A national laboratory of the U.S. Department of Energy

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National Renewable Energy Laboratory 

Innovation for Our Energy Future 

Preparation of Samples for 

Compositional Analysis 

Laboratory Analytical Procedure (LAP) 

Issue Date: 9/28/2005 

B. Hames, R. Ruiz, C. Scarlata, A. Sluiter, 

J. Sluiter, and D. Templeton 

Technical Report 

NREL/TP-510-42620 

January 2008 

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by Midwest Research Institute • Battelle 

Contract No. DE-AC36-99-GO10337

Technical Report 

NREL/TP-510-42620 

January 2008 

Preparation of Samples for 

Compositional Analysis 

Laboratory Analytical Procedure (LAP) 

Issue Date: 9/28/2005 

B. Hames, R. Ruiz, C. Scarlata, A. Sluiter, 

J. Sluiter, and D. Templeton 
DISCLAIMER 

These Standard Biomass Analytical Methods (“Methods”) are provided by the National 

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OF THESE METHODS. 
Procedure Title: Preparation of Samples for 

Compositional Analysis 

Laboratory Analytical Procedure 

1. Introduction 

1.1 This procedure describes a reproducible way to convert a variety of biomass samples 

into a uniform material suitable for compositional analysis. The National Renewable 

Energy Laboratory (NREL) LAPs for compositional analysis have been optimized using 

samples with a specific particle size range and moisture content. All NREL biomass 

compositional analysis procedures assume that the samples have been prepared to meet 

these specifications. Deviations from these parameters may invalidate assumptions 

made in those methods and thereby introduce errors in the analysis. Procedures are 

listed that are suitable for the preparation of biomass feedstocks and a variety of 

biomass-derived materials. Representative sampling of biomass samples is also 

addressed. 

1.2 This procedure is substantially similar to ASTM Standard Practice E 1757 – 01. Parts of 

this procedure are similar to TAPPI method number T264 and NFTA method A 1.1. 

1.3 This procedure describes methods for drying, size reduction, obtaining samples with a 

uniform particle size and representative sampling of biomass samples. 

2. Scope 

2.1 This method is appropriate for the preparation of most types of biomass and biomassderived solids for compositional analysis. 

2.2 This procedure is not intended for materials that will already pass through a 20-mesh 

sieve. 

2.3 This procedure is not intended for materials that cannot be dried by the described 

methods to a total solids content of greater than 85% of the sample’s oven dried weight. 

2.4 All analyses shall be performed according to the guidelines established in an appropriate 

Quality Assurance Plan (QAP). 

3. Terminology 

3.1 Ambient conditions: a temperature of 20°C to 30°C (68°F to 85°F), less than 50% 

relative humidity. 

3.2 Prepared biomass: biomass that has been prepared according to this practice. 

3.3 Oven dry weight (ODW)- the weight of biomass mathematically corrected for the 

amount of moisture present in the sample at the time of weighing 

4. Significance and Use 

4.1 This procedure is used, in conjunction with other procedures to determine the chemical 

composition of biomass samples, see LAP “Summative Mass Closure for Biomass 

Samples”. 

4.2 This procedure describes drying, size reduction and representative sampling methods that 

must be performed prior to analysis for many other constituents. 

 1
5. Interferences 

5.1 This procedure produces biomass samples with a particle size range. The NREL 

procedures for biomass compositional analysis have been optimized for samples with 

this particle size range. Deviation to a smaller particle size may result in a low bias in 

carbohydrate content (and consequent high lignin bias) due to excessive carbohydrate 

degradation. Deviation to a larger particle size may also result in a low bias in 

carbohydrate content (and consequent high lignin bias) due to incomplete hydrolysis of 

polymeric sugars to monomeric sugars. 

