Summative Mass Closure 

Laboratory Analytical Procedure (LAP) 

Review and Integration 

Issue Date: April 2010 

Revision Date: July 2011 

(Version 07-08-2011) 

J. Sluiter and A. Sluiter 

Technical Report 

NREL/TP-510-48087
 

Revised July 2011
 
 

 

 

Technical Report Summative Mass Closure 

NREL/TP-510-48087 

Revised July 2011 Laboratory Analytical Procedure (LAP) 

Review and Integration 

Issue Date: April 2010 

Revision Date: July 2011 

(Version 07-08-2011) 

J. Sluiter and A. Sluiter 

Prepared under Task No. BB072230 

NREL Laboratory Analytical Procedures for standard 

biomass analysis are available electronically at 

http://www.nrel.gov/biomass/analytical_procedures.html 

National Renewable Energy Laboratory

1617 Cole Boulevard, Golden, Colorado 80401-3393 

303-275-3000 • www.nrel.gov 

NREL is a national laboratory of the U.S. Department of Energy 

Office of Energy Efficiency and Renewable Energy 

Operated by the Alliance for Sustainable Energy, LLC 

Contract No. DE-AC36-08-GO28308 
 

 

 

 

 

 

 

 

 

 

 

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Summative Mass Closure – LAP Review and Integration: 

Feedstocks 

NREL has developed a suite of Laboratory Analytical Procedures (LAPs) for the analysis of 

biomass. Many of these methods build on years of research in the biomass analytical field. 

Choosing the appropriate combinations of LAPs allows for the summative mass closure of 

biomass feedstocks and process intermediates. By combining the appropriate LAPs, the goal is to 

break the biomass sample down into constituents that sum to 100% by weight. Some of these 

constituents are individual components, such as individual carbohydrates, and some are groups 

of compounds, such as extractable material. However, the goal of these analyses is to 

characterize all of the material in the sample. It is imperative to follow the LAPs as they are 

written, as small deviations can have a large detrimental impact on the constituent values. This 

document covers the summative mass closure for biomass feedstocks only. 

There are many subtle differences in the analytical suites for differing feedstocks. This LAP is 

designed to help in the decision making process. It will assist in the selection of appropriate 

LAPs and help organize the flow from one LAP to the next in the analysis sequence. It is not 

meant to replace the specific LAPs in any way, but to aid in the development of a comprehensive 

analysis specific to the feedstock. The LAPs have been optimized for corn stover and generally 

work well on woody feedstocks and herbaceous materials such as switchgrass, sorghum, and 

miscanthus, although minor adjustments may be necessary. Unusual feedstocks will typically 

require some method development to capture constituents not included in the LAP suite. 

There are several important points within the compositional analysis suite where decisions must 

be made to optimize the analysis. Some of these decisions are based on the type of biomass 

present, and some decisions must be made to obtain complete summative mass closure of all 

constituents. Discussions to aid these decisions are included with LAP discussions below. 

Following the LAP discussions is a flowchart of analyses and a walkthrough of the flowchart 

using an example feedstock, including some troubleshooting. 

Preparation of Samples for Compositional Analysis 

Biomass samples typically arrive from the field in an intact or semi-intact state that includes soil 

or other debris and significant moisture content. Proper sample preparation will minimize 

interferences in subsequent compositional analyses. Sample drying, particle size reduction, and 

potential sieving are discussed in this LAP. 

The first consideration for sample preparation is drying. If a sample contains greater than 10% 

moisture by weight, it must be dried to ensure compatibility with subsequent analyses. LAP 

“Preparation of Samples for Compositional Analysis” describes the appropriate drying 

procedures for the compositional analysis suite. Three options are presented within the LAP. One 

option is air-drying, most suitable for low ambient humidity. When ambient humidity is too high 

to permit this technique, samples must be monitored for degradation and microbial growth until 

the moisture content is less than 10% by weight. Convection oven drying at 45o

C, most suitable 

for samples likely to be unstable at ambient conditions or where ambient humidity does not 

allow for air-drying, is covered as well. Oven drying at 105o

C should be used with care, as many 

biomass types chemically change when exposed to prolonged heat. The last technique covered is 

1 
 

 

 

 

lyophilization drying, or freeze drying, which is suitable for samples that are at risk for 

degradation at elevated temperature, and therefore are not suitable for convection oven drying or 

air-drying. Lyophilization is an unsuitable technique for large or bulk samples and those with 

large pieces of biomass. 

