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Determination of Acid Soluble 

Lignin Concentration Curve by 

UV-Vis Spectroscopy 

Laboratory Analytical Procedure (LAP) 

Issue Date: 06/01/2007 

D. Hyman, A. Sluiter, D. Crocker, D. Johnson, 

J. Sluiter, S. Black, and C. Scarlata 

Technical Report 

NREL/TP-510-42617 

January 2008 

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by Midwest Research Institute • Battelle 

Contract No. DE-AC36-99-GO10337

Technical Report 

NREL/TP-510-42617 

January 2008 

Determination of Acid Soluble 

Lignin Concentration Curve by 

UV-Vis Spectroscopy 

Laboratory Analytical Procedure (LAP) 

Issue Date: 06/01/2007 

D. Hyman, A. Sluiter, D. Crocker, D. Johnson, 

J. Sluiter, S. Black, and C. Scarlata 
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Procedure Title: Determination of Acid Soluble Lignin 

Concentration Curve by UV-Vis Spectroscopy 

Laboratory Analytical Procedure 

1. Introduction 

1.1 Lignin is a complex phenolic polymer found in biomass feedstocks and biomass derived 

products. Lignin contributes a significant mass to the composition of biomass. A fraction 

of the lignin content can be solubilized during certain analytical or industrial procedures. 

Accurately measuring the lignin value is critical to mass closure. This procedure describes 

the isolation of lignin from an acidic solution, and measurement of a maximum peak and 

absorptivity constant specific to that sample or biomass type. These values can usually be 

applied to other samples of the same matrix. Ultraviolet-Visible spectroscopy (UV-Vis) is 

employed in this technique, and measurements are made at a peak maxima that minimizes 

the confounding influence of other analytes. Reference appendix 15.1 for further 

information on confounding analytes. 

1.2 The solubilized lignin fraction is extracted from a calibration liquor or other matrix using 

methyl isobutyl ketone (MIBK). The solvent is removed, and the fraction is dried. The 

extracted material is dissolved in water to produce a set of lignin concentration standards. A 

wavelength maximum is identified that minimizes interferences. A response curve is 

generated using a UV-Vis spectrometer. To analyze an unknown liquor sample, the sample 

is diluted until it is within the range of the response curve. The lignin concentration of the 

unknown liquor sample is determined using Beer’s Law and a dilution factor relative to the 

intact sample. 

2. Scope 

This procedure is appropriate for acidic hydrolyzate liquors from biomass. 

This procedure is appropriate for biomass containing the components listed throughout the 

procedure. Any biomass containing other interfering components not listed must be further 

investigated. 

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

Assurance Plan (QAP). 

3. Terminology 

3.1 HMF—hydroxymethyl furfural 

3.2 MIBK—Methyl Isobutyl Ketone 

3.3 Acid Soluble Lignin- The fraction of lignin solubilized during certain analytical procedures, 

such as LAP “Determination of Structural Carbohydrates and Lignin in Biomass”, or during 

certain industrial or pretreatment procedures 

3.4 Absorptivity constant- Reference section 11.1, also known as molar extinction coefficient 

4. Significance and Use 

This procedure is used, possibly in conjunction with other procedures, to determine the amount 

of lignin in a solid biomass sample or hydrolyzate liquor. 

 1
5. Interferences 

5.1 Methyl Isobutyl Ketone (MIBK) forms emulsions easily with water. Care must be taken not 

to shake such a mixture too vigorously. 

5.2 A neutralization step must be done after the MIBK extraction. This prevents the 

concentration of trace acid during solvent removal, which may cause sample degradation. 

This neutralization must be performed after the MIBK extraction to ensure that all of the 

lignin is extracted. (Lignin has a higher affinity for MIBK than it does for acidic solutions.) 

5.3 Sodium Hydrogen Carbonate (Sodium Bicarbonate) loses buffering capacity when exposed 

to oxygen for extended periods. Buffers for neutralization should be created each time an 

extraction is performed to keep the reagent fresh. 

5.4 Biomass samples tend to degrade when exposed to temperatures above 40°C. When 

removing solvent avoid raising the materials above this temperature. 

5.5 This method has been optimized to reduce interference from HMF and furfural that are 

likely to be present in the matrix by specifying an extended wavelength, such as 320 nm. 