5.2 This procedure produces samples with moisture contents below 10%. The NREL 

procedures for biomass compositional analysis have been optimized for samples with 

low moisture contents. Higher moisture content in biomass samples will alter the 

effective acid concentration in the concentrated acid hydrolysis steps. Lowering the acid 

concentration may result in a low bias in carbohydrate content due to incomplete 

hydrolysis of polymeric sugars to monomeric sugars. Incomplete hydrolysis leaves 

many oligomeric sugars, which are not soluble in 4% acid and are incorrectly counted as 

acid insoluble residue in these procedures, introducing a consequent high lignin bias. 

5.3 This procedure assumes that chemical fractionation does not occur during the sieving 

steps and that the chemical composition of any fines removed from the sample is 

substantially similar to the composition of the bulk sample. 

6. Apparatus 

6.1 Large table or drying rack for air drying biomass (method A only). 

6.2 Convection oven capable of maintaining 45 ±5oC (method B only). 

6.3 Freeze-Drier - System with vacuum chamber and pump capable of maintaining a 

pressure of <1 torr and a cold finger in the chamber capable of maintaining a temperature 

of -50°C (method C only). 

6.4 Balance, sensitive to 0.1 g. 

6.5 Standard laboratory knife mill with 2 mm screen. A Wiley Mill, size No. 4 with a 2-mm 

screen, is suitable for samples >20 g, and the intermediate model Wiley Mill, with 1-mm 

screen, is suitable for samples <20 g that will not be sieved. Equivalent knife mills are 

acceptable. 

6.6 Sieve Shaker that provides motion in both horizontal and vertical axes. 

6.7 Sieve Set, No. 20 (850 μm), No. 80 (180 μm) stackable sieves with lid and bottom pan. 

Sieves and bottom pan should be 8.9 cm (31/2 in.) in height. Sieves should conform to 

ASTM Specification E 11. 

6.8 Riffle Sampler with Pans–– A manual sample divider that splits the milled biomass into 

two or more equivalent sub-samples. Riffle divisions should be between 6.4 mm and 

12.7 mm (1/4 to 1/2 in.) with at least twenty-four riffle openings. The feed chute and 

riffles should have a slope of at least 60°. Collection pans, one to pour the sample into 

the riffler, and two or more to collect the sub–samples. 

7. Reagents and materials 

7.1 Reagents 

7.1.1 Acetone, electronic grade. 

7.1.2 Dry ice, ground. 

 2
7.2 Materials 

7.2.1 Assorted trays and containers as appropriate for the selected drying method. 

8. ES&H Considerations and Hazards 

8.1 Milling and sieving actions both produce large amounts of dust. This dust can be a 

nuisance, hazard and irritant. Use appropriate respiratory protection and eye protection as 

needed. 

8.2 If excessive amounts of dust are allowed to become airborne, a potential explosion 

hazard is possible. Provide appropriate dust control measures as needed. 

8.3 Follow all applicable NREL chemical handling procedures. 

8.4 This standard does not purport to address all of the safety concerns, if any, associated 

with its use. It is the responsibility of the user of this standard to establish appropriate 

safety and health practices and determine the applicability of regulatory limitations prior 

to use. 

9. Sampling, Test Specimens and Test Units 

9.1 This procedure describes sampling, and sample preparation required prior to analysis for 

ash, extractives, carbohydrates, lignin, and protein. 

10. Procedure 

10.1 Prepare the biomass sample using one of the three methods described below. 

10.2 Sample Preparation Method A: Air-drying. This method is suitable for the preparation 

of large quantities (>20 g) of field-collected samples into a form appropriate for 

compositional analysis. This method is suitable for drying materials where ambient 

humidity allows the sample to air-dry to a moisture content below 10% as measured 

using LAP “Determination of Total Solids in Biomass” (2004). 

10.2.1 Biomass samples must first be available as pieces with overall dimensions less 

than 5 by 5 by 0.6 cm (2 by 2 by 1/4 in.). Stems or twigs should not exceed 0.6 

cm (1/4 in.) diameter. It is recommended that wastepaper should be shredded into 

pieces less then 1 cm (3/8 in.) wide. Twigs, straw and wastepaper should not 

exceed 20 cm (8 in.) in length to facilitate milling. 

