Determination of Carbohydrates in Acid Hydrolysates of Wood Lipika Basumallick and Jeffrey Rohrer Thermo Fisher Scienti� c, Sunnyvale, CA, USA

Po

ster No

te 71636

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Determination of Carbohydrates in Acid Hydrolysates of Wood Lipika Basumallick and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA

Introduction To prepare wood for the production of alcohol based biofuels by fermentation, the wood must first be hydrolyzed in strong acid with applied heat to yield the constituent monosaccharides needed for fermentation. Quantification of those monosaccharides allows an estimate of the biofuel yield. This poster describes a liquid chromatography (LC) method for quantifying monosaccharides from acid hydrolysates of wood that resolves all the released monosaccharides and cellobiose in less than ten minutes using high-performance anion-exchange chromatography. The separated carbohydrates, fucose, arabinose, rhamnose, glucose, mannose, xylose, fructose, and cellobiose are detected by pulsed amperometry. The method requires only sample dilution prior to LC analysis and the mobile phase for chromatography is prepared automatically by the instrument, which reduces labor and increases method accuracy. Using the same LC technology for carbohydrate analysis but with a different high-performance anion-exchange column, we also developed two methods to determine the glucuronic and galacturonic acid contents of the same acid hydrolysate of wood (loblolly pine). One method determines the two uronic acids, while the other method determines the two uronic acids as well as the monosaccharides. Analytical details (e.g., reproducibility, accuracy, etc.) will be presented for each of the three methods.

Methods Experimental details can be found in references 1 and 2. Three different high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods were used depending on the goal(s) of the analysis. These methods are shown in the table below. A Thermo Scientific™ Dionex™ ICS-5000+ system was used for all HPAE-PAD analysis. Acid-hydrolyzed wood samples were kindly provided by the National Renewable Energy Lab (NREL) in Golden Colorado. The liquor sample contained 1% sulfuric acid. Centrifuge wood acid hydrolysate samples (liquor and rinsate from lodge pole pine) at 16,000 g for 10 min to ensure elimination of particulates. Samples were diluted with deionized water.

Results and Discussion Figure 1 shows the separation of a mixed carbohydrate standard and one of the wood hydrolysate samples using Method 1. These conditions allow the separation of galactose and rhamnose at the expense of the fructose/mannose separation. To achieve this separation the column temperature is reduced to 30 oC from 45 and the flow rate is reduced to 1.2 from 1.5 mL/min. This results in an increased analysis time. Flow rate was decreased so that the backpressure was in the allowed range for the eluent generator, which produces the mobile phase. If the galactose/rhamnose separation is not needed, a faster separation can be achieved (Figure 2). As these samples have high concentrations of carbohydrates, in order to reduce the need to dilute for the sensitive PAD, we used a small injection volume and a thick gasket in the electrochemical cell.

FIGURE 1: Determination of small carbohydrates in an acid hydrolysate of wood with Method 1.

FIGURE 2: HPAE-PAD chromatograms of three wood hydrolysate samples These use Method 1 conditions with the exceptions that the temperature is 45 oC and the flow rate is 1.5 mL/min.

Figure 2 shows the separation of all three samples provided by the NREL using conditions more typical of biofuel carbohydrate analysis, particularly for corn stover acid hydrolysates.4 Not that with these conditions there is no resolution of galactose and rhamnose and that cellobiose elutes in < 10 min.

FIGURE 3: Determination of small carbohydrates and uronic acids in acid hydrolysates of wood with Method 2.

The acid hydrolysates of wood can also contain galacturonic and glucuronic acids. These uronic acids require a stronger mobile phase than hydroxide to elute them from an anion-exchange column. Figure 3 shows that by using Method 2, we can first elute the small carbohydrates from the column using a hydroxide eluent and then elute the uronic acids by adding sodium acetate to the eluent. Although, using the sample dilution needed for the small carbohydrates, we can not determine if the samples have uronic acids.

FIGURE 4: Injection of a more concentrated sample of acid hydrolysates of wood to determine uronic acids using Method 2.

To determine if the samples in Figure 3 contain uronic acids, the samples were diluted less and then analyzed with Method 2. Figure 4 shows that while the monosaccharide peaks are overloaded there is evidence of uronic acids in both samples. To better view the uronic acids, Figure 5 shows an enlargement of the region between 35 and 40 min. While galacturonic acid is easily identified and quantified in both samples, there is a peak that compromises glucuronic acid quantification in the hydrolysate liquor sample. Another approach to determining uronic acids in these samples is to design a method that elutes the smaller carbohydrates quickly and only aims to resolve the uronic acids. Figure 6 shows the results of using such a method (Method 3). Note that both uronic acids are resolved from neighboring peaks.

While all the carbohydrates of interest can be measured with Method 2, one analysis for small carbohydrates using Method 1, and another for uronic acids using Method 3 may be more effective for long term success as Methods 1 and 3 are chromatographically simpler than Method 2. The table below show relevant analytical data for the three methods.

Conclusions HPAE-PAD is an effective tool for determining carbohydrate composition of

samples used to produced biofuels.

Monosaccharides can be determined in < 10 min (though without resolving rhamnose and galactose).

Two approaches for determining monosaccharides and uronic acids from wood acid hydrolysates:

Single injection on a Dionex CarboPac PA20 column.

1 injection on a Dionex CarboPac SA10 column for monosaccharides and a 2nd injection on a Dionex CarboPac PA200 column for uronic acids.

References 1. Thermo Scientific Application Note 1089: Determination of Carbohydrates in Acid

Hydrolysates of Wood. Sunnyvale, CA, 2014.

2. Thermo Scientific Application Note 1091: Determination of Uronic Acids and Wood Sugars in Wood-Based Hydrolysates. Sunnyvale, CA, 2014.

3. Thermo Scientific Technical Note 21: Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemcial Detector. Sunnyvale, CA, 2013.

4. Thermo Scientific Application Update 192: Carbohydrate Determination in Biofuel Samples. Sunnyvale, CA, 2014.

Figure 6: Determination of uronic acids in acid hydrolysates of wood using Method 3.

Figure 5: Enlargement of the uronic acid elution time period from Figure 4.

0 4 8 12 16 20 24 10

100

Minutes

nC

Peaks 1. Fucose 2. Sucrose

3. Arabinose 4. Galactose 5. Rhamnose

6. Glucose 7. Xylose

8. Mannose 9. Fructose

10.Cellobiose 11.Maltose

1

2

3

4

6

7

8,9

10 5

11

A. Mix of Sugar Standards (1 g/L) B. Sample 3 (dilution 2)

Chromatographic Conditions Method 1

Small Carbohydrates

Method 2 Small Carbohydrates and

Uronic Acids

Method 3 Uronic Acids

Column

Thermo Scientific™ Dionex™ CarboPac™ SA10-4µm Guard, 4 × 50 mm, and Analytical, 4 × 250 mm

Dionex CarboPac PA20 Guard, 3 × 30 mm, and Analytical, 3 × 150 mm

Dionex CarboPac PA200 Guard, 3 × 30 mm, and Analytical, 3 × 250 mm

Mobile Phases KOH (EGC III KOH) – by Eluent Generation

A. DI Water, B. 100 mM NaOH, C. 100 mM NaOH 1 00 mM Sodium Acetate

A. 1M Sodium Acetate 100 mM NaOH,

B. 100 mM NaOH

Total Run Time 24 min 60 min 30 min

Flow Rate 1.2 mL/min 0.4 mL/min 0.5 mL/min

Column Temperature 30 ºC 30 ºC 30 ºC

Detection

PAD, Au working electrode, 62 mil gasket, Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

Injection Volume 0.4 μL (internal loop) 10 μL 10 μL

Mobile Phase Conditions 1 mM KOH

0-18 min (98.8 A/1.2 B/0 C), 20-30 min (50/50/0), 30.1-46 min (0/0/100), 46.1-50 min

(100/0/0), 50.1-60 min (98.8/1/2/0)

