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Determination of Insoluble Technical Report 

NREL/TP-510-42627 Solids in Pretreated Biomass March 2008 

Material 

Laboratory Analytical Procedure (LAP) 

Issue Date: 03/21/2008 

A. Sluiter, D. Hyman, C. Payne, and J. Wolfe 

NREL is operated by Midwest Research Institute ● Battelle Contract No. DE-AC36-99-GO10337 
Determination of Insoluble Technical Report 

NREL/TP-510-42627 Solids in Pretreated Biomass March 2008 

Material 

Laboratory Analytical Procedure (LAP) 

Issue Date: 03/21/2008 

A. Sluiter, D. Hyman, C. Payne, and J. Wolfe 

National Renewable Energy Laboratory 

1617 Cole Boulevard, Golden, Colorado 80401-3393 

303-275-3000 • www.nrel.gov 

Operated for the U.S. Department of Energy 

Office of Energy Efficiency and Renewable Energy 

by Midwest Research Institute • Battelle 

Contract No. DE-AC36-99-GO10337 
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Procedure Title: Determination of Insoluble Solids in 

Pretreated Biomass 

Laboratory Analytical Procedure 

1. Introduction 

1.1	 Pretreating biomass samples results in slurry, a mixture of liquid and solid material. For 

analytical purposes the liquid and solid material must be separated. The analytical results 

on each fraction are mathematically combined at the conclusion of the analyses to obtain 

mass balance. The following procedure covers two methods of separating these fractions. 

The centrifugation method is useful for severely pretreated samples, while the filtration 

method can be used for mildly pretreated “chunky” material. 

1.2	 The solid fraction must also be water washed to remove any residual liquor prior to 

analysis, so as to not confound the solids analysis. 

1.3	 This method may also be applied to enzymatically digested pretreated solids to close mass 

balance around enzymatic digestion. 

1.4	 This procedure may also be referred to as Fraction Insoluble Solids (FIS). 

2. Scope 

2.1 This procedure is used to separate the liquid and solid fractions of a pretreated biomass 

slurry. It is used to calculate the weight percent of insoluble material in biomass slurry. It 

is also used to remove any residual liquor from the solid fraction. 

2.2 All analyses should be performed in accordance with an appropriate laboratory specific 

Quality Assurance Plan (QAP). 

3. Terminology 

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

moisture present in the sample at the time of weighing 

3.2	 Pretreated biomass- biomass that has been chemically or thermally altered, possibly 

changing the structural composition 

3.3	 Slurry- the combined liquid and solid material resulting from biomass pretreatment 

3.4	 Liquor- the liquid fraction of a biomass slurry 

3.5	 Washed solids- the water insoluble portion of a biomass slurry, water rinsed to remove any 

residual liquor to less than 0.05 g/L glucose. 

3.6	 Fraction insoluble solids (FIS)- the oven dry weight of the washed solids, referred to as a 

weight percentage of the slurry 

3.6.1	 Fraction insoluble solids dry (FISD)- the oven dry weight of the washed solids, referred 

to as a weight percentage of the dry slurry weight 

3.6.2	 Fraction insoluble solids slurry(FISS)- the oven dry weight of the washed solids, 

referred to as a weight percentage of the wet slurry weight 

4. Significance and Use 

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

composition of biomass materials. 

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5. Interferences 

5.1 Slurry can separate quickly. Thoroughly mix slurry prior to sampling to ensure a
 

representative sample. 

5.2 Centrifugation method is recommended unless slurry sample is composed of large chunky
 

material, as the filter can quickly clog with fine material when using the filtration method.
 

Extensive filtering time can concentrate the liquor and should be avoided. If filtration is 

difficult, the centrifugation technique described in 10.1 should be used. 

5.3 Technique is critical to minimizing the amount of material lost during solids washing.
 

When decanting liquid for the centrifugation method, minimize the loss of fine solid 

material. 