Samples must be milled prior to compositional analysis if particle size is greater than 2 mm. 

Since the subsequent methods are optimized for a 2-mm or less particle size, milling the sample 

will likely be necessary. Prior to milling, the sample must meet moisture requirements discussed 

above. Milling wet samples can result in the degradation of the sample during milling. The mill 

must be monitored to ensure that it is operating at optimal temperature. An overheated mill can 

cause extractable material to separate from the biomass and deposit on the heated metal portions 

of the mill. Milling with dry ice is not recommended, as the oils in dry ice leave a residue on the 

biomass. 

Sieving of a sample is sometimes necessary to accurately analyze a biomass feedstock but may 

interfere with representative sampling. The acid hydrolysis steps have been optimized for a 

-20/+80 particle size, and deviation to a larger particle size distribution will cause structural 

carbohydrates to be incompletely dissolved into solution. Such deviation will result in higher 

acid insoluble lignin values and lower overall structural carbohydrates, especially cellulose. 

Deviation from the recommended particle size to a smaller particle size may result in over 

hydrolysis of the structural carbohydrates, contributing to an overproduction of sugar 

degradation products, which can complicate the acid soluble lignin measurement. 

Sieving was originally developed for the analysis of very homogeneous materials, such as wood 

samples. However, in herbaceous feedstocks, fines (-80 mesh) often contain a disproportionately 

large percentage of inorganic materials as compared to the bulk sample. Removal of the fines 

will cause an overall change in composition. In such cases sieving is not recommended, to ensure 

the integrity of the sample. Alternately, sieving may be performed, but the constituent values of 

the fines should be mathematically added back into the whole value before reporting; this 

calculation is not discussed. Sieving can be performed to remove a portion of the higher ash 

content fraction. This is only the case when the ash content of the extracted biomass is high 

enough to interfere with hydrolysis. Further discussion of ash interference is included with the 

hydrolysis discussions. 

Determination of Total Solids in Biomass 

Due to the high variability of moisture content in biomass feedstocks, all biomass compositional 

analysis results are reported on dry weight basis. This allows for comparison of biomass samples 

on a consistent baseline. At several points in the compositional analysis suite, a %Total Solids 

determination is required. The LAP “Determination of Total Solids in Biomass and Total 

Dissolved Solids in Liquid Process Samples” details appropriate procedures for such 

determinations. The LAP describes drying by convection oven at 105o

C and drying by infrared 

moisture balance. 

This determination should be done at the same time as the corresponding procedure that calls for 

a %Total Solids determination, as the moisture content of a biomass sample can change quickly. 

The values obtained during this analysis are used to mathematically correct the sample back to a 

dry weight basis. The sample aliquot used for %Total Solids analysis has been exposed to 

2 
 

 

 

 

 

 

 

elevated temperatures and should not be used in further analyses, with the exception of 

determining ash content on an extremely limited sample quantity. 

Calculation of %Total Solids is shown below. 

ሺܹ݁݅ ݄݃ ݐௗ௥௬ ௣௔௡ ௣௟௨௦ ௗ௥௬ ௦௢௟௜ௗ௦ െ ܹ݁

݅ܵ݋݈݈ݐ݋ܽ%ܶ ݏ݀ ൌ ܹ݄݁݅݃ݐ௦௔௠௣௟௘ ௔௦ ௥௘௖௘ ௩௘ௗ ௜

݄݃݅ ݐௗ௥௬ ௣௔௡ሻ

ൈ 100 

Calculation of the dry weight of a sample or its Oven Dried Weight (ODW) is described in the 

following formula. 

௔௜௥ ௗ௥ ሺܹ݄݁݅݃ݐ

ܹܱܦ ௦௔௠௣௟௘ ൌ ௜௘ௗ ௦௔௠௣௟௘ ൈ % ݋ܶ ݐܽ

100 

ሻ ݈ܵ݋݈݀݅ݏ

Determination of Ash in Biomass 

Inorganic materials are present in both whole and structural, or extracted, biomass samples. In 

addition to contributing significantly to total mass closure, inorganic material may interfere with 

acid hydrolysis. LAP “Determination of Ash in Biomass” describes two methods for the 

determination of %Ash in biomass. The LAP provides instructions for ash determination in a 

muffle furnace set to 575oC with prior preignition and describes the use of a ramping muffle 

furnace with no preignition. The calculation for determining %Ash is below. 