0-18 min 2%A, 18.1 – 22 min 50% A, 22.1-30 min 2%A

0 2 4 6 8 10 12 14 0

140

Minutes

nC

Signal Offset 10%

A. Mix of Sugar Standards (1g/L) B. Sample 1 (dilution 2) C. Sample 2 (dilution 2) D. Sample 3 (dilution 50 )

Peaks 1. Fucose 2. Sucrose 3. Arabinose 4. Galactose 5. Rhamnose 6. Glucose 7. Xylose 8. Mannose 9. Fructose 10.Cellobiose 11.Maltose

1

2

3

4,5

6

7 8

9

10 11

A

B

C

D

0 5 10 15 20 25 30 35 40 8

90

Minutes

nC

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic

acid standards 0.5 mg/L B. Wood hydrolysate liquor (diluted

1000 fold) C. Wood hydrolysate rinsate (diluted

200 fold)

B A

C

1 2

3

4

5 7 8

9

10

12

6

11

System peak

0 5 10 15 20 25 30 35 40 0

300

Minutes

nC

1 2

3 4 5

6

7 8

9 10 11 12

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic acid standards 5 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

A

B

C

Signal Offset 10%

System Peak

35 36 37 38 12

77

Minutes

nC

A

B

C

Samples A. Mix of wood sugars and uronic acid standards 1 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

Peaks mg/L A B C 11. Galacturonic acid 5 0.5 0.5 12. Glucuronic acid 5 0.2 0.2

11 12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0

90

Minutes

nC

Signal Offset 20%

A

B

C

Samples A. Mix of uronic acid

standards 10 mg/L B. Wood hydrolysate

liquor (diluted 10 fold) C. Wood hydrolysate

rinsate (diluted 2 fold) 1

2

Peaks 1. Galacturonic acid 2. Glucuronic acid

Analytical Data Method 11 Method 2 2 Method 32

Linearity

Range – 0.05 – 2.4 g/L For 10 sugars r2 ranged from 0.9980- 0.9998

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9997 Glucuronic acid r2 = 0.9999

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9982 Glucuronic acid r2 = 0.9990

Retention Time Precision

For 7 sugars in standards and samples RSD ranged from 0.07- 0.27%

For 12 sugars (six injections) the range was 0.02-0.09%

For the 2 uronic acids (6 injections) the values were 0.06 and 0.08%

Retention Area Precision

For 7 sugars in standards and samples RSD ranged from 1.18- 3.75%

For 12 sugars (six injections) the range was 0.68-3.53%

For the 2 uronic acids (6 injections) the values were 2.06 and 1.38%

Spike Recovery from

Samples

For 7 sugars in 2 samples the range was

81-103% Not evaluated

For the 2 uronic acids in 2 samples at in triplicate the range was 86-96% and 81-90%

PO71636-EN 0415S

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Determination of Carbohydrates in Acid Hydrolysates of Wood Lipika Basumallick and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA

Introduction To prepare wood for the production of alcohol based biofuels by fermentation, the wood must first be hydrolyzed in strong acid with applied heat to yield the constituent monosaccharides needed for fermentation. Quantification of those monosaccharides allows an estimate of the biofuel yield. This poster describes a liquid chromatography (LC) method for quantifying monosaccharides from acid hydrolysates of wood that resolves all the released monosaccharides and cellobiose in less than ten minutes using high-performance anion-exchange chromatography. The separated carbohydrates, fucose, arabinose, rhamnose, glucose, mannose, xylose, fructose, and cellobiose are detected by pulsed amperometry. The method requires only sample dilution prior to LC analysis and the mobile phase for chromatography is prepared automatically by the instrument, which reduces labor and increases method accuracy. Using the same LC technology for carbohydrate analysis but with a different high-performance anion-exchange column, we also developed two methods to determine the glucuronic and galacturonic acid contents of the same acid hydrolysate of wood (loblolly pine). One method determines the two uronic acids, while the other method determines the two uronic acids as well as the monosaccharides. Analytical details (e.g., reproducibility, accuracy, etc.) will be presented for each of the three methods.

Methods Experimental details can be found in references 1 and 2. Three different high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods were used depending on the goal(s) of the analysis. These methods are shown in the table below. A Thermo Scientific™ Dionex™ ICS-5000+ system was used for all HPAE-PAD analysis. Acid-hydrolyzed wood samples were kindly provided by the National Renewable Energy Lab (NREL) in Golden Colorado. The liquor sample contained 1% sulfuric acid. Centrifuge wood acid hydrolysate samples (liquor and rinsate from lodge pole pine) at 16,000 g for 10 min to ensure elimination of particulates. Samples were diluted with deionized water.

Results and Discussion Figure 1 shows the separation of a mixed carbohydrate standard and one of the wood hydrolysate samples using Method 1. These conditions allow the separation of galactose and rhamnose at the expense of the fructose/mannose separation. To achieve this separation the column temperature is reduced to 30 oC from 45 and the flow rate is reduced to 1.2 from 1.5 mL/min. This results in an increased analysis time. Flow rate was decreased so that the backpressure was in the allowed range for the eluent generator, which produces the mobile phase. If the galactose/rhamnose separation is not needed, a faster separation can be achieved (Figure 2). As these samples have high concentrations of carbohydrates, in order to reduce the need to dilute for the sensitive PAD, we used a small injection volume and a thick gasket in the electrochemical cell.

FIGURE 1: Determination of small carbohydrates in an acid hydrolysate of wood with Method 1.

FIGURE 2: HPAE-PAD chromatograms of three wood hydrolysate samples These use Method 1 conditions with the exceptions that the temperature is 45 oC and the flow rate is 1.5 mL/min.

Figure 2 shows the separation of all three samples provided by the NREL using conditions more typical of biofuel carbohydrate analysis, particularly for corn stover acid hydrolysates.4 Not that with these conditions there is no resolution of galactose and rhamnose and that cellobiose elutes in < 10 min.

FIGURE 3: Determination of small carbohydrates and uronic acids in acid hydrolysates of wood with Method 2.

The acid hydrolysates of wood can also contain galacturonic and glucuronic acids. These uronic acids require a stronger mobile phase than hydroxide to elute them from an anion-exchange column. Figure 3 shows that by using Method 2, we can first elute the small carbohydrates from the column using a hydroxide eluent and then elute the uronic acids by adding sodium acetate to the eluent. Although, using the sample dilution needed for the small carbohydrates, we can not determine if the samples have uronic acids.

FIGURE 4: Injection of a more concentrated sample of acid hydrolysates of wood to determine uronic acids using Method 2.

To determine if the samples in Figure 3 contain uronic acids, the samples were diluted less and then analyzed with Method 2. Figure 4 shows that while the monosaccharide peaks are overloaded there is evidence of uronic acids in both samples. To better view the uronic acids, Figure 5 shows an enlargement of the region between 35 and 40 min. While galacturonic acid is easily identified and quantified in both samples, there is a peak that compromises glucuronic acid quantification in the hydrolysate liquor sample. Another approach to determining uronic acids in these samples is to design a method that elutes the smaller carbohydrates quickly and only aims to resolve the uronic acids. Figure 6 shows the results of using such a method (Method 3). Note that both uronic acids are resolved from neighboring peaks.

While all the carbohydrates of interest can be measured with Method 2, one analysis for small carbohydrates using Method 1, and another for uronic acids using Method 3 may be more effective for long term success as Methods 1 and 3 are chromatographically simpler than Method 2. The table below show relevant analytical data for the three methods.

Conclusions HPAE-PAD is an effective tool for determining carbohydrate composition of

samples used to produced biofuels.

Monosaccharides can be determined in < 10 min (though without resolving rhamnose and galactose).

Two approaches for determining monosaccharides and uronic acids from wood acid hydrolysates:

Single injection on a Dionex CarboPac PA20 column.

1 injection on a Dionex CarboPac SA10 column for monosaccharides and a 2nd injection on a Dionex CarboPac PA200 column for uronic acids.

References 1. Thermo Scientific Application Note 1089: Determination of Carbohydrates in Acid

Hydrolysates of Wood. Sunnyvale, CA, 2014.