5.6 MIBK can be difficult to evaporate. Adding small amounts of water to a solution of MIBK 

creates an azeotrope with a lower boiling point than MIBK, thus aiding evaporation. 

6. Apparatus 

6.1 Analytical balance, accurate to 0.1 mg 

6.2 Diode array UV-Vis spectrometer 

6.3 Vacuum solvent evaporation system, such as a Bucchi Rotavapor 

7. Reagents and materials 

7.1 Reagents 

7.1.1 Methyl Isobutyl Ketone (MIBK) a.k.a. 4-Methyl-2-pentanone, HPLC grade 

7.1.2 Nanopure water 

7.1.3 Sodium Hydrogen Carbonate (NaHCO3) a.k.a. Sodium Bicarbonate 

7.1.4 Acetone, spectroscopic grade 

7.1.5 Ethyl alcohol, HPLC grade 

7.1.6 Sodium Bicarbonate Buffer 

7.1.6.1 Add approximately 2 grams of Sodium Bicarbonate to 100 mL water. 

7.1.6.2 Stir until dissolved. 

7.1.6.3 Prepare fresh buffer each day. 

7.2 Materials 

7.2.1 Graduated cylinder, 100 mL 

7.2.2 Beakers, 250 mL, or sized to accommodate sample volume 

7.2.3 pH paper, range 2-10 

7.2.4 Round bottom flasks, 50 mL, or sized to accomodate sample volume 

7.2.5 Separation funnel, 250 mL, or sized to accommodate sample volume 

7.2.6 Erlenmeyer flasks, 250 mL 

7.2.7 Volumetric flask, sized to accommodate sample amount, refer to step 10.3.5 

7.2.8 Adjustable pipettors, covering appropriate ranges needed for dilutions 

8. ES&H Considerations and Hazards 

8.1 Acidic compounds can be corrosive and should be handled with care. 

 2
8.2 MIBK has moderate health risks and is extremely flammable. Refer to MSDS for more 

information. 

8.3 Operate all equipment with the appropriate manual and NREL Safe Operating Procedures. 

8.4 Follow all applicable NREL chemical handling procedures. 

9. Sampling, Test Specimens and Test Units 

9.1 Care must be taken to ensure a representative sample is taken for analysis. 

10. Procedure 

10.1 Separate the lignin fraction 

10.1.1 Add the hydrolyzate sample (or liquor) to an appropriately sized separation funnel. 

10.1.2 Add 25 mL MIBK to separation funnel for every 100 mL of sample. 

10.1.3 Agitate gently, at about one inversion per second. Avoid forming an emulsion by 

mixing too vigorously. Vent the separation funnel during each inversion. 

10.1.4 Allow mixture to separate for at least 30 minutes. 

10.1.5 If an emulsion is still present then add several drops of high purity acetone and 

repeat step 10.1.4, or let the mixture sit for a longer period of time. 

10.1.6 Separate the fractions into labeled beakers. 

10.1.7 Repeat steps 10.1.1-10.1.6 at least two more times with aqueous phase. The MIBK 

phases from each separation can be combined. 

10.2 Neutralize the MIBK fraction 

10.2.1 Place the MIBK fraction in clean separation funnel. This fraction now contains the 

soluble lignin. 

10.2.2 Wash the MIBK fraction by adding a 10 mL aliquot of the Sodium Bicarbonate 

solution to the separation funnel and inverting several times. Vent the separation 

funnel during each inversion. 

10.2.3 Separate the fractions into labeled beakers. Using pH paper, check the pH of the 

aqueous phase as it drips from the separation funnel. Collect the wash water, but do 

NOT mix it with the original hydrolyzate liquor. 

10.2.4 Repeat steps 10.2.1-10.2.3 until the pH of the aqueous phase is greater than 6.5. 

10.3 Dry the MIBK fraction and prepare the stock solution 

10.3.1 Weigh an appropriately sized oven dry round bottom flask and place the MIBK 

extract in the flask. 

10.3.2 Remove the solvent from the MIBK fraction using a Rotavapor solvent removal 

system or similar technique. The water bath heating the round bottom flask should 

be set at 30-35 °C. MIBK can be difficult to evaporate. When only several 

milliliters of solvent are left, add small amounts of water (a few milliliters at a time) 

to the MIBK to create an azeotrope with a lower boiling point than MIBK, until all 

solvent is gone. 