ሺܹ݁݅ ݄݃ ݐ௥௖௨௖௜௕௟௘ ௣௟௨௦ ௔௦௛ െ ܹ ݄݃݅݁ ݐ௥௖௨௖௜௕௟௘ሻ %ݏܣ݄ ൌ ܹܱܦ ௦௔௠௣௟௘

ൈ 100 

Determination of Protein in Biomass 

Herbaceous feedstocks can contain a significant amount of protein in the stalks and leaves, 

which will interfere with lignin measurements in subsequent analyses. Quantification of the 

protein will allow this interference to be mathematically minimized. Measurement of protein in 

biomass is performed indirectly by measurement of nitrogen content and use of a nitrogen-toprotein conversion multiplier. The typically used nitrogen-to-protein conversion value of 6.25 is 

not generally applicable to biomass proteins. Instead, calculation of the conversion factor is done 

by measurement of individual amino acids in the feedstock of interest. 

Because a portion of the protein is often removed during the extraction process, protein analysis 

is performed on both whole and extractives-free materials. 

Determination of Extractives in Biomass 

Nonstructural materials in biomass often contribute significantly to the mass closure and will 

interfere with the characterization of carbohydrates and lignin. LAP “Determination of 

Extractives in Biomass” describes extraction processes to both remove and quantify the 

extractable portion of a biomass feedstock using successive ethanol and water extractions. 

3 
Extraction with ethanol is required for all biomass types to ensure the removal of waxy materials 

that co-precipitate during filtration of the acid hydrolysate. When analyzing woody feedstocks, 

ethanol extraction alone is generally sufficient to remove interfering extractable material, 

including sap and resins. Herbaceous feedstocks require a water extraction prior to the ethanol 

extraction to allow for additional quantification of components more commonly found in 

herbaceous feedstocks. Nonstructural water soluble components commonly include inorganic 

material in the form of soil or fertilizers, proteins that are easily washed from the biomass, and a 

diverse array of carbohydrates, especially sucrose. Sucrose, a dimer of glucose and fructose, is of 

particular interest to fermentation and can be abundant in an herbaceous plant, but it is easily lost 

during the hydrolysis stages. Early removal and analysis of sucrose allows for better 

quantification of the structural glucose present in a feedstock as well. The LAP describes the 

sampling of the water extractable material to determine sucrose concentration. 

Once a sample has been extracted, subsequent analytical values must be corrected for that 

removal to bring all values to a whole biomass basis. That calculation is included in the 

calculation list below. 

Note that herbaceous feedstocks are typically higher in inorganic materials (commonly soil or 

fertilizer) and protein than woody feedstocks are. The water extraction process will remove some 

of these materials; therefore ash and protein measurements are recommended both before and 

after extraction. 

The formula below should be used in calculating %Extractives content for both water and 

ethanol extractives. 

 ܹ݄݁݅݃ݐ௔௟௙௦௞ ௣௟௨௦ ௘௫௧௥௔௖௧௜௩௘௦ െ ܹ ݄݃݅݁ ݐ௔௟௙௦௞ %ݎݐݔܧܽܿݐ݅ݒ݁ݏ ൌ ܹܱܦ ௦௔௠௣௟௘

ൈ 100 

The formula below is used when a portion of the extractives are removed for sucrose analysis. 

ܹ݁ െ ௦௘௩ ൌ ݏ݁ݒ ௣ ௞௦ ௙௟௔ܹ݁݅ ݃ݐ݄ ݐݔܧ%ݎܽݐ݅ܿ

௣௠௔௦ ௘௟ܹܦܱ

݄݃݅ ݐ௔௟௙௦௞ ൈ 

௔௧௢௧ ௟݉݁ݑ݈݋ܸ

ܸ݋݈ݑ݉݁௜ ௨௧௜௢௡ ௠௟௦௢ ௡௨௦ ௦௔௠௣௟௘ ௥௘௠௢௩௘ௗ 

௟௨௦ ௘௫௧௥௔௖௧௜ ௦௢௟௨௧௜௢௡ ൈ 100 

The calculation to correct from an extractives free basis to a whole biomass basis for any 

component measured on the extracted biomass is the following: 