2. Thermo Scientific Application Note 1091: Determination of Uronic Acids and Wood Sugars in Wood-Based Hydrolysates. Sunnyvale, CA, 2014.

3. Thermo Scientific Technical Note 21: Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemcial Detector. Sunnyvale, CA, 2013.

4. Thermo Scientific Application Update 192: Carbohydrate Determination in Biofuel Samples. Sunnyvale, CA, 2014.

Figure 6: Determination of uronic acids in acid hydrolysates of wood using Method 3.

Figure 5: Enlargement of the uronic acid elution time period from Figure 4.

0 4 8 12 16 20 24 10

100

Minutes

nC

Peaks 1. Fucose 2. Sucrose

3. Arabinose 4. Galactose 5. Rhamnose

6. Glucose 7. Xylose

8. Mannose 9. Fructose

10.Cellobiose 11.Maltose

1

2

3

4

6

7

8,9

10 5

11

A. Mix of Sugar Standards (1 g/L) B. Sample 3 (dilution 2)

Chromatographic Conditions Method 1

Small Carbohydrates

Method 2 Small Carbohydrates and

Uronic Acids

Method 3 Uronic Acids

Column

Thermo Scientific™ Dionex™ CarboPac™ SA10-4µm Guard, 4 × 50 mm, and Analytical, 4 × 250 mm

Dionex CarboPac PA20 Guard, 3 × 30 mm, and Analytical, 3 × 150 mm

Dionex CarboPac PA200 Guard, 3 × 30 mm, and Analytical, 3 × 250 mm

Mobile Phases KOH (EGC III KOH) – by Eluent Generation

A. DI Water, B. 100 mM NaOH, C. 100 mM NaOH 1 00 mM Sodium Acetate

A. 1M Sodium Acetate 100 mM NaOH,

B. 100 mM NaOH

Total Run Time 24 min 60 min 30 min

Flow Rate 1.2 mL/min 0.4 mL/min 0.5 mL/min

Column Temperature 30 ºC 30 ºC 30 ºC

Detection

PAD, Au working electrode, 62 mil gasket, Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

Injection Volume 0.4 μL (internal loop) 10 μL 10 μL

Mobile Phase Conditions 1 mM KOH

0-18 min (98.8 A/1.2 B/0 C), 20-30 min (50/50/0), 30.1-46 min (0/0/100), 46.1-50 min

(100/0/0), 50.1-60 min (98.8/1/2/0)

0-18 min 2%A, 18.1 – 22 min 50% A, 22.1-30 min 2%A

0 2 4 6 8 10 12 14 0

140

Minutes

nC

Signal Offset 10%

A. Mix of Sugar Standards (1g/L) B. Sample 1 (dilution 2) C. Sample 2 (dilution 2) D. Sample 3 (dilution 50 )

Peaks 1. Fucose 2. Sucrose 3. Arabinose 4. Galactose 5. Rhamnose 6. Glucose 7. Xylose 8. Mannose 9. Fructose 10.Cellobiose 11.Maltose

1

2

3

4,5

6

7 8

9

10 11

A

B

C

D

0 5 10 15 20 25 30 35 40 8

90

Minutes

nC

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic

acid standards 0.5 mg/L B. Wood hydrolysate liquor (diluted

1000 fold) C. Wood hydrolysate rinsate (diluted

200 fold)

B A

C

1 2

3

4

5 7 8

9

10

12

6

11

System peak

0 5 10 15 20 25 30 35 40 0

300

Minutes

nC

1 2

3 4 5

6

7 8

9 10 11 12

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic acid standards 5 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

A

B

C

Signal Offset 10%

System Peak

35 36 37 38 12

77

Minutes

nC

A

B

C

Samples A. Mix of wood sugars and uronic acid standards 1 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

Peaks mg/L A B C 11. Galacturonic acid 5 0.5 0.5 12. Glucuronic acid 5 0.2 0.2

11 12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0

90

Minutes

nC

Signal Offset 20%

A

B

C

Samples A. Mix of uronic acid

standards 10 mg/L B. Wood hydrolysate

liquor (diluted 10 fold) C. Wood hydrolysate

rinsate (diluted 2 fold) 1

2

Peaks 1. Galacturonic acid 2. Glucuronic acid

Analytical Data Method 11 Method 2 2 Method 32

Linearity

Range – 0.05 – 2.4 g/L For 10 sugars r2 ranged from 0.9980- 0.9998

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9997 Glucuronic acid r2 = 0.9999

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9982 Glucuronic acid r2 = 0.9990

Retention Time Precision

For 7 sugars in standards and samples RSD ranged from 0.07- 0.27%

For 12 sugars (six injections) the range was 0.02-0.09%

For the 2 uronic acids (6 injections) the values were 0.06 and 0.08%

Retention Area Precision

For 7 sugars in standards and samples RSD ranged from 1.18- 3.75%

For 12 sugars (six injections) the range was 0.68-3.53%

For the 2 uronic acids (6 injections) the values were 2.06 and 1.38%

Spike Recovery from

Samples

For 7 sugars in 2 samples the range was

81-103% Not evaluated

For the 2 uronic acids in 2 samples at in triplicate the range was 86-96% and 81-90%

PO71636-EN 0415S

2 Determination of Carbohydrates in Acid Hydrolysates of Wood

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Determination of Carbohydrates in Acid Hydrolysates of Wood Lipika Basumallick and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA

Introduction To prepare wood for the production of alcohol based biofuels by fermentation, the wood must first be hydrolyzed in strong acid with applied heat to yield the constituent monosaccharides needed for fermentation. Quantification of those monosaccharides allows an estimate of the biofuel yield. This poster describes a liquid chromatography (LC) method for quantifying monosaccharides from acid hydrolysates of wood that resolves all the released monosaccharides and cellobiose in less than ten minutes using high-performance anion-exchange chromatography. The separated carbohydrates, fucose, arabinose, rhamnose, glucose, mannose, xylose, fructose, and cellobiose are detected by pulsed amperometry. The method requires only sample dilution prior to LC analysis and the mobile phase for chromatography is prepared automatically by the instrument, which reduces labor and increases method accuracy. Using the same LC technology for carbohydrate analysis but with a different high-performance anion-exchange column, we also developed two methods to determine the glucuronic and galacturonic acid contents of the same acid hydrolysate of wood (loblolly pine). One method determines the two uronic acids, while the other method determines the two uronic acids as well as the monosaccharides. Analytical details (e.g., reproducibility, accuracy, etc.) will be presented for each of the three methods.

Methods Experimental details can be found in references 1 and 2. Three different high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods were used depending on the goal(s) of the analysis. These methods are shown in the table below. A Thermo Scientific™ Dionex™ ICS-5000+ system was used for all HPAE-PAD analysis. Acid-hydrolyzed wood samples were kindly provided by the National Renewable Energy Lab (NREL) in Golden Colorado. The liquor sample contained 1% sulfuric acid. Centrifuge wood acid hydrolysate samples (liquor and rinsate from lodge pole pine) at 16,000 g for 10 min to ensure elimination of particulates. Samples were diluted with deionized water.

Results and Discussion Figure 1 shows the separation of a mixed carbohydrate standard and one of the wood hydrolysate samples using Method 1. These conditions allow the separation of galactose and rhamnose at the expense of the fructose/mannose separation. To achieve this separation the column temperature is reduced to 30 oC from 45 and the flow rate is reduced to 1.2 from 1.5 mL/min. This results in an increased analysis time. Flow rate was decreased so that the backpressure was in the allowed range for the eluent generator, which produces the mobile phase. If the galactose/rhamnose separation is not needed, a faster separation can be achieved (Figure 2). As these samples have high concentrations of carbohydrates, in order to reduce the need to dilute for the sensitive PAD, we used a small injection volume and a thick gasket in the electrochemical cell.

FIGURE 1: Determination of small carbohydrates in an acid hydrolysate of wood with Method 1.