6. Apparatus and Materials 

6.1 Analytical balance, accurate to 0.1mg
 

6.2 Convection drying oven, with temperature control of 45 ± 3o

C 

6.3 Convection drying oven, with temperature control of 105 ± 3o

C 

6.4 Desiccator containing desiccant
 

6.5 YSI analyzer with appropriate membranes or equivalent glucose quantification method 

6.6 Additional equipment required for centrifugation method 

6.6.1	 Centrifuge refrigerated to 4o

C with rotor specified to hold 500 ml bottles and rated at 

least 9,000 rpm 

6.6.2	 Centrifuge bottles, 500 ml capacity, with wide mouth, and caps with seals. 

6.6.3	 2L flask or beaker for wash water collection 

6.6.4	 Aluminum foil weigh pans 

6.6.5	 Glass pipettes with bulbs 

6.7 Additional equipment required for filtration method 

6.7.1	 Buchner funnel filters (extra large size) with glass microfiber filter paper, 30 μm or less 

pore size
 

6.7.2	 1 L vacuum flasks 

6.7.3	 Vacuum source and funnel adaptors 

6.7.4	 Spatulas 

7. Reagents 

7.1 Reagents 

7.1.1	 Water, 18 megaohm deionized 

8. ES&H Considerations and Hazards 

8.1 Slurry may contain hazardous material. Handle accordingly.
 

8.2 Ensure that centrifuge bottles are well balanced to prevent centrifuge malfunction. Inspect 

each bottle and rubber o-ring for cracks prior to use to prevent leaking.
 

8.3 Follow all applicable NREL chemical handling procedures 

9. Sampling, Test Specimens and Test Units 

9.1 Vigorously shake or vortex the sample to suspend any solids. Transfer the whole slurry to a 

larger volume container if necessary to ensure proper mixing to achieve sample homogeneity. 

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9.2 Care must be taken to ensure a representative sample is taken for analysis at each step. 

When measuring volumes for analysis, the sample should be at room temperature. 

9.3 Store samples in sealed containers so the volatile component concentration remains 

consistent. Samples should be stored in a refrigerator until ready to use. 

10. Procedure 

Separate the slurry into liquid and solid components. Perform either 10.1 or 10.2. 

Note: Centrifugation method is recommended unless slurry sample is composed of large chunky 

material, as the filter can quickly clog with fine material when using the filtration method. 

Extensive filtering time can concentrate the liquor and should be avoided. If filtration is 

difficult, the centrifugation technique described in 10.1 should be used. 

If LAP “Determination of Total Solids in Biomass” is to be performed on whole slurry, aliquot a 

portion of whole slurry and set aside for this analysis. 

10.1 Centrifugation method. 

10.1.1 Record the weight of a labeled centrifuge bottle with labeled cap to the nearest 0.01 g on 

an analytical balance. Tare the balance. Add 100-150 g of homogenized slurry sample. 

Record the weight of the sample to the nearest 0.01 g. Tightly cap bottle. 

10.1.2 Repeat step 10.1.1 for the remaining centrifuge bottles, ensuring that the total weight of 

each bottle with sample is equal to all other centrifuge bottles. Check centrifuge 

specifications for allowable weight differences between bottles. Analyze samples in 

duplicate. 

10.1.3 Spin bottles in centrifuge at 4o

C for 20 minutes at 9,000 rpm. Bottles can be spun for 10 

minutes at 10,000 rpm if centrifuge is rated at least 10,000 rpm. Gently remove the 

bottles, taking care to not disturb the solid pellet. 

10.1.4 Decant the liquor without disturbing the pellet. Residual liquid may be removed with a 

pipette. Save and refrigerate the liquor for later analysis. If little or no liquor is obtained 

from centrifugation, a pneumatic or hydraulic hand press may be used to press liquor out 

of the whole slurry. 

10.1.5 To wash the solids, rinse the solids from the side of the bottle into the bottom of the 

bottle. Break up the pellet with a spatula; rinse the solids off of the spatula into the 

bottle. Add 100-400 ml deionized water to each bottle, ensuring that the total weight of 

each bottle and cap with sample is equal to all other centrifuge bottles. Each bottle 

should be no more than 3⁄4 full. Cap tightly and shake each bottle vigorously for at least 

30 seconds. 

10.1.6 Repeat step 10.1.3. Decant the liquor without disturbing the pellet. Residual liquid may 

be removed with a pipette. Dispose of the wash water appropriately. 

10.1.7 Repeat steps 10.1.5 and 10.1.6 until glucose remaining in wash is less than 0.05 g/L, as 

determined by YSI or equivalent method. 

10.1.8 Record the weight of the bottle, cap, and washed sample. Perform LAP “Determination 

of Total Solids in Biomass”, convection oven method, on a portion of the homogenized 

solids. Store remaining solids in refrigerator or freezer, or dry, as appropriate. 