௕ ௘௟௢௛௪ݐ ݋ܥ݌݉݋݊݁݊

݁ݏ ݒ ݔܧݎݐ݅ܿܽݐ % െ 100

௢௠௔௦௦ ௜ ൌ ݊ݐ݊݁݌݋݉݋ ܥ௘ ௫௧௥௔௖௧௜௩௘௦ ௙௥௘௘ ൈ

100 

݊݋ܽݐ݅ݎݐ݊ ݋ܥ݊ܿ݁௟௚ ௨௖௢௦௘ ൈ 1.9 ൈ ݈݋ ܸ ݑ ݁݉ ௧௢௧௔௟ ௦௢௟௨௧௜௢௡ ൅

௘௟௣௠௔௦ܹܦܱ ௘௟௣௠௔௦ܹܦܱ ൌ ݁ݏ݋ݎܿݑ%ܵ ௡௢௜௨௧௢௟௦௔௟ ௧௢௧ ݁݉ ݑ݈݋ܸ ൈ௘ ௦௖௥௢௦௨ ݊ܿ݁݋ܥ ݊ݎݐ݅ݐܽ݋݊

ൈ 100 

4 
Determination of Structural Carbohydrates and Lignin in Biomass

Structural carbohydrates and lignin make up the bulk of most feedstocks and often represent the 

most interesting portions. LAP “Determination of Structural Carbohydrates and Lignin in 

Biomass” describes the acid hydrolysis and subsequent analyses of acid soluble and acid 

insoluble portions. It describes the preparation and two-stage sulfuric acid hydrolysis of the 

sample and includes the use of sugar recovery standards, which are used to correct for loss of 

carbohydrates during hydrolysis. The LAP also describes carbohydrate analysis, including 

preparation of standards, hydrolysate neutralization, HPLC method setup, and acetyl analysis. 

The determination of carbohydrates using this method requires that all carbohydrates be in 

monomeric form. The presence of polymers indicates incomplete hydrolysis, and those 

carbohydrates will not be captured. During hydrolysis, the conversion of polymers to monomers 

in the carbohydrates results in the addition of a hydrogen and a hydroxyl group to each 

monomer. An anhydro correction is used to mathematically convert the monomeric values back 

to a structural polymeric value. 

Sugar recovery standards (SRSs) are used to account for sample sugar degradation during the 

dilute sulfuric acid step. SRSs are used to mimic the behavior and degradation of sample 

monomers. Since these values can fluctuate depending on a variety of factors, SRSs are included 

with every sample analysis. They are independent from the sample but are run in parallel. 

Because carbohydrate concentration will affect degradation levels, it is imperative to mimic the 

sample carbohydrate concentrations as closely as possible in the SRSs. Since this correction is 

critical, duplicate or triplicate SRSs are recommended. 

The LAP details the steps necessary to determine acid insoluble residue, including filtration of 

the hydrolysate and ash determination of the residue. Acid insoluble residue, once corrected for 

ash and protein content if necessary, is considered high molecular weight lignin. This definition 

of lignin is considered a behavior-based lignin definition, as opposed to a chemical based 

definition, which would include further characterization of the material. Acid insoluble residue 

must be corrected for ash, as a significant portion of the ash in the whole biomass is acid 

insoluble. Some herbaceous feedstocks may need to have the acid insoluble residue corrected for 

protein as well, as a significant portion of the protein from the feedstock can condense into that 

fraction. The specific amount of protein that will co-condense can vary between feedstocks. 

Individual feedstocks need to be evaluated for protein condensation into the acid insoluble 

residue. This evaluation is not included in the method. 

Acid soluble lignin is low molecular weight lignin that is solubilized in the acidic hydrolysis 

solution. Inclusion of acid soluble lignin concentration in the total lignin value is necessary, as 

acid soluble lignin can represent an important portion of the lignin. The LAP describes the 

measurement of acid soluble lignin but does not detail the determination of the proper extinction 

coefficient for feedstocks. A short list of common extinction coefficients is included in the LAP. 

This LAP contains several notable interferences, such as high moisture or ash content in the 

sample. High moisture content, above 10% by weight, dilutes the acid concentration beyond the 

specifications of the LAP, possibly resulting in incomplete hydrolysis. Similarly, ash content 

above 10% by weight may buffer the acid, causing an effective reduction in acid concentration. 