FIGURE 2: HPAE-PAD chromatograms of three wood hydrolysate samples These use Method 1 conditions with the exceptions that the temperature is 45 oC and the flow rate is 1.5 mL/min.

Figure 2 shows the separation of all three samples provided by the NREL using conditions more typical of biofuel carbohydrate analysis, particularly for corn stover acid hydrolysates.4 Not that with these conditions there is no resolution of galactose and rhamnose and that cellobiose elutes in < 10 min.

FIGURE 3: Determination of small carbohydrates and uronic acids in acid hydrolysates of wood with Method 2.

The acid hydrolysates of wood can also contain galacturonic and glucuronic acids. These uronic acids require a stronger mobile phase than hydroxide to elute them from an anion-exchange column. Figure 3 shows that by using Method 2, we can first elute the small carbohydrates from the column using a hydroxide eluent and then elute the uronic acids by adding sodium acetate to the eluent. Although, using the sample dilution needed for the small carbohydrates, we can not determine if the samples have uronic acids.

FIGURE 4: Injection of a more concentrated sample of acid hydrolysates of wood to determine uronic acids using Method 2.

To determine if the samples in Figure 3 contain uronic acids, the samples were diluted less and then analyzed with Method 2. Figure 4 shows that while the monosaccharide peaks are overloaded there is evidence of uronic acids in both samples. To better view the uronic acids, Figure 5 shows an enlargement of the region between 35 and 40 min. While galacturonic acid is easily identified and quantified in both samples, there is a peak that compromises glucuronic acid quantification in the hydrolysate liquor sample. Another approach to determining uronic acids in these samples is to design a method that elutes the smaller carbohydrates quickly and only aims to resolve the uronic acids. Figure 6 shows the results of using such a method (Method 3). Note that both uronic acids are resolved from neighboring peaks.

While all the carbohydrates of interest can be measured with Method 2, one analysis for small carbohydrates using Method 1, and another for uronic acids using Method 3 may be more effective for long term success as Methods 1 and 3 are chromatographically simpler than Method 2. The table below show relevant analytical data for the three methods.

Conclusions HPAE-PAD is an effective tool for determining carbohydrate composition of

samples used to produced biofuels.

Monosaccharides can be determined in < 10 min (though without resolving rhamnose and galactose).

Two approaches for determining monosaccharides and uronic acids from wood acid hydrolysates:

Single injection on a Dionex CarboPac PA20 column.

1 injection on a Dionex CarboPac SA10 column for monosaccharides and a 2nd injection on a Dionex CarboPac PA200 column for uronic acids.

References 1. Thermo Scientific Application Note 1089: Determination of Carbohydrates in Acid

Hydrolysates of Wood. Sunnyvale, CA, 2014.

2. Thermo Scientific Application Note 1091: Determination of Uronic Acids and Wood Sugars in Wood-Based Hydrolysates. Sunnyvale, CA, 2014.

3. Thermo Scientific Technical Note 21: Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemcial Detector. Sunnyvale, CA, 2013.

4. Thermo Scientific Application Update 192: Carbohydrate Determination in Biofuel Samples. Sunnyvale, CA, 2014.

Figure 6: Determination of uronic acids in acid hydrolysates of wood using Method 3.

Figure 5: Enlargement of the uronic acid elution time period from Figure 4.

0 4 8 12 16 20 24 10

100

Minutes

nC

Peaks 1. Fucose 2. Sucrose

3. Arabinose 4. Galactose 5. Rhamnose

6. Glucose 7. Xylose

8. Mannose 9. Fructose

10.Cellobiose 11.Maltose

1

2

3

4

6

7

8,9

10 5

11

A. Mix of Sugar Standards (1 g/L) B. Sample 3 (dilution 2)

Chromatographic Conditions Method 1

Small Carbohydrates

Method 2 Small Carbohydrates and

Uronic Acids

Method 3 Uronic Acids

Column

Thermo Scientific™ Dionex™ CarboPac™ SA10-4µm Guard, 4 × 50 mm, and Analytical, 4 × 250 mm

Dionex CarboPac PA20 Guard, 3 × 30 mm, and Analytical, 3 × 150 mm

Dionex CarboPac PA200 Guard, 3 × 30 mm, and Analytical, 3 × 250 mm

Mobile Phases KOH (EGC III KOH) – by Eluent Generation

A. DI Water, B. 100 mM NaOH, C. 100 mM NaOH 1 00 mM Sodium Acetate

A. 1M Sodium Acetate 100 mM NaOH,

B. 100 mM NaOH

Total Run Time 24 min 60 min 30 min

Flow Rate 1.2 mL/min 0.4 mL/min 0.5 mL/min

Column Temperature 30 ºC 30 ºC 30 ºC

Detection

PAD, Au working electrode, 62 mil gasket, Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

Injection Volume 0.4 μL (internal loop) 10 μL 10 μL

Mobile Phase Conditions 1 mM KOH

0-18 min (98.8 A/1.2 B/0 C), 20-30 min (50/50/0), 30.1-46 min (0/0/100), 46.1-50 min

(100/0/0), 50.1-60 min (98.8/1/2/0)

0-18 min 2%A, 18.1 – 22 min 50% A, 22.1-30 min 2%A

0 2 4 6 8 10 12 14 0

140

Minutes

nC

Signal Offset 10%

A. Mix of Sugar Standards (1g/L) B. Sample 1 (dilution 2) C. Sample 2 (dilution 2) D. Sample 3 (dilution 50 )

Peaks 1. Fucose 2. Sucrose 3. Arabinose 4. Galactose 5. Rhamnose 6. Glucose 7. Xylose 8. Mannose 9. Fructose 10.Cellobiose 11.Maltose

1

2

3

4,5

6

7 8

9

10 11

A

B

C

D

0 5 10 15 20 25 30 35 40 8

90

Minutes

nC

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic

acid standards 0.5 mg/L B. Wood hydrolysate liquor (diluted

1000 fold) C. Wood hydrolysate rinsate (diluted

200 fold)

B A

C

1 2

3

4

5 7 8

9

10

12

6

11

System peak

0 5 10 15 20 25 30 35 40 0

300

Minutes

nC

1 2

3 4 5

6

7 8

9 10 11 12

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic acid standards 5 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

A

B

C

Signal Offset 10%

System Peak

35 36 37 38 12

77

Minutes

nC

A

B

C

Samples A. Mix of wood sugars and uronic acid standards 1 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

Peaks mg/L A B C 11. Galacturonic acid 5 0.5 0.5 12. Glucuronic acid 5 0.2 0.2

11 12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0

90

Minutes

nC

Signal Offset 20%

A

B

C

Samples A. Mix of uronic acid

standards 10 mg/L B. Wood hydrolysate

liquor (diluted 10 fold) C. Wood hydrolysate

rinsate (diluted 2 fold) 1

2

Peaks 1. Galacturonic acid 2. Glucuronic acid

Analytical Data Method 11 Method 2 2 Method 32

Linearity

Range – 0.05 – 2.4 g/L For 10 sugars r2 ranged from 0.9980- 0.9998

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9997 Glucuronic acid r2 = 0.9999

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9982 Glucuronic acid r2 = 0.9990

Retention Time Precision

For 7 sugars in standards and samples RSD ranged from 0.07- 0.27%

For 12 sugars (six injections) the range was 0.02-0.09%

For the 2 uronic acids (6 injections) the values were 0.06 and 0.08%

Retention Area Precision

For 7 sugars in standards and samples RSD ranged from 1.18- 3.75%

For 12 sugars (six injections) the range was 0.68-3.53%

For the 2 uronic acids (6 injections) the values were 2.06 and 1.38%

Spike Recovery from

Samples

For 7 sugars in 2 samples the range was

81-103% Not evaluated

For the 2 uronic acids in 2 samples at in triplicate the range was 86-96% and 81-90%

PO71636-EN 0415S

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Determination of Carbohydrates in Acid Hydrolysates of Wood Lipika Basumallick and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA

Introduction To prepare wood for the production of alcohol based biofuels by fermentation, the wood must first be hydrolyzed in strong acid with applied heat to yield the constituent monosaccharides needed for fermentation. Quantification of those monosaccharides allows an estimate of the biofuel yield. This poster describes a liquid chromatography (LC) method for quantifying monosaccharides from acid hydrolysates of wood that resolves all the released monosaccharides and cellobiose in less than ten minutes using high-performance anion-exchange chromatography. The separated carbohydrates, fucose, arabinose, rhamnose, glucose, mannose, xylose, fructose, and cellobiose are detected by pulsed amperometry. The method requires only sample dilution prior to LC analysis and the mobile phase for chromatography is prepared automatically by the instrument, which reduces labor and increases method accuracy. Using the same LC technology for carbohydrate analysis but with a different high-performance anion-exchange column, we also developed two methods to determine the glucuronic and galacturonic acid contents of the same acid hydrolysate of wood (loblolly pine). One method determines the two uronic acids, while the other method determines the two uronic acids as well as the monosaccharides. Analytical details (e.g., reproducibility, accuracy, etc.) will be presented for each of the three methods.