10.2.2 The biomass material should be spread out on a suitable surface and allowed to 

air-dry prior to any milling. Do not pile the material deeper than 15 cm. Turn the 

material at least once per day to ensure even drying and Microbial growth in 

samples. 

10.2.3 Following NREL “LAP Determination of Total Solids in Biomass” (2004) 

measure the solids content of the biomass sample once every 24 hours. 

10.2.4 The material is considered dried when the moisture content is less than 10% and 

the change in weight is less than 1% in 24 h. 

10.2.5 Feed the air-dried biomass into the knife-mill and mill until the entire sample 

passes through the 2 mm screen in the bottom of the mill. Laboratory mills can 

generate enough heat to damage biomass samples. Monitor the mill closely and 

allow the mill to cool to room temperature between batches if necessary. 

10.2.6 Stack the sieves in the following order, starting at the bottom: the bottom pan, 80-

mesh sieve, 20-mesh sieve. 

 3
10.2.7 Place the milled biomass in the 20-mesh sieve. The sample should be no more 

than 7 cm deep in the 20-mesh sieve. The milled sample may be processed in 

batches if necessary. 

10.2.8 Place the cover on the sieve stack and secure the stack in the sieve shaker. 

10.2.9 Shake the sieves for 15 ± 1 min. 

10.2.10 The fraction retained on the 20-mesh sieve (+20 mesh fraction) should be 

reprocessed (steps 10.2.5 through 10.2.9) until no biomass remains on the 20-

mesh sieve. 

10.2.11 The fraction retained on the 80-mesh sieve (-20/+80 mesh fraction) should be 

retained for compositional analysis. 

10.2.12 The material in the bottom pan is the fines (-80mesh) fraction. Retain this material 

for ash analysis. 

10.2.13 Combine all of the -20/+80 mesh batches. Weigh the combined -20/+80 mesh 

fraction to the nearest 0.1 g. Record the weight of the -20/+80 mesh fraction as 

Wt20/80. 

10.2.14 Combine all of -80 mesh batches. Weigh the combined fines to the nearest 0.1 g. 

Record the weight of fines fraction as Wt80. 

10.2.15 If multiple sieved samples were combined they must homogenized. Pour the -

20/+80 mesh fraction into the riffle sampler. 

10.2.15.1 The sample must be distributed evenly onto all the riffle openings. A pan, as 

wide as the riffle opening, should be used. Pour the sample evenly off the 

entire side of the pan and not from the end or the corner. Do not transfer the 

biomass sample from a narrow-mouth container such as a jar. 

10.2.16 Recombine the riffled sub-samples. 

10.2.17 Repeat steps 10.2.15 through 10.2.16 a total of four times 

10.2.18 Determine the total solids content (TS) of both the –20/+80 mesh fraction and the 

fines fraction using LAP “Determination of Total Solids in Biomass” (2004). 

Record the total solids of the -20/+80 mesh fraction as TS20/80. Record the total 

solids content of the fines as TS-80. 

10.2.19 Determine the ash content of each fraction using LAP “Determination of Ash in 

Biomass” (2004). Record the ash content of the -20/+80 mesh fraction as 

Ash20/80. Record the ash content of the fines as Ash-80. 

10.2.20 Using the equations in Section 11, calculate the percent of each fraction in the 

original, biomass sample. The fraction weight percent is used to reconstruct the 

composition of the original biomass sample. In most cases, the composition of 

non-ash biomass in the fines can be assumed to be the same as the composition of 

the non-ash biomass in the –20/+80 fraction. 

10.2.21 If the total sample needs to be subdivided into smaller samples, use the riffler to 

make this separation. 

10.2.22 If the prepared sample is not analyzed immediately after sieving and riffling, the 

sample should be stored in an airtight container or sealable polyethylene bag and 

kept at -20°C until needed. 