10.3.3 Place the round bottom flask and residue from step 10.3.2 in a vacuum oven at 

40°C for a minimum of 12 hours. Repeat until there is no further weight loss. 

10.3.4 Weigh the flask to determine the total mass of lignin isolated in the MIBK fraction. 

Quantitatively transfer the lignin from the dried round bottom into a pre-weighed 

volumetric flask. The size of the flask depends on the amount of lignin present. (A 

resulting solution of at least 1 mg/mL is desirable.) Use just enough ethanol to 

dissolve the lignin in the round bottom flask. It may be necessary to mix this for a 

 3
prolonged amount of time to ensure complete dissolution. Quantitatively transfer 

the ethanol/lignin solution to the volumetric flask. Evaporate off the ethanol under 

a stream of nitrogen until dry. Dry in a vacuum oven at 40°C for at least 12 hours. 

10.3.5 Weigh the volumetric flask with lignin. This step is necessary because not all of the 

lignin transfers from the round bottom flask to the volumetric flask. 

10.3.6 Re-dissolve the lignin in ethanol in the volumetric flask, bringing the solution to 

volume. This is the stock solution that will be used to make dilutions for the 

standard curve. A stock solution of at least 1 mg/mL is desirable to perform serial 

dilutions. 

10.4 Build A Standard Curve 

10.4.1 Create a set of aqueous standards using the stock solution made in step 10.3.7. 

Formulate standards at concentrations that give a linear response versus absorbance, 

usually within an absorbance range of 0.1 to 2.5 absorbance units, maximum. See 

Table 1 for example dilutions and concentrations. 

10.4.2 Measure the absorbance of each standard at an appropriate wavelength on a UVVisible spectrophotometer. See appendix A for an example scenario of appropriate 

wavelength determination. Record the absorbance to three decimal places. 

Reproducibility should be + 0.05 absorbance units. Analyze each sample in 

duplicate, at minimum. 

10.4.3 Use good analytical techniques to create a curve of concentration versus UV-VIS 

absorption at an appropriate wavelength, e.g. 240, 280, or 320 nm. Determine the 

extinction coefficient (ε) for lignin by taking the slope of the curve. 

Table 1 

Table 1 example is based on the extraction of ASL from a solids analysis hydrolysis liquor 

% ASL is calculated for the stock solution using the equation: 

(Conc. of stock soln. from step 10.3.7)*((Volume hydrolyzate)/(Dry weight solid sample))*100) 

Assuming that the stock solution is 100.0 mg lignin in 100 mL water, 

%ASL = 1.000 mg/mL *( 87.00 mL / 290.0 mg) *100 = 30.0% 

Standard 

# 

% ASL 

stock 

Dilution 

factor 

mL of 

stock in 25 

mL water 

% ASL 

with 

dilution 

Final Conc. 

of standard 

(mg/ml) 

1 30.0 10 2.5 3.00 0.100 

2 30.0 20 1.25 1.50 0.050 

3 30.0 50 0.5 0.60 0.020 

4 30.0 65 0.385 0.46 0.015 

5 30.0 80 0.313 0.38 0.013 

6 30.0 100 0.25 0.30 0.010 

7 30.0 120 0.208 0.25 0.008 

8 30.0 160 0.156 0.19 0.006 

9 30.0 250 0.1 0.12 0.004 

10 30.0 500 0.05 0.06 0.002 

11 30.0 1000 0.025 0.03 0.001 

12 30.0 2000 0.0125 0.02 0.0005 

 4
10.5 Measure An Unknown 

10.5.1 Dilute the sample so that the UV-VIS absorbance at the wavelength selected in step 

10.4.2 is within the calibration range. Record the absorbance to three decimal 

places. Reproducibility should be + 0.05 absorbance units. Analyze each sample in 

duplicate, at minimum. 

10.5.2 Calculate the amount of acid soluble lignin present using calculation 11.1 for 

process liquors, and 11.2 for liquors from solids analyses. 