Methods Experimental details can be found in references 1 and 2. Three different high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods were used depending on the goal(s) of the analysis. These methods are shown in the table below. A Thermo Scientific™ Dionex™ ICS-5000+ system was used for all HPAE-PAD analysis. Acid-hydrolyzed wood samples were kindly provided by the National Renewable Energy Lab (NREL) in Golden Colorado. The liquor sample contained 1% sulfuric acid. Centrifuge wood acid hydrolysate samples (liquor and rinsate from lodge pole pine) at 16,000 g for 10 min to ensure elimination of particulates. Samples were diluted with deionized water.

Results and Discussion Figure 1 shows the separation of a mixed carbohydrate standard and one of the wood hydrolysate samples using Method 1. These conditions allow the separation of galactose and rhamnose at the expense of the fructose/mannose separation. To achieve this separation the column temperature is reduced to 30 oC from 45 and the flow rate is reduced to 1.2 from 1.5 mL/min. This results in an increased analysis time. Flow rate was decreased so that the backpressure was in the allowed range for the eluent generator, which produces the mobile phase. If the galactose/rhamnose separation is not needed, a faster separation can be achieved (Figure 2). As these samples have high concentrations of carbohydrates, in order to reduce the need to dilute for the sensitive PAD, we used a small injection volume and a thick gasket in the electrochemical cell.

FIGURE 1: Determination of small carbohydrates in an acid hydrolysate of wood with Method 1.

FIGURE 2: HPAE-PAD chromatograms of three wood hydrolysate samples These use Method 1 conditions with the exceptions that the temperature is 45 oC and the flow rate is 1.5 mL/min.

Figure 2 shows the separation of all three samples provided by the NREL using conditions more typical of biofuel carbohydrate analysis, particularly for corn stover acid hydrolysates.4 Not that with these conditions there is no resolution of galactose and rhamnose and that cellobiose elutes in < 10 min.

FIGURE 3: Determination of small carbohydrates and uronic acids in acid hydrolysates of wood with Method 2.

The acid hydrolysates of wood can also contain galacturonic and glucuronic acids. These uronic acids require a stronger mobile phase than hydroxide to elute them from an anion-exchange column. Figure 3 shows that by using Method 2, we can first elute the small carbohydrates from the column using a hydroxide eluent and then elute the uronic acids by adding sodium acetate to the eluent. Although, using the sample dilution needed for the small carbohydrates, we can not determine if the samples have uronic acids.

FIGURE 4: Injection of a more concentrated sample of acid hydrolysates of wood to determine uronic acids using Method 2.

To determine if the samples in Figure 3 contain uronic acids, the samples were diluted less and then analyzed with Method 2. Figure 4 shows that while the monosaccharide peaks are overloaded there is evidence of uronic acids in both samples. To better view the uronic acids, Figure 5 shows an enlargement of the region between 35 and 40 min. While galacturonic acid is easily identified and quantified in both samples, there is a peak that compromises glucuronic acid quantification in the hydrolysate liquor sample. Another approach to determining uronic acids in these samples is to design a method that elutes the smaller carbohydrates quickly and only aims to resolve the uronic acids. Figure 6 shows the results of using such a method (Method 3). Note that both uronic acids are resolved from neighboring peaks.

While all the carbohydrates of interest can be measured with Method 2, one analysis for small carbohydrates using Method 1, and another for uronic acids using Method 3 may be more effective for long term success as Methods 1 and 3 are chromatographically simpler than Method 2. The table below show relevant analytical data for the three methods.

Conclusions HPAE-PAD is an effective tool for determining carbohydrate composition of

samples used to produced biofuels.

Monosaccharides can be determined in < 10 min (though without resolving rhamnose and galactose).

Two approaches for determining monosaccharides and uronic acids from wood acid hydrolysates:

Single injection on a Dionex CarboPac PA20 column.

1 injection on a Dionex CarboPac SA10 column for monosaccharides and a 2nd injection on a Dionex CarboPac PA200 column for uronic acids.

References 1. Thermo Scientific Application Note 1089: Determination of Carbohydrates in Acid

Hydrolysates of Wood. Sunnyvale, CA, 2014.

2. Thermo Scientific Application Note 1091: Determination of Uronic Acids and Wood Sugars in Wood-Based Hydrolysates. Sunnyvale, CA, 2014.

3. Thermo Scientific Technical Note 21: Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemcial Detector. Sunnyvale, CA, 2013.

4. Thermo Scientific Application Update 192: Carbohydrate Determination in Biofuel Samples. Sunnyvale, CA, 2014.

Figure 6: Determination of uronic acids in acid hydrolysates of wood using Method 3.

Figure 5: Enlargement of the uronic acid elution time period from Figure 4.

0 4 8 12 16 20 24 10

100

Minutes

nC

Peaks 1. Fucose 2. Sucrose

3. Arabinose 4. Galactose 5. Rhamnose

6. Glucose 7. Xylose

8. Mannose 9. Fructose

10.Cellobiose 11.Maltose

1

2

3

4

6

7

8,9

10 5

11

A. Mix of Sugar Standards (1 g/L) B. Sample 3 (dilution 2)

Chromatographic Conditions Method 1

Small Carbohydrates

Method 2 Small Carbohydrates and

Uronic Acids

Method 3 Uronic Acids

Column

Thermo Scientific™ Dionex™ CarboPac™ SA10-4µm Guard, 4 × 50 mm, and Analytical, 4 × 250 mm

Dionex CarboPac PA20 Guard, 3 × 30 mm, and Analytical, 3 × 150 mm

Dionex CarboPac PA200 Guard, 3 × 30 mm, and Analytical, 3 × 250 mm

Mobile Phases KOH (EGC III KOH) – by Eluent Generation

A. DI Water, B. 100 mM NaOH, C. 100 mM NaOH 1 00 mM Sodium Acetate

A. 1M Sodium Acetate 100 mM NaOH,

B. 100 mM NaOH

Total Run Time 24 min 60 min 30 min

Flow Rate 1.2 mL/min 0.4 mL/min 0.5 mL/min

Column Temperature 30 ºC 30 ºC 30 ºC

Detection

PAD, Au working electrode, 62 mil gasket, Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

Injection Volume 0.4 μL (internal loop) 10 μL 10 μL

Mobile Phase Conditions 1 mM KOH

0-18 min (98.8 A/1.2 B/0 C), 20-30 min (50/50/0), 30.1-46 min (0/0/100), 46.1-50 min

(100/0/0), 50.1-60 min (98.8/1/2/0)

0-18 min 2%A, 18.1 – 22 min 50% A, 22.1-30 min 2%A

0 2 4 6 8 10 12 14 0

140

Minutes

nC

Signal Offset 10%

A. Mix of Sugar Standards (1g/L) B. Sample 1 (dilution 2) C. Sample 2 (dilution 2) D. Sample 3 (dilution 50 )

Peaks 1. Fucose 2. Sucrose 3. Arabinose 4. Galactose 5. Rhamnose 6. Glucose 7. Xylose 8. Mannose 9. Fructose 10.Cellobiose 11.Maltose