10.3 Sample Preparation Method B: Convection oven drying. An acceptable alternative to 

air-drying is to dry the biomass sample in a convection oven at temperatures no greater 

than 45°C. This method is suitable for very wet biomass that is at risk for microbial 

growth during drying, wet pretreated biomass, samples that would not be stable during 

 4
prolonged exposure to ambient conditions, or for drying materials when ambient 

humidity does not allow the sample to air-dry to a moisture content below 10% as 

measured using LAP “Determination of Total Solids in Biomass” (2004). This drying 

method is suitable for small samples of biomass (<20 g). 

10.3.1 Select a container suitable for oven drying the biomass sample and dry this 

container at 45± 3°C for a minimum of 3 h. 

10.3.2 Place the container in a dessicator and allow the container to cool to room 

temperature. 

10.3.3 Weigh the container to the nearest 0.1 g and record this weight as Wt. 

10.3.4 Place the biomass material into the dried container to a maximum depth of 1 cm. 

10.3.5 Weigh the container and biomass to the nearest 0.1 g and record this weight as Wi. 

10.3.6 Place the container and biomass in a drying oven maintaining the temperature at 

45 ± 3°C. Allow the material to dry for 24 to 48 h. 

10.3.7 Remove the container and biomass from the drying oven, place in a desiccator and 

allow the sample to cool to room temperature. 

10.3.8 Weigh the container and biomass to the nearest 0.1 g and record this weight as Wf. 

10.3.9 Return the sample to the drying oven, maintaining the temperature at 45 ± 3°. 

Keep the sample in the drying oven at 45 ± 3°C for minimum of 4 h . 

10.3.10 Remove the container and biomass from the drying oven, place in a desiccator and 

allow the sample to cool to room temperature 

10.3.11 Weigh each sample to the nearest 0.1 mg and record this weight. 

10.3.12 Return the samples to the drying oven at 45°C for 1 h. 

10.3.13 Remove the container and biomass from the drying oven, place in a desiccator and 

allow the sample to cool to room temperature. 

10.3.14 Weigh each sample to the nearest 0.1 mg and record this weight. 

10.3.15 Repeat steps 10.3.12 through 10.3.14 until the change in the mass of the biomass 

is less than 1%. 

10.3.16 Mill the dried biomass sample using one of the following procedures. 

10.3.16.1 For small quantities (<20 g) containing material that would not pass through a 

20 mesh screen, reduce the particle size of the solid residue by knife-milling 

the entire sample through an intermediate size knife-mill with a 1 mm screen. 

If the sample size is sufficient, sieve the sample as described in Method A, 

steps 10.2.5 through 10.2.14. 

10.3.16.2 For larger quantities (>20 g) containing material that would not pass through a 

20 mesh screen, reduce the particle size of the solids by knife-milling, sieving 

and riffling the entire sample as described in Method A steps 10.2.5 through 

10.2.18. 

10.3.17 Using the equations in Section 11, calculate the percent of each fraction in the 

original biomass sample. The mass fraction is used to reconstruct the composition 

of the original biomass sample. In most cases, the composition of non-ash 

biomass in the fines can be assumed to be the same as the composition of the nonash biomass in the –20/+80 fraction. 

10.3.18 If the total sample needs to be subdivided into smaller samples, use the riffler to 

make this separation whenever possible. 

 5
10.3.19 If the prepared sample is not analyzed immediately after sieving and riffling, the 

sample should be stored in an airtight container or sealable polyethylene bag and 

kept at -20°C until needed. 

10.4 Sample Preparation Method C: Lyophilization. An acceptable alternative to air-drying 

(Method A) or drying in a convection oven (Method B) is lyophilization (freeze-drying) 

of the sample. This method is suitable for very wet biomass that is at risk for microbial 

growth during drying, wet pretreated biomass, samples that would not be stable during 

prolonged exposure to ambient conditions, or for drying materials when ambient 

humidity does not allow the sample to air-dry to a moisture content below 10% as 

measured using LAP “Determination of Total Solids in Biomass” (2004). This test 

method is also suitable for materials that are heat sensitive and would degrade if 

subjected to the drying oven in Test Method B. This drying method is suitable for small 

samples of biomass (<20 g). 