3
 
 

10.2 Filtration method. 

10.2.1 Record the weight of a Buchner funnel with filter paper to the nearest 0.01 g on an 

analytical balance. Tare the balance. Add 100-150 g of homogenized slurry sample. 

Record the weight of the sample to the nearest 0.01 g. Analyze samples in duplicate. 

10.2.2 Filter the sample using vacuum filtration. Filtration should last no longer than five 

minutes. Extensive filtering time can concentrate the liquor and should be avoided. If 

filtration is difficult, the centrifugation technique described in 10.1 should be used 

instead. Slurries with very little liquor may require a pneumatic or hydraulic hand press 

to press liquor out of the whole slurry. 

10.2.3 Save and refrigerate the liquor for later analysis. 

10.2.4 While sample is still under vacuum, add approximately 200 ml deionized water. Stir the 

sample, taking care to not disturb the filter paper. Filter the sample. 

10.2.5 Repeat step 10.2.3 until glucose remaining in wash is less than 0.05 g/L, as determined 

by YSI or equivalent method. 

10.2.6 Record the weight of the funnel, filter paper, and washed sample. Perform LAP 

“Determination of Total Solids in Biomass” on a portion of the homogenized solids. 

Store remaining solids in refrigerator or freezer, or dry, as appropriate. 

10.3 If FISD is required, perform LAP “Determination of Total Solids in Biomass”, convection 

oven method, on a portion of the whole slurry. 

11. Calculations 

11.1 Calculate the amount of fraction insoluble solids in the slurry, utilizing LAP 

“Determination of Total Solids in Biomass”, convection oven method, to obtain the oven 

dry weight (ODW) of the washed solids and the ODW of the slurry, if necessary. 

ODWwashed ⋅solids %FISS(Fraction Insouble Solids Slurry)=	 x 100

Weight bottle,cap,& slurry -Weight bottle&cap 

ODWwashed ⋅solids %FISD(Fraction Insouble Solids Dry)=	 x 100

ODWslurry 

11.2 To report or calculate the relative percent difference (RPD) between two samples, use the 

following calculation 

⎛(X1 − X 2) ⎞

RPD = ⎜

⎜ ⎟⎟×100 

⎝ X mean ⎠

Where:
 

X1 and X2 = measured values 

Xmean = the mean of X1 and X2 

11.3 To report or calculate the root mean square deviation (RMS deviation) or the standard 

deviation (st dev) of the samples, use the following calculations. 

First find the root mean square (RMS), of the sample using

 4 
 

 

 

n ⎛ ⎞2 

⎜ ∑x ⎟ 

⎜ 1 ⎟ RMS = x m = mean = ⎜ ⎟ n ⎜

⎜ ⎟⎟ ⎝ ⎠ 

Then find the root mean square deviation, or standard deviation, using 

n 

)

2

∑(xi − xm 

1 RMSdeviation = σ = stdev = 

n 

Where: 

xm=the root mean square of all x values in the set 

n=number of samples in set 

xi=a measured value from the set 

12. Report Format 

12.1 Report percent insoluble solids to two decimal places. RPD and RMS deviation may also 

be reported. 

13. Precision and Bias 

13.1 Determined by data quality objectives and laboratory specific Quality Assurance Plan. 

14. Quality Control 

14.1 Reported Significant Figures or Decimal Places: Determined by data quality objectives 

and laboratory specific Quality Assurance Plan. 

14.2 Replicates: Duplicate analyses, at minimum. 

14.3 Blank: None. 

14.4 Relative percent difference criteria: Determined by data quality objectives and laboratory 

specific Quality Assurance Plan. 

14.5 Calibration verification standard: None. 

14.6 Sample size: 50-200 g 

14.7 Sample storage: Store slurry and liquor samples in the refrigerator in sealed containers so 

the volatile component concentration remains consistent. Wet solids should be stored in 

sealed containers in the refrigerator or freezer. Solid samples with less than 10% moisture 

by weight may be stored in sealed containers at room temperature. 

14.8 Standard storage: None. 

14.9 Standard preparation: None. 

14.10Definition of a batch: Any number of samples which are analyzed and recorded together. 

The maximum size of a batch will be limited by equipment constraints. 

14.11Control charts: None. 

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15. Appendices 

15.1 None. 

16. References 

16.1 NREL LAP “Determination of Total Solids in Biomass”. 

16.2 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #018, “Determination 

of Insoluble Solids of Pretreated Biomass Material”. 

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