1

2

3

4,5

6

7 8

9

10 11

A

B

C

D

0 5 10 15 20 25 30 35 40 8

90

Minutes

nC

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic

acid standards 0.5 mg/L B. Wood hydrolysate liquor (diluted

1000 fold) C. Wood hydrolysate rinsate (diluted

200 fold)

B A

C

1 2

3

4

5 7 8

9

10

12

6

11

System peak

0 5 10 15 20 25 30 35 40 0

300

Minutes

nC

1 2

3 4 5

6

7 8

9 10 11 12

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic acid standards 5 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

A

B

C

Signal Offset 10%

System Peak

35 36 37 38 12

77

Minutes

nC

A

B

C

Samples A. Mix of wood sugars and uronic acid standards 1 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

Peaks mg/L A B C 11. Galacturonic acid 5 0.5 0.5 12. Glucuronic acid 5 0.2 0.2

11 12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0

90

Minutes

nC

Signal Offset 20%

A

B

C

Samples A. Mix of uronic acid

standards 10 mg/L B. Wood hydrolysate

liquor (diluted 10 fold) C. Wood hydrolysate

rinsate (diluted 2 fold) 1

2

Peaks 1. Galacturonic acid 2. Glucuronic acid

Analytical Data Method 11 Method 2 2 Method 32

Linearity

Range – 0.05 – 2.4 g/L For 10 sugars r2 ranged from 0.9980- 0.9998

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9997 Glucuronic acid r2 = 0.9999

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9982 Glucuronic acid r2 = 0.9990

Retention Time Precision

For 7 sugars in standards and samples RSD ranged from 0.07- 0.27%

For 12 sugars (six injections) the range was 0.02-0.09%

For the 2 uronic acids (6 injections) the values were 0.06 and 0.08%

Retention Area Precision

For 7 sugars in standards and samples RSD ranged from 1.18- 3.75%

For 12 sugars (six injections) the range was 0.68-3.53%

For the 2 uronic acids (6 injections) the values were 2.06 and 1.38%

Spike Recovery from

Samples

For 7 sugars in 2 samples the range was

81-103% Not evaluated

For the 2 uronic acids in 2 samples at in triplicate the range was 86-96% and 81-90%

PO71636-EN 0415S

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Determination of Carbohydrates in Acid Hydrolysates of Wood Lipika Basumallick and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA

Introduction To prepare wood for the production of alcohol based biofuels by fermentation, the wood must first be hydrolyzed in strong acid with applied heat to yield the constituent monosaccharides needed for fermentation. Quantification of those monosaccharides allows an estimate of the biofuel yield. This poster describes a liquid chromatography (LC) method for quantifying monosaccharides from acid hydrolysates of wood that resolves all the released monosaccharides and cellobiose in less than ten minutes using high-performance anion-exchange chromatography. The separated carbohydrates, fucose, arabinose, rhamnose, glucose, mannose, xylose, fructose, and cellobiose are detected by pulsed amperometry. The method requires only sample dilution prior to LC analysis and the mobile phase for chromatography is prepared automatically by the instrument, which reduces labor and increases method accuracy. Using the same LC technology for carbohydrate analysis but with a different high-performance anion-exchange column, we also developed two methods to determine the glucuronic and galacturonic acid contents of the same acid hydrolysate of wood (loblolly pine). One method determines the two uronic acids, while the other method determines the two uronic acids as well as the monosaccharides. Analytical details (e.g., reproducibility, accuracy, etc.) will be presented for each of the three methods.

Methods Experimental details can be found in references 1 and 2. Three different high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods were used depending on the goal(s) of the analysis. These methods are shown in the table below. A Thermo Scientific™ Dionex™ ICS-5000+ system was used for all HPAE-PAD analysis. Acid-hydrolyzed wood samples were kindly provided by the National Renewable Energy Lab (NREL) in Golden Colorado. The liquor sample contained 1% sulfuric acid. Centrifuge wood acid hydrolysate samples (liquor and rinsate from lodge pole pine) at 16,000 g for 10 min to ensure elimination of particulates. Samples were diluted with deionized water.

Results and Discussion Figure 1 shows the separation of a mixed carbohydrate standard and one of the wood hydrolysate samples using Method 1. These conditions allow the separation of galactose and rhamnose at the expense of the fructose/mannose separation. To achieve this separation the column temperature is reduced to 30 oC from 45 and the flow rate is reduced to 1.2 from 1.5 mL/min. This results in an increased analysis time. Flow rate was decreased so that the backpressure was in the allowed range for the eluent generator, which produces the mobile phase. If the galactose/rhamnose separation is not needed, a faster separation can be achieved (Figure 2). As these samples have high concentrations of carbohydrates, in order to reduce the need to dilute for the sensitive PAD, we used a small injection volume and a thick gasket in the electrochemical cell.

FIGURE 1: Determination of small carbohydrates in an acid hydrolysate of wood with Method 1.

FIGURE 2: HPAE-PAD chromatograms of three wood hydrolysate samples These use Method 1 conditions with the exceptions that the temperature is 45 oC and the flow rate is 1.5 mL/min.

Figure 2 shows the separation of all three samples provided by the NREL using conditions more typical of biofuel carbohydrate analysis, particularly for corn stover acid hydrolysates.4 Not that with these conditions there is no resolution of galactose and rhamnose and that cellobiose elutes in < 10 min.

FIGURE 3: Determination of small carbohydrates and uronic acids in acid hydrolysates of wood with Method 2.

The acid hydrolysates of wood can also contain galacturonic and glucuronic acids. These uronic acids require a stronger mobile phase than hydroxide to elute them from an anion-exchange column. Figure 3 shows that by using Method 2, we can first elute the small carbohydrates from the column using a hydroxide eluent and then elute the uronic acids by adding sodium acetate to the eluent. Although, using the sample dilution needed for the small carbohydrates, we can not determine if the samples have uronic acids.

FIGURE 4: Injection of a more concentrated sample of acid hydrolysates of wood to determine uronic acids using Method 2.

To determine if the samples in Figure 3 contain uronic acids, the samples were diluted less and then analyzed with Method 2. Figure 4 shows that while the monosaccharide peaks are overloaded there is evidence of uronic acids in both samples. To better view the uronic acids, Figure 5 shows an enlargement of the region between 35 and 40 min. While galacturonic acid is easily identified and quantified in both samples, there is a peak that compromises glucuronic acid quantification in the hydrolysate liquor sample. Another approach to determining uronic acids in these samples is to design a method that elutes the smaller carbohydrates quickly and only aims to resolve the uronic acids. Figure 6 shows the results of using such a method (Method 3). Note that both uronic acids are resolved from neighboring peaks.

While all the carbohydrates of interest can be measured with Method 2, one analysis for small carbohydrates using Method 1, and another for uronic acids using Method 3 may be more effective for long term success as Methods 1 and 3 are chromatographically simpler than Method 2. The table below show relevant analytical data for the three methods.

Conclusions HPAE-PAD is an effective tool for determining carbohydrate composition of

samples used to produced biofuels.

Monosaccharides can be determined in < 10 min (though without resolving rhamnose and galactose).

Two approaches for determining monosaccharides and uronic acids from wood acid hydrolysates:

Single injection on a Dionex CarboPac PA20 column.

1 injection on a Dionex CarboPac SA10 column for monosaccharides and a 2nd injection on a Dionex CarboPac PA200 column for uronic acids.

References 1. Thermo Scientific Application Note 1089: Determination of Carbohydrates in Acid

Hydrolysates of Wood. Sunnyvale, CA, 2014.

2. Thermo Scientific Application Note 1091: Determination of Uronic Acids and Wood Sugars in Wood-Based Hydrolysates. Sunnyvale, CA, 2014.

3. Thermo Scientific Technical Note 21: Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemcial Detector. Sunnyvale, CA, 2013.