5 
 

 

 

However, not all inorganic material in biomass has this buffering effect. The buffering effect of 

excessive inorganic material should be determined prior to analysis if this problem is suspected. 

Unextracted biomass feedstock will interfere with this method. Extractives can deposit on the 

filter during separation of the acid soluble and acid insoluble fractions, resulting in excessive 

filtration time and potential concentration of the liquid fraction. Extractives can also partition 

irreproducibly between the acid soluble and acid insoluble fractions, compromising the lignin 

values. 

The calculations for %Acid insoluble lignin (%AIL) and %Acid soluble lignin (%ASL) are 

included below along with necessary carbohydrate calculations. 

௣௥௢௧௘ ൯ െ ݄݃݅݁ ܹ ݐ௥௖ ௨௖௜௕௟௘ െ ܹ ݄݃݅݁ ݐ ൫ܹ݁ ݄݃ݐ݅௥௖ ௨௖௜௕௟௘ ௣௟௨௦ ஺ூோ െ ܹ ݄݃݅݁ ݐ௥௖௨௖௜௕௟௘൯െ൫ ݄ܹ݃݅݁ ݐ௥௖௨௖௜௕௟௘ ௣௟௨௦ ௔௦௛ ܮ%ܫܣ ൌ ܱܦܹ௦௔௠௣௟௘௜௡ ൈ 100 

Where Weightprotein = amount of protein present in the acid insoluble residue, as determined in 

LAP “Determination of Protein Content in Biomass.” This measurement is only necessary for 

biomass containing high amounts of protein. 

௣௠௔௦ ܱ ܦܹ ൈ ߝ

݋݊݅ ݐ ܦ ݑ݈݅ ൈ௢௥ ௨ ௟௜௤௦௜௬௦௥௢௟௬ௗ௛݉݁ݑ݈݋ܸൈ ݏܷܸܾܽ 

 ൌ ܮܵܣ%

௘ ሺ௠௚ሻ௟ ൈ ܲܽݐ ݈݄݃݊݁ ݐ݄ ൈ 100 

Where: 

UVabs = average UV-Vis absorbance for the sample at the appropriate wavelength 

Volumehydrolysis liquor = volume of filtrate, 87mL 

ݑ ݁݉ ௗ௜௟௨௧௜௡௚ ௦௢௟௩௘௡௧ ݈݉݁ݑ ܸ݋ ௦௔௠௣௟௘ ൅ ܸ ݈݋

Dilution ൌ ݈݉݁ݑ ܸ݋ ௦௔௠௣௟௘

ߝ ൌ Absorptivity of biomass at specific wavelength ሺsee table belowሻ. 

Table 1. Absorptivity Constants for Acid Soluble Lignin Measurement for Select Biomass Types 

Biomass type Lambda max (nm) Absorptivity at 

lambda max (L/g 

cm) 

Recommended 

wavelength (nm) 

Absorptivity at 

recommended 

wavelength (L/g 

cm) 

Pinus radiate 198 25 240 12 

Sugarcane 

bagasse 

198 40 240 25 

Corn stover 197 55 320 30 

Populus deltoids 197 60 240 25 

6 
 

 

 

 

 

Calculate the total amount of lignin on an extractive free basis as below: 

%ܮ݅݃݊݅݊௘௫௧௥௔௖௧௜௩௘௦ ௙௥௘௘ ൌ % ܣܫܮ൅ % ܮܵܣ

For the determination of carbohydrates, first correct the sugar concentrations for degradation 

during hydrolysis: 

ܮ/݉ ݉ ݃ , ݕܾ ܥܮܲܪ ݏ݅ݏݕ݈݋ݎ݀ݕ ݎ݁ݐ݂ܽ ݄ ݋ ݂ݎ݃ܽݑݏ ݊ܿ.݋ܿ 

%ܴ௦௨௚௔௥ ൌ ൈ 100 

݉݃ ܮ/݉ , ݕܾ ܥܮܲܪ ݏ݅ݏ݈݋ݎ݀ݕ ݁ݎ݋ܾ݂݁ ݄ ݋ ݂ݎ݃ܽݑݏ ݊ܿ.݋ܿ 

/100௥௔௚௦௨%ܴ ൌ௚௔௥ ௦௨ ݋ܥܿ݊ ݋݊݅ݐ݂ܽܿ ݋ݎ ݐ ݀ݑ݈݅ ൈ௉௅஼ ு ݋ܥ ܿ݊

Where:

 ConcHPLC = concentration of an individual sugar as determined by HPLC after hydrolysis, 

 mg/mL.

