Biomass Compositional Analysis Method Variability

David W. Templeton, Christopher J. Scarlata, Justin Sluiter, and Ed Wolfrum

National Bioenergy Center National Renewable Energy Laboratory 1617 Cole Blvd Golden CO 80401

Office of Energy Efficiency and Renewable Energy

Operated by the Alliance for Sustainable Energy, LLC

National Bioenergy Center, National Renewable Energy Laboratory, 1617 Cole Blvd. Golden, CO 80401

Motivation

• Compositional analysis data used in all steps of biomass 

to biofuels process research.

• We would like to determine “typical” feedstock analytical 

wet chemistry errors.

• Better able to match NIR calibration models to wet 

chemistry errors.

• Identify and then control important sources of error.

Method Background

W t h i t th d d t d f

Corn Stover Wet Chemistry Statistics Sample Description % Whole Ash %Structural Inorganics %Non- structural inorganics %Structural Protein % Sucrose %Water Extractable Others %Ethanol Extractives % Lignin

% Glucan% Xylan% Galactan% Arabinan% Acetyl% Total Component Closure

Within Batch Repeatability

Grand average 4.78 1.67 3.11 1.08 7.24 10.14 2.58 12.29 33.96 19.15 1.05 2.50 2.88 97.41

pooled sd 0.10 0.09 0.14 0.04 0.37 0.56 0.12 0.12 0.36 0.23 0.05 0.10 0.14 0.62

Pooled n 136 136 136 115 135 134 136 124 128 131 135 135 123 130

pooled CV 2.10% 5.23% 4.39% 4.06% 5.08% 5.50% 4.72% 0.95% 1.06% 1.19% 4.61% 4.10% 4.78% 0.63%

Between Analyst Reproducibility (one sample per analyst) Wet chemistry methods adapted from 

wood lignin methods. 

1920’s -1940’s: USDA Forest Products Lab (FPL)

Applied H2SO4 lignin methods for US wood samples

1954: Adapted as TAPPI lignin standard method

1967: Procedures for the Chemical Analysis of Wood and Wood 

Products by Moore and Johnson (FPL)

1970’s -1990’s: Methods adapted to dietary fiber analyses 

(Uppsala Method for dietary fiber)

1990’s: FPL/TAPPI/Uppsala methods applied to wood analysis at 

NREL for biofuels research (NREL LAPs)

1993: IEA/NREL round-robin compositional analysis 

Table 1. Table showing within and between batch compositional analysis summary statistics for corn stover. Values for NIST Bagasse 

(SRM 8491) were similar (data not shown)

Fig 1. Charts of corn stover water 

soluble material (upper) and wholebasis lignin (lower) shown in run 

order. The samples are grouped in 

batches run together by an analyst. 

Error bars are set at ± 1sd around

Fig 4. Chart of corn 

stover glucan in run 

order (shown without 

extractives outliers). 

The average and ± 1 

38

40

Corn Stover Glucan (w/o extractives outliers)

AFUF

FTLB

Open 

Symbols 

Grubbs 

outliers 

Between Analyst Reproducibility (one sample per analyst)

Reproducibility ave 4.78 1.67 3.11 1.08 7.24 10.14 2.58 12.29 33.96 19.16 1.05 2.50 2.87 97.43

Reproducibility sd 0.21 0.10 0.25 0.05 0.42 1.23 0.10 0.16 0.46 0.29 0.11 0.22 0.27 1.57

Reproducibility n 12 12 12 12 12 12 12 12 12 12 12 12 12 12

Reproducibility CV 4.36% 6.28% 8.20% 4.92% 5.78% 12.16% 4.07% 1.30% 1.35% 1.53% 10.78% 8.85% 9.52% 1.62%

16

18

20

22

% dry wt.

Corn Stover Total Water Extractives

AFUF

FTLB

1995: ASTM adapted NREL LAPs as standard method

2000’s: NREL adapted LAPs to herbaceous residues (corn 

stover). Continue to improve methods

Experimental Conditions

Analysis material: Corn Stover (Pioneer 33B51)

•Harvested 2003 from Northeast Colorado

•Milled (2mm) and sieved (-20/+80 mesh) 

•900g sample coned and quartered 3X

•NIR check confirmed homogeneity

11.5

12.5

13.5

0 25 50 75 100 125 150 175

% dry wt.

Run order

Corn Stover Lignin

AFUF

FTLB

Open symbols

excluded from

statistics

Error bars are set at ± 1sd around

each batch average. The error in 

water solubles dominated the 

overall errors. Samples with 

unusually low water extractives had 

high lignin values. The same effect 

is seen in glucan and xylan (data 

not shown). For water extractives, 

the average and ± 3 sd lines are 

shown. Samples (n=29) with water 

extractives below 3 sd are not 

included in final statistics.

sd lines are shown. 