4. Thermo Scientific Application Update 192: Carbohydrate Determination in Biofuel Samples. Sunnyvale, CA, 2014.

Figure 6: Determination of uronic acids in acid hydrolysates of wood using Method 3.

Figure 5: Enlargement of the uronic acid elution time period from Figure 4.

0 4 8 12 16 20 24 10

100

Minutes

nC

Peaks 1. Fucose 2. Sucrose

3. Arabinose 4. Galactose 5. Rhamnose

6. Glucose 7. Xylose

8. Mannose 9. Fructose

10.Cellobiose 11.Maltose

1

2

3

4

6

7

8,9

10 5

11

A. Mix of Sugar Standards (1 g/L) B. Sample 3 (dilution 2)

Chromatographic Conditions Method 1

Small Carbohydrates

Method 2 Small Carbohydrates and

Uronic Acids

Method 3 Uronic Acids

Column

Thermo Scientific™ Dionex™ CarboPac™ SA10-4µm Guard, 4 × 50 mm, and Analytical, 4 × 250 mm

Dionex CarboPac PA20 Guard, 3 × 30 mm, and Analytical, 3 × 150 mm

Dionex CarboPac PA200 Guard, 3 × 30 mm, and Analytical, 3 × 250 mm

Mobile Phases KOH (EGC III KOH) – by Eluent Generation

A. DI Water, B. 100 mM NaOH, C. 100 mM NaOH 1 00 mM Sodium Acetate

A. 1M Sodium Acetate 100 mM NaOH,

B. 100 mM NaOH

Total Run Time 24 min 60 min 30 min

Flow Rate 1.2 mL/min 0.4 mL/min 0.5 mL/min

Column Temperature 30 ºC 30 ºC 30 ºC

Detection

PAD, Au working electrode, 62 mil gasket, Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

Injection Volume 0.4 μL (internal loop) 10 μL 10 μL

Mobile Phase Conditions 1 mM KOH

0-18 min (98.8 A/1.2 B/0 C), 20-30 min (50/50/0), 30.1-46 min (0/0/100), 46.1-50 min

(100/0/0), 50.1-60 min (98.8/1/2/0)

0-18 min 2%A, 18.1 – 22 min 50% A, 22.1-30 min 2%A

0 2 4 6 8 10 12 14 0

140

Minutes

nC

Signal Offset 10%

A. Mix of Sugar Standards (1g/L) B. Sample 1 (dilution 2) C. Sample 2 (dilution 2) D. Sample 3 (dilution 50 )

Peaks 1. Fucose 2. Sucrose 3. Arabinose 4. Galactose 5. Rhamnose 6. Glucose 7. Xylose 8. Mannose 9. Fructose 10.Cellobiose 11.Maltose

1

2

3

4,5

6

7 8

9

10 11

A

B

C

D

0 5 10 15 20 25 30 35 40 8

90

Minutes

nC

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic

acid standards 0.5 mg/L B. Wood hydrolysate liquor (diluted

1000 fold) C. Wood hydrolysate rinsate (diluted

200 fold)

B A

C

1 2

3

4

5 7 8

9

10

12

6

11

System peak

0 5 10 15 20 25 30 35 40 0

300

Minutes

nC

1 2

3 4 5

6

7 8

9 10 11 12

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic acid standards 5 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

A

B

C

Signal Offset 10%

System Peak

35 36 37 38 12

77

Minutes

nC

A

B

C

Samples A. Mix of wood sugars and uronic acid standards 1 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

Peaks mg/L A B C 11. Galacturonic acid 5 0.5 0.5 12. Glucuronic acid 5 0.2 0.2

11 12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0

90

Minutes

nC

Signal Offset 20%

A

B

C

Samples A. Mix of uronic acid

standards 10 mg/L B. Wood hydrolysate

liquor (diluted 10 fold) C. Wood hydrolysate

rinsate (diluted 2 fold) 1

2

Peaks 1. Galacturonic acid 2. Glucuronic acid

Analytical Data Method 11 Method 2 2 Method 32

Linearity

Range – 0.05 – 2.4 g/L For 10 sugars r2 ranged from 0.9980- 0.9998

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9997 Glucuronic acid r2 = 0.9999

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9982 Glucuronic acid r2 = 0.9990

Retention Time Precision

For 7 sugars in standards and samples RSD ranged from 0.07- 0.27%

For 12 sugars (six injections) the range was 0.02-0.09%

For the 2 uronic acids (6 injections) the values were 0.06 and 0.08%

Retention Area Precision

For 7 sugars in standards and samples RSD ranged from 1.18- 3.75%

For 12 sugars (six injections) the range was 0.68-3.53%

For the 2 uronic acids (6 injections) the values were 2.06 and 1.38%

Spike Recovery from

Samples

For 7 sugars in 2 samples the range was

81-103% Not evaluated

For the 2 uronic acids in 2 samples at in triplicate the range was 86-96% and 81-90%

PO71636-EN 0415S

PN71636-EN 0415S

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Determination of Carbohydrates in Acid Hydrolysates of Wood Lipika Basumallick and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA

Introduction To prepare wood for the production of alcohol based biofuels by fermentation, the wood must first be hydrolyzed in strong acid with applied heat to yield the constituent monosaccharides needed for fermentation. Quantification of those monosaccharides allows an estimate of the biofuel yield. This poster describes a liquid chromatography (LC) method for quantifying monosaccharides from acid hydrolysates of wood that resolves all the released monosaccharides and cellobiose in less than ten minutes using high-performance anion-exchange chromatography. The separated carbohydrates, fucose, arabinose, rhamnose, glucose, mannose, xylose, fructose, and cellobiose are detected by pulsed amperometry. The method requires only sample dilution prior to LC analysis and the mobile phase for chromatography is prepared automatically by the instrument, which reduces labor and increases method accuracy. Using the same LC technology for carbohydrate analysis but with a different high-performance anion-exchange column, we also developed two methods to determine the glucuronic and galacturonic acid contents of the same acid hydrolysate of wood (loblolly pine). One method determines the two uronic acids, while the other method determines the two uronic acids as well as the monosaccharides. Analytical details (e.g., reproducibility, accuracy, etc.) will be presented for each of the three methods.

Methods Experimental details can be found in references 1 and 2. Three different high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) methods were used depending on the goal(s) of the analysis. These methods are shown in the table below. A Thermo Scientific™ Dionex™ ICS-5000+ system was used for all HPAE-PAD analysis. Acid-hydrolyzed wood samples were kindly provided by the National Renewable Energy Lab (NREL) in Golden Colorado. The liquor sample contained 1% sulfuric acid. Centrifuge wood acid hydrolysate samples (liquor and rinsate from lodge pole pine) at 16,000 g for 10 min to ensure elimination of particulates. Samples were diluted with deionized water.

Results and Discussion Figure 1 shows the separation of a mixed carbohydrate standard and one of the wood hydrolysate samples using Method 1. These conditions allow the separation of galactose and rhamnose at the expense of the fructose/mannose separation. To achieve this separation the column temperature is reduced to 30 oC from 45 and the flow rate is reduced to 1.2 from 1.5 mL/min. This results in an increased analysis time. Flow rate was decreased so that the backpressure was in the allowed range for the eluent generator, which produces the mobile phase. If the galactose/rhamnose separation is not needed, a faster separation can be achieved (Figure 2). As these samples have high concentrations of carbohydrates, in order to reduce the need to dilute for the sensitive PAD, we used a small injection volume and a thick gasket in the electrochemical cell.

FIGURE 1: Determination of small carbohydrates in an acid hydrolysate of wood with Method 1.

FIGURE 2: HPAE-PAD chromatograms of three wood hydrolysate samples These use Method 1 conditions with the exceptions that the temperature is 45 oC and the flow rate is 1.5 mL/min.

Figure 2 shows the separation of all three samples provided by the NREL using conditions more typical of biofuel carbohydrate analysis, particularly for corn stover acid hydrolysates.4 Not that with these conditions there is no resolution of galactose and rhamnose and that cellobiose elutes in < 10 min.