11. Calculations 

11.1 Beer’s law can be expressed as 

εbcA , λ =

Where: 

Aλ = average UV-Vis absorbance at a specific wavelength 

ε = absorptivity constant at a specific wavelength in L/g-cm 

b is the path length through the sample in cm 

c is the concentration of a single analyte in mg/ml 

11.2 Calculate the amount of acid soluble lignin (ASL) on an extractives free basis 

% ASL = 

UVabs x Volume filtrate x Dilution

ε x ODWsample

 x 100

where: 

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

Volumehydrolysis liquor = volume of filtrate, 87 mL 

Dilution = 

Volumesample + Volume diluting solvent

Volumesample

ε = absorptivity constant of biomass at specific wavelength in L/g-cm 

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

following calculation 

RPD = 1 (X − X2)

X mean

⎛

⎝ ⎜ ⎞⎠ ⎟ × 100

Where: 

X1 and X2 = measured values 

Xmean = the mean of X1 and X2 

11.4 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 

 5
RMS = x m = mean = 

x

1

n

∑

 n 

⎛ 

⎝ 

⎜ 

⎜ 

⎜ 

⎜ ⎜ 

⎞ 

⎠ 

⎟ 

⎟ 

⎟ 

⎟ ⎟ 

2

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

RMSdeviation = σ = stdev =

xi − x ( ) m

2

1

n

∑

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 the wavelength used as well as the absorptivity constant. 

12.2 Report the acid soluble lignin on a mg/mL basis, or a dry weight percent if using LAP 

“Determination of Structural Carbohydrates and Lignin in Biomass”. 

12.3 For replicate analyses, report the average and relative percent difference. 

13. Precision and Bias 

13.1 None to report 

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: Run independent duplicates from two different dilutions, at minimum. 

14.3 Blank: nanopure water 

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: Minimum volume of sample sufficient to yield enough lignin to create a 

calibration curve that falls above the lower detection limit of the UV-Vis instrument 

14.7 Sample storage: Acidic hydrolysis liquors may be stored in a refrigerator for up to two weeks. 

Lignin can precipitate and adhere to containers during storage. 

14.8 Standard storage: Acidic hydrolysis liquors may be stored in a refrigerator for up to two 

weeks. Neutral lignin solutions may be stored in a refrigerator for up to four days. Lignin can 

precipitate and adhere to containers during storage. 

14.9 Standard preparation: Refer to section 10. 

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

14.11Control charts: All values may be control charted if desired. 

 6
15. Appendices 

15.1 

Influence of Carbohydrate Degradation Products on the Measurement of Acid Soluble Lignin 

David K. Johnson 

Furfural and hydroxymethyl furfural (HMF) are the main acid catalyzed degradation products 

of pentoses and hexoses. Hydrolyzates produced in the analysis of biomass samples always 

contain small amounts of these furans. Both furans are very strongly absorbing in the UV 

spectral range from 190 to 300 nm. The analysis of biomass also results in the dissolution of a 

small fraction of the lignin in biomass. 

The current method for determining the quantity of lignin dissolved in an acid hydrolyzate is to 

measure the UV absorbance of the hydrolyzate at 205nm and apply an absorbtivity of 110 L/gcm to calculate the concentration of acid soluble lignin in the sample. The wavelength, 205nm, 

is chosen because it corresponds to a minimum in the UV spectrum of furfural and HMF, and 

is in a region where lignin components absorb strongly. However, the absorption of the furans 

is still significant at 205 nm (see Table 1). Recently new methods have been explored using 

different wavelengths and absorbtivities determined for specific biomass samples. The 

absorbtivity of acid soluble lignins from pine, sugarcane bagasse, corn stover, poplar, and other 

feedstocks have been determined at their λmax (197 – 202 nm) and at 240 nm. The latter 

wavelength corresponds to another minimum in the spectra of the furans, but again there is still 

a significant absorbance by the furans at these wavelengths (Table 1). Table 1 shows the λmax

and the relative absorbtivities of furfural, HMF and some lignin-derived compounds found in 

acid hydrolyzates by reversed-phase HPLC. The relative absorbtivity in the table is the 

absorbtivity of a substance at a given wavelength compared to its absorbtivity at its λmax. A 

comparison of the spectra of the furfurals and lignin-derived compounds found in biomass 

hydrolyzates is shown in Figure 1. 