Seven samples (open 

symbols) are identified 

as outliers using the 

Grubbs test and not 

included in final 

statistics.

Six samples are significantly above batch average. Sugars 

may have been concentrated in the lignin vacuum filtration 

step. A similar trend was seen in xylan and (weakly) in 

acetyl (data not shown), but not lignin (Fig 2).

32

34

36

0 25 50 75 100 125 150 175

% dry wt.

Run order

10

12

14

0 25 50 75 100 125 150 175

Run order

Samples

improperly dried 

at 105oC

Open symbols excluded 

from summary statistics.

c ec co ed o oge e ty

Run in batches of 12 + NIST Bagasse (SRM #8491)

•Limited by number of ASE positions

14 batches (168) run by 8 analysts in 2 labs at NREL

•Two analysts ran 4 batches split between both AFUF 

and FTLB labs

•Six analysts ran one batch each split between labs

•Designated autoclaves in AFUF (larger, built in) and 

FTLB (smaller, benchtop size)

•Triplicate sugar recovery standards (SRS) run with each 

batch to correct for losses during hydrolysis

Complete solids composition including extractives

Fig 2. Effect of variable 

extractives values on wholebasis glucan and lignin 

calculations. Extractives-free 

glucan and lignin (red bars) 

show narrow distribution. 

When converted to whole 

basis the glucan and lignin 

(yellow bars) take on the 

water extractives errors (blue 

bars) This shows the error 75

Extractives Effect on Lignin Calculation

0

25

50

75

12 16 20 24 28 32 36 40 44 48

F

r

e

q

u

e

n

c

y

% dry weight

Extractives Effect on Glucan Calculation

Extractives Glucan Ext. Free Glucan

Glucan = EF Glucan/(100%‐Extractives)

Fig 5. Run chart of 

sugar recovery 

standards (SRS) run in 

triplicate with each 

batch. Samples run in 

AFUF (closed symbols) 

in larger autoclave 

showed significantly 

lower SRS recovery for 

both glucan and xylan. 

No differences were seen by lab for corn stover composition. 

SRS seem to be effectively correcting for autoclave 

85%

95%

105%

0 10 20 30 40 50

% recovery

Run order

Sugar Recovery Standard

Glucose SRS

Xylose SRS

Closed symbols

run at AFUF

Washing, Drying, Chopping, 

Milling, Homogenizing

Whole Corn 

Stover

(as received)

H2O Extraction and

EtOH Extraction

Weigh Solids

2-Stage Acid Hydrolysis

Package 

li id

Acid insoluble 

solids transfer 

to filtering 

crucible 

Crucibles

Wash 

solids

Filter 

liquids

Neutralize? Y/N

Sample preparation

•Drying then milling

Extraction

•Water then ethanol

•Accelerated Solvent Extraction

Two-stage hydrolysis

•72% H2SO4 1hr, 30°C

•4% H2SO4

•Autoclave 1hr, 121°C

SRS t t f l

Method Scheme

50

75

F

r

Extractives Tests

Ethanol Extractives

Sucrose

Water Extractible "Others"

Fig 3. Histograms of extractible 

components in corn stover. 

Ethanol extractives and sucrose 

show narrow distributions, while

•Reproducible results seen from 8 analysts in 2 labs.

•No compositional difference seen between labs.

•Errors in water extract not seen in ethanol extraction.

•Error in water extractives dominated overall errors.

•This effect was computational rather than physical as 

extractives free values showed good reproducibility.

•Carbohydrates may be concentrated in hydrolysate 

liquor during lignin vacuum filtration step.

Conclusions

bars). This shows the error

comes from the extractives 

test not the glucan/lignin test. 

No effect of poor extraction 

seen in extractives free data.

0

25

50

11 13 15 17 19 21 23

F

r

e

q

u

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n

c

y

% dry weight

Lignin EF Lignin Extractives

Lignin = EF Lignin/(100%‐Extractives)

y g

differences.

The information contained in this poster is subject to a government license.

31st Symposium on Biotechnology for Fuels and Chemicals • San Francisco, CA

May 3-6, 2009 • NREL/PO-510-45799

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.

HPLC 

analysis

Drying, 

ashing, 

weighing

liquid 

samples in 

barcode 

labeled vials

Crucibles

with solids go 

on for further 

analysis

•SRS to correct for loss 

Gravimetric analysis

•lignin

HPLC analysis

•As monomers

Available at: 

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

0

25

0%2%4%6%8%10%12%

r

e

q

u

e

n

c

y

% dry weight

show narrow distributions, while

water extractible “others” shows 

wide distribution. Errors in water 

extraction not seen in sucrose 

and ethanol extractives.

•SRS effectively corrects for different autoclaves.

Analysts:

Crissa Doeppke, Erik Fisk, Deb Hyman, Ryan Ness, 

Courtney Payne, Darren Peterson, Kristen Reichel, 

Jeff Wolfe

Acknowledgments