FIGURE 3: Determination of small carbohydrates and uronic acids in acid hydrolysates of wood with Method 2.

The acid hydrolysates of wood can also contain galacturonic and glucuronic acids. These uronic acids require a stronger mobile phase than hydroxide to elute them from an anion-exchange column. Figure 3 shows that by using Method 2, we can first elute the small carbohydrates from the column using a hydroxide eluent and then elute the uronic acids by adding sodium acetate to the eluent. Although, using the sample dilution needed for the small carbohydrates, we can not determine if the samples have uronic acids.

FIGURE 4: Injection of a more concentrated sample of acid hydrolysates of wood to determine uronic acids using Method 2.

To determine if the samples in Figure 3 contain uronic acids, the samples were diluted less and then analyzed with Method 2. Figure 4 shows that while the monosaccharide peaks are overloaded there is evidence of uronic acids in both samples. To better view the uronic acids, Figure 5 shows an enlargement of the region between 35 and 40 min. While galacturonic acid is easily identified and quantified in both samples, there is a peak that compromises glucuronic acid quantification in the hydrolysate liquor sample. Another approach to determining uronic acids in these samples is to design a method that elutes the smaller carbohydrates quickly and only aims to resolve the uronic acids. Figure 6 shows the results of using such a method (Method 3). Note that both uronic acids are resolved from neighboring peaks.

While all the carbohydrates of interest can be measured with Method 2, one analysis for small carbohydrates using Method 1, and another for uronic acids using Method 3 may be more effective for long term success as Methods 1 and 3 are chromatographically simpler than Method 2. The table below show relevant analytical data for the three methods.

Conclusions HPAE-PAD is an effective tool for determining carbohydrate composition of

samples used to produced biofuels.

Monosaccharides can be determined in < 10 min (though without resolving rhamnose and galactose).

Two approaches for determining monosaccharides and uronic acids from wood acid hydrolysates:

Single injection on a Dionex CarboPac PA20 column.

1 injection on a Dionex CarboPac SA10 column for monosaccharides and a 2nd injection on a Dionex CarboPac PA200 column for uronic acids.

References 1. Thermo Scientific Application Note 1089: Determination of Carbohydrates in Acid

Hydrolysates of Wood. Sunnyvale, CA, 2014.

2. Thermo Scientific Application Note 1091: Determination of Uronic Acids and Wood Sugars in Wood-Based Hydrolysates. Sunnyvale, CA, 2014.

3. Thermo Scientific Technical Note 21: Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemcial Detector. Sunnyvale, CA, 2013.

4. Thermo Scientific Application Update 192: Carbohydrate Determination in Biofuel Samples. Sunnyvale, CA, 2014.

Figure 6: Determination of uronic acids in acid hydrolysates of wood using Method 3.

Figure 5: Enlargement of the uronic acid elution time period from Figure 4.

0 4 8 12 16 20 24 10

100

Minutes

nC

Peaks 1. Fucose 2. Sucrose

3. Arabinose 4. Galactose 5. Rhamnose

6. Glucose 7. Xylose

8. Mannose 9. Fructose

10.Cellobiose 11.Maltose

1

2

3

4

6

7

8,9

10 5

11

A. Mix of Sugar Standards (1 g/L) B. Sample 3 (dilution 2)

Chromatographic Conditions Method 1

Small Carbohydrates

Method 2 Small Carbohydrates and

Uronic Acids

Method 3 Uronic Acids

Column

Thermo Scientific™ Dionex™ CarboPac™ SA10-4µm Guard, 4 × 50 mm, and Analytical, 4 × 250 mm

Dionex CarboPac PA20 Guard, 3 × 30 mm, and Analytical, 3 × 150 mm

Dionex CarboPac PA200 Guard, 3 × 30 mm, and Analytical, 3 × 250 mm

Mobile Phases KOH (EGC III KOH) – by Eluent Generation

A. DI Water, B. 100 mM NaOH, C. 100 mM NaOH 1 00 mM Sodium Acetate

A. 1M Sodium Acetate 100 mM NaOH,

B. 100 mM NaOH

Total Run Time 24 min 60 min 30 min

Flow Rate 1.2 mL/min 0.4 mL/min 0.5 mL/min

Column Temperature 30 ºC 30 ºC 30 ºC

Detection

PAD, Au working electrode, 62 mil gasket, Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

PAD, Au working electrode, 2 mil gasket Ag/Ag/Cl ref. electrode, 4-potential waveform3

Injection Volume 0.4 μL (internal loop) 10 μL 10 μL

Mobile Phase Conditions 1 mM KOH

0-18 min (98.8 A/1.2 B/0 C), 20-30 min (50/50/0), 30.1-46 min (0/0/100), 46.1-50 min

(100/0/0), 50.1-60 min (98.8/1/2/0)

0-18 min 2%A, 18.1 – 22 min 50% A, 22.1-30 min 2%A

0 2 4 6 8 10 12 14 0

140

Minutes

nC

Signal Offset 10%

A. Mix of Sugar Standards (1g/L) B. Sample 1 (dilution 2) C. Sample 2 (dilution 2) D. Sample 3 (dilution 50 )

Peaks 1. Fucose 2. Sucrose 3. Arabinose 4. Galactose 5. Rhamnose 6. Glucose 7. Xylose 8. Mannose 9. Fructose 10.Cellobiose 11.Maltose

1

2

3

4,5

6

7 8

9

10 11

A

B

C

D

0 5 10 15 20 25 30 35 40 8

90

Minutes

nC

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic

acid standards 0.5 mg/L B. Wood hydrolysate liquor (diluted

1000 fold) C. Wood hydrolysate rinsate (diluted

200 fold)

B A

C

1 2

3

4

5 7 8

9

10

12

6

11

System peak

0 5 10 15 20 25 30 35 40 0

300

Minutes

nC

1 2

3 4 5

6

7 8

9 10 11 12

Peaks 1. Fucose 2. Rhamnose 3. Arabinose 4. Galactose 5. Glucose 6. Sucrose 7. Xylose 8. Mannose 9. Fructose 10. Cellobiose 11. Galacturonic acid 12. Glucuronic acid

Samples A. Mix of wood sugars and uronic acid standards 5 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

A

B

C

Signal Offset 10%

System Peak

35 36 37 38 12

77

Minutes

nC

A

B

C

Samples A. Mix of wood sugars and uronic acid standards 1 mg/L B. Wood hydrolysate liquor (diluted 100 fold) C. Wood hydrolysate rinsate (diluted 20 fold)

Peaks mg/L A B C 11. Galacturonic acid 5 0.5 0.5 12. Glucuronic acid 5 0.2 0.2

11 12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0

90

Minutes

nC

Signal Offset 20%

A

B

C

Samples A. Mix of uronic acid

standards 10 mg/L B. Wood hydrolysate

liquor (diluted 10 fold) C. Wood hydrolysate

rinsate (diluted 2 fold) 1

2

Peaks 1. Galacturonic acid 2. Glucuronic acid

Analytical Data Method 11 Method 2 2 Method 32

Linearity

Range – 0.05 – 2.4 g/L For 10 sugars r2 ranged from 0.9980- 0.9998

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9997 Glucuronic acid r2 = 0.9999

Range – 0.01 – 10 mg/L Galacturonic acid r2 = 0.9982 Glucuronic acid r2 = 0.9990

Retention Time Precision

For 7 sugars in standards and samples RSD ranged from 0.07- 0.27%

For 12 sugars (six injections) the range was 0.02-0.09%

For the 2 uronic acids (6 injections) the values were 0.06 and 0.08%

Retention Area Precision

For 7 sugars in standards and samples RSD ranged from 1.18- 3.75%

For 12 sugars (six injections) the range was 0.68-3.53%

For the 2 uronic acids (6 injections) the values were 2.06 and 1.38%

Spike Recovery from

Samples

For 7 sugars in 2 samples the range was

81-103% Not evaluated

For the 2 uronic acids in 2 samples at in triplicate the range was 86-96% and 81-90%

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