Table 2. λmax and Relative Absorbtivities of Furfurals and Lignin-derived Components Found in 

Biomass Hydrolyzates 

 

Relative Absorbtivies at 

Wavelengths Shown 

 λmax (nm) 200 nm 205 nm 240 nm 320 nm 

Furfural 278 11% 6% 16% 1% 

HMF 284 26% 13% 12% 3% 

p-Coumaric acid 308 35% 39% 19% 83% 

Ferulic Acid 322 58% 53% 60% 99% 

Vanillin 230 90% 98% 45% 55% 

Hydrolyzate 190 77% 61% 28% 11.2% 

Hydrolyzate-Furfural 190 64% 52% 18% 10.5% 

 7
Because of the high level of absorbance of the lignin-derived compounds at high wavelengths 

(300 nm and above) at the end of the absorption of the furans, a high wavelength absorbance 

should also be considered for the measurement of acid-soluble lignin. 

Figure 2 shows a reversed-phase HPLC chromatogram for a biomass hydrolyzate from a dilute 

acid pretreatment of corn stover. The hydrolyzate contains a large peak for furfural from 

degradation of pentoses in the corn stover and a HMF peak from degradation of the hexoses. 

The lignin-derived components appear later in the chromatogram and contain verifiable peaks 

for vanillin, p-coumaric acid and ferulic acid. p-Coumaric acid is normally the largest peak in 

biomass hydrolyzates. Figure 2 also contains the chromatogram for the hydrolyzate after it 

was azeotropically rotovapped with n-octane to remove furfural (water was also removed). 

After rotovapping the sample was brought back to its original weight by adding water to return 

the retained components back to their original concentrations. The chromatogram indicates 

that while 96% of the furfural was removed all the other components remained in the solution 

including the HMF. 

200 225 250 275 300 325 350 375 nm

Norm.

0

200

400

600

800

1000

1200

1400

1600

Figure 1. UV Spectra of Biomass Hydrolyzate Components 

Vanillin 

p-Coumaric 

Acid 

Ferulic Acid 

HMF

 8
Figure 3 contains the UV spectra of the hydrolyzate before and after evaporation. As can be 

seen from Figure 3 removal of furfural had a significant affect on the spectrum throughout the 

wavelength range from 190 nm to about 320 nm. From 320 nm, however, the spectrum was 

virtually unchanged. Table 1 lists the relative absorbtivities of the corn stover hydrolyzates. 

The relative absorbtivities for the hydrolyzate-furfural sample were calculated using the 

absorbtivity at the λmax of the hydrolyzate before furfural was removed to show the influence 

that furfural had on what would have been the measurements taken for assessing the acidsoluble lignin content. It is clear that furfural had a significant affect on the absorbance at 200 

and 205 nm, and the largest affect at 240 nm, however, the affect at 320 nm was minimal. 

mAU

0

100

200

300

400

500

600

 DAD1 B, Sig=230,4 Ref=550,100 (F:\HPCHEM\2\DATA\DJ030502\015-0701.D) HMF Furfural

 Vanillin

 p-Coumaric acid Ferulic Acid

 DAD1 B, Sig=230,4 Ref=550,100 (F:\HPCHEM\2\DATA\DJ030502\016-0601.D)

Before Evaporation 

After Evaporation 

2.5 5 7.5 10 12.5 15 17.5 20 22.5 min

Figure 2. Reversed-phase HPLC of Biomass Hydrolyzate Before and 

After Removal of Furfural by Evaporation 

 9
0

0.2

0.4

0.6

0.8

1

1.2

200 220 240 260 280 300 320 340 360 380 400

Wavelength (nm)

Absorbance (AU)

Figure 3. UV Spectra of a Corn Stover Prehydrolyzate Before and After Evaporation to Remove 

Furfural 

I would like to recommend that a higher wavelength, such as 320 nm, be assessed for the 

measurement of acid-soluble lignin, especially for feedstocks such as corn stover that have the 

potential for generating larger amounts of furfural and HMF than is seen in some other 

feedstocks such as wood. 

Internal NREL publication, 2005. 

16. References 

16.1. Johnson, David K., Influence of Carbohydrate Degradation Products on the 

Measurement of Acid-Soluble Lignin, internal publication, 2005. 

Before Evaporation 

After Evaporation 

 10