin when handling crucibles Vacuum filter the autoclaved hydrolysis solution through one of the previously ignited filtercrucibles into a vacuum flask For carbohydrate analysis and/or an acid-soluble lignin analysis transfer the undiluted filtrate into a sample storage bottle. If the hydrolysis liquor is not used immediately for further analysis, store in refrigerator at 4 o C. The acidic hydrolysis liquor can be stored in the refrigerator for a maximum of 2 weeks. Note: Acid-soluble lignin should be analyzed within 6 hours to prevent loss due to precipitation of soluble lignin. Caution: Undiluted filtrate must be collected before proceeding to the washing step Use hot deionized water to wash all solids into the crucible and rinse the solids with at least 50 ml of hot water using vacuum filtration. Note1: The rinsing step removes residual acid before the 105 ºC drying step. Note 2: Hot water rinsing increases filtration rate. Room temperature water may be used Dry the crucible and contents at 105 ± 3 o C until constant weight is achieved (±0.3 mg) 2-5 hours Cool in desiccator. Record the weight, W 3, of the crucible and acid-insoluble residue to the nearest 0.1 mg Note1: acid-insoluble residue includes acid insoluble lignin, acid-insoluble ash and acid insoluble protein. Note2: The amount of acid insoluble protein is assumed to be equal to the amount of protein in the extractives-free stover. The protein content of the extractives-free
6 sample is determined using LAP 20CS Determination of Protein Content in Corn Stover Place the crucible and contents in a muffle furnace set at 105 C. Heat at 10 o C/min to 250 C and hold for one hour. Continue to increase heat at 10 o C/min to 575 ± 25 o C and hold for a minimum of 3 hours and maximum of 24 hours. After ashing, the furnace should be held at 105 o C until samples are removed. Note 1:Heating above the maximum stated temperature or longer than the maximum time may result in a low ash bias due to loss of volatile inorganics.. Note 2: Protect the test container from drafts at all times to avoid mechanical loss of the test specimen Cool in desiccator and record the weight, W 4, of the crucible and acid-insoluble ash, to the nearest 0.1 mg. Note: The amount of acid-insoluble ash remaining in the crucible is not equal to the total ash content of the original biomass sample. Refer to Laboratory Analytical Procedure #005 if total ash is to be determined Since this procedure always starts with extracted samples, all recorded weights are on an extractives-free 105 o C dry weight basis. Convert these values to an as received (whole sample) 105 o C dry weight basis, using the equations below. 11. Calculations 11.1 Calculate % acid-insoluble residue on an extractives-free basis as follows: ODW (oven dried weight) = W 1 * (%T final /100) %AIR (acid-insoluble residue) =((W 3 -W 2 )/ ODW) * % AI Ash = ((W 4 -W 2 )/ODW) * % AIL extr-free (acid insoluble lignin) = %AIR - %AI Ash - %P extr-free Where: W 1 = initial air dried weight of extractives-free sample sample W 2 = weight of ashed crucible W 3 = weight of crucible and acid-insoluble residue W 4 = weight of ashed crucible and AI Ash %T final = % total solids in the extractives-free sample determined at 105 o C using LAP 01CS Determination of Total Solids in Corn Stover. %P extr-free = % protein in the extractives-free sample determined using
7 LAP 20CS Determination of Protein in Corn Stover 11.2 Convert the acid-insoluble residue value, calculated above on an extractives-free basis, to an as-received (whole sample) 105 o C dry weight basis: % whole sample extractives- free acid -insoluble residue = % AIR Where: % AIR extractives-free = % acid-insoluble residue on an extractives-free 105 o C dry weight basis, as determined in the previous step % extractives = % extractives in the prepared biomass sample as determined using LAP 10 Determination of Extractives in Biomass x (100% - % extractives) 100% 12. Report 12.1 Report the % acid-insoluble lignin, to no more than two decimal places, on a 105 o C dry weight basis, and cite the reporting basis (extractives-free or as-received sample) For replicate analyses of the same sample, report the average, standard deviation, and relative percent difference (RPD) when the sample is run in duplicate. %RPD = (absolute value of (measurement 1 measurement 2) /average (meas1:meas 2)) * Precision and Bias 13.2 Data obtained by replicate testing of NIST Standard Reference Material 8492 for hybrid poplar, in one laboratory gave a standard deviation in Acid insoluble Residue content of 0.32% and a Coefficient of Variation% of 1.26% Data obtained by replicate testing of NIST Standard Reference Material 8491 for Bagasse in different laboratories gave a standard deviation of 2.4% and a Coefficient of Variation% of 2.5% Total lignin number includes both acid insoluble lignin and acid soluble lignin. Due to inconsistent partitioning of the lignin between these fractions, the total lignin values should have a smaller Standard Deviation than either fraction. 14. Quality Control Procedure #xxx Issue Date: Supersedes: Page 7 of **
8 14.1 Reported significant figures: The acid-insoluble lignin results will be reported with no more than two decimal places, on a 105 o C dry weight basis Replicates: All samples and all method verification standards are to be analyzed in duplicate, at minimum Blank: A blank crucible may be processed through the procedure. The difference in weight should be less than the equivalent of a 0.5% error Relative percent difference criteria: The RPD must be less than 7 %. If the RPD is too large, the sample should be rerun QA standard: A QA standard must be run in duplicate with every batch of samples Calibration verification standard: Not applicable Sample size: Approximately 3.0 grams of sample are required for conducting duplicate analyses. If there is insufficient sample, the results should be flagged and the lack of precision information should be noted Sample storage: Not applicable 14.9 Standard preparation: Not applicable Definition of a batch: Any number of samples that are analyzed together and recorded together Control charts: The result of each replicate analysis of the QA standard is recorded along with the average, RPD, and a laboratory book/page reference. The average value obtained for each analysis of the QA standard is to be control charted Others: (Delete this section if there is no "other" to list.) 15. Appendixes None 16. References 16.1 Ehrman, C.I., and M.E. Himmel "Simultaneous Saccharification and Fermentation of Pretreated Biomass: Improving Mass Balance Closure." Biotechnology Techniques, 8(2): Procedure #xxx Issue Date: Supersedes: Page 8 of **
9 16.2 Moore, W.E., and D.B. Johnson Procedures for the Chemical Analysis of Wood and Wood Products. Madison, WI: U.S. Forest Products Laboratory, U.S. Department of Agriculture NREL Laboratory Analytical Procedure #001, "Standard Method for the Determination of Total Solids in Biomass" NREL Laboratory Analytical Procedure #002CS, "Determination of Carbohydrates in Corn Stover by HPLC" NREL Laboratory Analytical Procedure #004CS, "Determination of Acid-Soluble Lignin in Corn Stover" NREL Laboratory Analytical Procedure #010, "Determination of Extractives in Corn Stover" TAPPI Test Method T222 om-88, "Acid-Insoluble Lignin in Wood and Pulp." In Tappi Test Methods. Atlanta, GA: Technical Association of the Pulp and Paper Industry 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 Vinzant, T.B., L. Ponfick, N.J. Nagle, C.I. Ehrman, J.B. Reynolds, and M.E. Himmel "SSF Comparison of Selected Woods From Southern Sawmills." Appl. Biochem. Biotechnol., 45/46: Procedure #xxx Issue Date: Supersedes: Page 9 of **
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