 %Rave.sugar = average recovery of a specific SRS component (%Rsugar) 

Concsugar = Conccorr.sample, concentration in mg/mL of a sugar in the hydrolysate sample 

after correction for loss on 4% hydrolysis. 

Next, calculate the concentration of the polymeric sugars from the concentration of the 

corresponding monomeric sugars, using an anhydro correction of 0.88 (or 132/150) for C-5 

sugars (xylose and arabinose) and a correction of 0.90 (or162/180) for C-6 sugars (glucose, 

galactose, and mannose). 

݊݋݅ݐ݁ܿݎݎ݋ܿ ܣ ݋ݎ݀ݕ݄݊ ൈ ஼௢௥௥ ݋ܿ݊ ܥ ൌ௥௢ ௬ௗ ௔௡௛݋ܥ ܿ݊

ൈ 100 

1݃

1000 ݃݉

ܿ݊݋ܥ௛௡௔ ௬ௗ௥௢ ൈ ܸ݋݈௟௜௙௧௥௔௧௘ ൈ

௘௟௣௠௔௦ ܦܹܱ

ൌ ௙௥௘௘ ௫௧௘ݎ݃ܽݑ%ܵ

ௗ,ு௉௅஼ ൈ ܸ ݈݋௟௜௙ ௧௥௔௧௘ ൈ 0.983 

௘௟௣௠௔௦ ܦܱ ܹ

ൌ ௙௥௘௘ ௫௧௘݈ݕݐܿ݁ܣ% ௜௖௔ ௖௜௧ ௔௖௘݋ܥ ܿ݊

Determination of Starch in Biomass 

Starch, a non-crystalline glucose polymer, is often found in biomass feedstock that contains 

grain. LAP “Determination of Starch in Biomass” is a procedure that is based heavily on the 

7 
 

 

 

 

ௌ௢௟௨௧௜௢௡

ܹ

݉݁

ܦܱ

ൈ௉௅஼ ு ݈ܩ ݁ݏ݋ܿݑܥ ݋݊ܿ ݋ܸ ݑ݈

%ܵݐܽݎ݄ܿ ൌ 1.11 ൈ % ݋ܴ݁ܿ ݒ ݕݎ݁ ௦௧௔௥௖௛ ௦௧௔௡ௗ௔௥ௗ

Megazyme Total Starch Assay (amyloglucosidase/α-amylase method). The major difference in 

the NREL adaptation is the analysis for quantification of glucose. The LAP changes from a 

derivatization for colorimetric detection to HPLC glucose analysis. Because the quantification of 

glucose is nonspecific to starch, extraction of the biomass is recommended prior to the starch 

assay to remove any nonstructural free glucose. Failure to remove free glucose will artificially 

elevate the starch content of the biomass sample. If this procedure is performed in conjunction 

with carbohydrate (cellulose and hemicelluloses) determination, the contribution of glucose from 

starch will be included in the total glucose value. 

ൈ 100 

Example of Flowchart Use and Decisions 

The flow chart of analysis (Figure 1) provides an example of a complete biomass feedstock 

analysis. The paragraphs below will step through an analysis of a whole feedstock sample, 

detailing the decisions necessary at each step. 

For the purposes of demonstrating the flow chart and decision making steps, a hypothetical 

herbaceous feedstock will be used as an example. This feedstock is a potential energy crop that is 

harvested off of the ground. The plant is known to produce grain late in life, but this particular 

sample should not contain grain, according to the sample source. Before being shipped for 

analysis the sample was dried and milled through a 2-mm screen and sealed in a plastic bag. 

As this sample has already been milled to an appropriate particle size distribution, the first 

decision to be considered for this sample is whether sieving is necessary. Initial ash 

measurements indicate an ash content of 12%, which is greater than the recommended 10%, and 

may interfere with the acid hydrolysis steps. Sieving could be tested to determine if some of the 

ash could be partitioned into the fines (-80 mesh), but as the plant was harvested off of the 

ground, the high ash content is likely soil and can be reduced by water extraction. Since the 

sample is herbaceous, water extraction is already part of the analysis suite. 