10.4.1 Weigh a suitable freeze-drier container to the nearest 0.1 g and record this weight 

as Wt. 

10.4.2 Place the biomass material in the container. For solid samples, do not fill the 

container more than half full. For liquid or slurry materials, limit the sample to the 

amount of material that gives a uniform coating of around 0.5 cm on the walls of 

the container when the sample is frozen. 

10.4.3 Weigh the container and biomass to the nearest 0.1 g and record this weight as Wi.

10.4.4 Combine the dry ice and acetone in a shallow container suitable for shell freezing. 

10.4.5 Place the freeze dry flask containing the biomass sample in the dry ice acetone 

mixture. Slowly turn the container (10 r/min) to freeze the material into a uniform 

layer on the walls of the container. 

10.4.6 Immediately place the container on the freeze-drier and allow the material to dry 

until all visible traces of ice and frost are gone from the sample. This process 

typically takes 12 h for small (<20 g) samples, and can extend to more than 96 h 

for large samples (>250 g). 

10.4.7 Remove the container and biomass from the freeze drier. 

10.4.8 Allow the sample to warm to room temperature. 

10.4.9 Weigh the container and biomass to the nearest 0.1 g and record this weight as Wf.

10.4.10 Mill the dried biomass sample using one of the following procedures. 

10.4.10.1 For small quantities (<20 g) containing material that would not pass through a 

20 mesh screen, reduce the particle size of the solid residue by knife-milling 

the entire sample through an intermediate size knife-mill with a 1 mm screen. 

If the sample size is sufficient, sieve the sample as described in Method A, 

steps 10.2.5 through 10.2.14. 

10.4.10.2 For larger quantities (>20 g) containing material that would not pass through a 

20 mesh screen, reduce the particle size of the solids by knife-milling, sieving 

and riffling the entire sample as described in Method A steps 10.2.5 through 

10.2.18. 

10.4.11 Using the equations in Section 11, calculate the percent of each fraction in the 

original, biomass sample. The mass fraction is used to reconstruct the 

composition of the original biomass sample. In most cases, the composition of 

non-ash biomass in the fines can be assumed to be the same as the composition of 

the non-ash biomass in the –20/+80 fraction. 

 6
10.4.12 If the total sample needs to be subdivided into smaller samples, use the riffler to 

make this separation whenever possible. 

10.4.13 If the prepared biomass sample is not analyzed immediately after sieving and 

riffling, the sample should be stored in an airtight container or sealable 

polyethylene bag and kept at -20°C until needed. 

11. Calculations 

11.1 To calculate the fraction percent of –20/+80 mesh fraction, use the following equation: 

100

( )

( %

80/20 80

80/20

80/20 ×⎟

⎟

⎠

⎞

⎜

⎜

⎝

⎛

+WtWt

Wt Fraction = 

Where: 

Wt 20/80 = weight of –20/+80 mesh fraction (g) 

Wt80 = weight of fines fraction (g) 

11.2 To calculate the fraction percent of -80 mesh fraction, use the following equation: 

100

( )

( %

80/20 80

80

80 ×⎟

⎟

⎠

⎞

⎜

⎜

⎝

⎛

+WtWt

Wt Fraction = 

Where: 

Wt 20/80 = weight of –20/+80 mesh fraction (g) 

Wt80 = weight of fines fraction (g) 

11.3 To calculate the percent of total solids obtained by drying at 45oC (Method B), use the 

following equation: 

100

WW

WW

 = T

ti

tf ×⎟

⎟

⎠

⎞

⎜

⎜

⎝

⎛

−

−

)(

)( % 45

Where: 

% T45 = percent total solids of a sample oven dried at 45oC, 

Wt = tare weight of freeze-drier container, 

Wi = initial weight of container and sample 

Wf = final weight of container and sample. 