Extraction is the next major consideration for the sample. As discussed above, all herbaceous 

materials are extracted with water as well as with ethanol. Prior to extraction with water, samples 

must have protein and total solids measurements performed, in addition to the ash measurement, 

which has already been done. Water extraction is required to quantify sucrose levels for 

herbaceous materials, but it also proves to be efficient at reducing the ash content to 5%. If it had 

not, sieving would need to be reconsidered to reduce the ash content to less than 10% before 

hydrolysis. Ethanol extraction follows the water extraction to ensure no complications regarding 

acid insoluble lignin measurements. The sample is now extractives-free and ready for hydrolysis. 

Prior to hydrolysis the sample must again have total solids, protein, and ash measurements 

performed. Total solids will be used to convert values to a dry weight basis. Protein and ash 

measurements are used to determine the amount of these constituents removed during the 

extraction process. 

8 
Hydrolysis is run as described in the LAP, but the sample seems to have difficulty filtering while 

removing the acid insoluble lignin. Carbohydrate analysis, acetyl analysis, and acid soluble 

lignin measurement proceed as expected. 

Once the data is compiled, the glucan measurement seems disproportionately high and the mass 

closure is significantly below 100%, while the other constituent values are what can be expected 

in similar feedstocks. Additionally, the acid soluble lignin results seem to have higher than 

expected errors. These problems may stem from one issue or a combination of problems. Two 

potential areas of interference will be examined. 

If the plant did contain grain that was not detected in the field, the sample may have starch that 

was not accounted for. Since starch is measured as glucan in the analysis, starch content would 

explain the high glucan values, but not the low mass closure. 

The unusually slow filtration of the acid insoluble lignin and the high acid insoluble lignin errors 

are commonly related. It is likely that there is something in the hydrolysate solution that the 

LAPs have not been optimized for, and an additional step is needed. In this case, further solvent 

extractions would be a good consideration to remove additional fractions of extractable material. 

Closing

The LAPs described above cover the summative mass closure for biomass feedstocks only, and 

some are not appropriate for chemically or thermally altered materials. The goal is to break the 

biomass sample down into constituents that sum to 100% by weight. If the constituents do not 

sum to 100%, the LAP flow chart should be revisited to determine missing or incorrectly 

quantified components. There are many subtleties in the analytical suites for differing feedstocks, 

and this LAP is not meant to replace the specific LAPs in any way. 

List of Revisions 

Version 07-08-2011: 

	 Page 1: Drying temperature in a convection oven in sample preparation section has been 

corrected from 105o

C to 45oC. 

	 Page 6: %ASL equation has been corrected. 

	 Page 7: Acetic acid to acetate modifier has been corrected. 

Original version (April 2010). 

9 
Start 1 ) Determination of Total Solids in Biomass and Total Dissolved Solids in Liquid Process Samples 

2 ) Preparation of Samples for Compositional Analysis 

> 10 % 

Moisture? Yes Dry

> 2 mm? 

No 

Mill No 

Sieving? 

Yes 

Yes Sieve 

Process 

Feedstock 

No 

Ash 

Protein Yes Herbaceous ? % Solids 

Water Extraction 

Sucrose 

Drying 

Ethanol 

Extraction 

No 

Solution Drying 

Extracted Solids 

Solids 

% Solids 

Separate by mesh 

size Mill 

Ash % Solids 

-20/+80 

Ash % Solids 

-80 

+20 

Solution Solids 

3 ) Determination of Ash in Biomass 

4 ) Determination of Extractives in Biomass 

5 ) Determination of Carbohydrates and Lignin in Biomass 

6 ) Determination of Protein Content in Biomass 

7 ) Determination of Starch in Biomass by HPLC 

1 

1 

1 

1 

2 

2 

2 

3 

3 

3 

4 

4 

4 

4 

4 

6 

% Solids Herbaceous? 

Protein 

Yes 

Starch? 

Starch 

Yes 

Hydrolysis Ash 

Filter 

Acid Insoluble 

Lignin Solids 

Ash 

Solution Solution 

Acids Carbo Acid Soluble 

Lignin 

Protein 

1 5 3

5 

5 5

5 5 5 

5 

6 7 

Figure 1. Flow chart of analysis 

10