11.3.1 To calculate the percent of total solids obtained by freeze drying (Method C), use 

the following equation: 

100

WW

WW T = 

ti

tf

fd ×⎟

⎟

⎠

⎞

⎜

⎜

⎝

⎛

−

−

)(

)( %

Where: 

% Tfd = percent total solids of a freeze-dried sample, 

Wt = tare weight of freeze-drier container, 

 7
Wi = initial weight of container and sample 

Wf = final weight of container and sample. 

11.4 For larger biomass samples that must be prepared in batches, record the total percent 

solids of each fraction as well as the combined weight of the biomass in each fraction, 

(Wf – Wt). 

11.5 To calculate the composition of constituent X in the original biomass sample, use the 

following equation: 

( ) ⎜

⎜

⎝

⎛ ⎟×

⎠

⎞ ⎜

⎝

⎛ +−

× + × − +−

+− +− +− 100

%100 % % % %% 80 80/20

80/20 80/20 80/20 Fra

Ash Ash

Origianl X= X Fraction X

12. Report Format 

12.1 Report weight percent prepared biomass and weight percent fines. Report results on an 

oven dry weight basis. 

13. Precision and Bias 

13.1 Repeatability – Duplicate determinations on splits of the gross sample, by the same 

operator, using the same sieves, should duplicate the percent mass fractions within 2% 

absolute. 

13.2 Reproducibility - Duplicate determinations on splits of the gross sample, by different 

operators, using the same sieves, should duplicate the percent mass fractions within 2% 

absolute. 

13.3 Bias - Since there is not an appropriate standard reference material, no statement about 

bias can be made. 

13.4 Round robin testing – For a report documenting an international round robin test of 

biomass analysis methods, including this procedure, see Milne et al., 1992. 

14. Quality Control (all items bellow, missing) 

14.1 Reported Significant Figures or decimal places: Determined by data quality 

objectives and laboratory specific Quality Assurance Plan, see LAP “Rounding and 

Significant Figures”. 

14.2 Replicates: not applicable 

14.3 Blank: not applicable 

14.4 Relative percent difference criteria: not applicable 

14.5 Calibration verification standard: not applicable 

14.6 Sample size: not applicable 

14.7 Sample storage: If the prepared biomass sample is not analyzed immediately after 

sieving and riffling, the sample should be stored in an air-tight container or sealable 

polyethylene bag and kept at –20oC until needed. 

14.8 Standard storage: not applicable 

14.9 Standard preparation: not applicable 

 8
14.10 Definition of a batch: not applicable 

14.11 Control charts: not applicable 

15. Appendices 

15.1 None 

16. References 

16.1 Moore, W., and D. Johnson. 1967. Procedures for the Chemical Analysis of Wood and 

Wood Products. Madison, WI: U.S. Forest Products Laboratory, U.S. Department of 

Agriculture

16.2 ASTM E 1757 - 01 “Standard Practice for Preparation of Biomass for Compositional 

Analysis" In 2003 Annual Book of ASTM Standards, Volume 11.05. Philadelphia, PA: 

American Society for Testing and Materials, International. 

16.3 NREL BAT Team Laboratory Analytical Procedure "Standard Method for the 

Determination of Total Solids in Biomass." 

16.4 TAPPI Test Method T264 cm-97, "Preparation of wood for chemical analysis" In Tappi 

Test Methosd 2002-2003. Atlanta, GA: Technical Association of the Pulp and Paper 

Industry 

16.5 National Forage Testing Association Methods, available on-line at 

http://www.foragetesting.org/lab_procedures.php, Omaha, Nebraska, National Forage 

Testing Association, Accessed April 2004 

16.6 Milne, T. A.; Chum, H. L.; Agblevor, F. A.; Johnson, D. K. (1992). “Standardized 

Analytical Methods” Biomass & Bioenergy. Proceedings of International Energy 

Agency Bioenergy Agreement Seminar”, 2-3 April 1992, Edinburgh, U.K.. Vol. 2(1-6), 

1992; pp. 341-366 

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