Proton NMR Methods in the Compositional Characterization of

Chapter 9

Proton NMR Methods in the Compositional Characterization of Polysaccharides

Downloaded by UNIV OF ARIZONA on January 11, 2013 | http://pubs.acs.org Publication Date: October 7, 2003 | doi: 10.1021/bk-2004-0864.ch009

David J. Kiemle, Arthur J. Stipanovic, and Kelly E. Mayo Faculty of Chemistry, College of Environmental Science and Forestry (SUNY-ESF), E. C. Jahn Chemistry Laboratory, State University of New York, One Forestry Drive, Syracuse, NY 13210

1

We have developed a new analytical method based on H-Nuclear Magnetic Resonance (proton NMR) spectroscopy to quantify the monosaccharide sugars released from lignocellulosic biomass (wood, pulp, agricultural residues, etc.) upon acid hydrolysis. Compared to other carbohydrate analysis procedures, the NMR protocol is relatively fast since actual acidic hydrolyzates are used, it requires no sample derivatization and provides excellent resolution of complex sugar mixtures. Using the αand βanomeric C1 protons of specific sugars as "probes" of their concentration, we have developed a computational algorithm which enables us to quantify the relative molar concentration of the following sugars in complex hydrolyzates: glucose, mannose, galactose, xylose, rhamnose, arabinose, glucuronic acid.

Introduction st

The 21 century is envisioned to become the "age of biology" as renewable biomass resources begin to replace petroleum in the production of energy and industrial products including fuels, chemicals and new biodegradable materials (7). Despite its relative abundance and renewability, the hemicellulosic fraction of woody plants has not been fully developed into a commercially attractive feedstock for the production of biobased products. Although differences exist between hardwoods, softwoods and other plants, the hemicellulose component of

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© 2004 American Chemical Society In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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woody materials typically comprises 20-40 dry wt% (2-4) and contains 2-3 different polysaccharides, each with a unique chemical composition capable of providing a different distribution of constituent sugars when hydrolyzed by acids or enzymes. As a result, the ultimate application of hemicelluloses as a feedstock for the production of fuels, chemicals and materials requires a careful analysis of the carbohydrates contained within this resource. Further, the accurate analysis of the hemicellulose fraction of wood pulp has important implications in papermaking, especially as fungal or enzyme biodelignification and bleaching evolve into commercial processes. The carbohydrate composition of woody feedstocks is typically analyzed by first treating the solid materials with high concentrations of sulfuric acid (72% H S 0 ) to hydrolyze the native polysaccharides (cellulose and species specific hemicelluloses) into their constituent sugars (D-glucose, D-xylose, D-galactose, D-mannose, D-glucuronic acid, L-arabinose, L-rhamnose, etc.). In most cases, these sugars are then quantified using High Pressure Liquid Chromatography (HPLC; 5-7) or Gas Chromatography / Mass Spectrometry (GC/MS) techniques (5). This report represents a preliminary study aimed at determining the utility of proton (*H) N M R spectroscopy in the quantification of sugars resulting from the acid hydrolysis of complex polysaccharides without the need to neutralize the hydrolysis reaction products, separate the sugars chromatographically, or to prepare volatile derivatives for G C / M S analysis. 2

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!

Sugar Analysis by Proton ( H) NMR Proton N M R methods have been widely employed to probe the composition, stereochemistry and mutarotation kinetics of sugars in aqueous solution for many years (8,9). Although various one and two dimensional (ID, 2D) N M R techniques have been applied in the elucidation of monomeric sugar composition and linkage stereochemistry for oligosaccharides (5,10), less attention appears to have been focussed on using simple ID proton N M R to quantify individual sugars in complex mixtures resulting from the acid or enzymatic hydrolysis of biomass. In part, this may be due to the fact that residual water (H 0) contributes a strong resonance in the anomeric proton region of the N M R spectrum for such hydrolyzates (7/). In addition, certain N M R resonances that result from common sugars may overlap, as shown in Figure 1, and a resolution algorithm is needed. As a result, the N M R method under development in our laboratory, which exploits acidic sugar solutions and an empirical quantification protocol (based on standard sugar model compounds), appears to be a unique approach in carbohydrate analysis. 2

As discussed above, the N M R method offers many advantages compared to techniques such as H P L C and a related technique, High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAECPAD) which are widely applied in the analysis of lignocellulosic hydrolyzates (5). Since most carbohydrate chromatography columns separate molecules based

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. J

Figure 1. 600 MHz HNMR

2

spectrum of hydrolyzed willow wood (Salix sp.) in acidic D 0.

(ppm)



125 on the slightly acidic character of sugars in neutral or alkaline solution (72), acidic biomass hydrolyzates must be neutralized before analysis and columns must be flushed with alkali after chromatography to release the bound sugars. Although H P L C and H P A E C - P A D generally provide excellent results for abundant sugars such glucose and xylose, in some cases they offer only modest separation of minor sugars such as arabinose, rhamnose, galactose and mannose and quantification of these sugars may be limited by detector sensitivity (72). In addition, analysis times are typically long (up to 60 minutes) and column flushing between runs requires additional time for sugar desorption and reequilibration with mobile phase. Using a so-called "pusher ion" solution following elution with the basic mobile phase, several groups have observed both an increase in resolution and faster elution times for carbohydrates (6,7). In these procedures, the chromatography experiment is significantly more complex. G C / M S has also been widely used to characterize the carbohydrate profile of lignocellulosic materials (5). However, this technique requires that sugar analytes be relatively volatile so manpower-intensive chemical derivatization is needed prior to analysis. Typical volatile derivatives include alditol acetates, silyl derivatives and methylated sugars (5). Compared to all of the chromatography-based techniques discussed above, the N M R method is an attractive alternative because it provides excellent resolution of sugars, including minor components, does not require sample derivatization or neutralization, and is very fast (instrument time 15-30 minutes). Disadvantages of the N M R method compared to other techniques include: (1) the very high cost of a N M R instrument compared to HPLC or G C / M S systems, (2) some signal overlap of sugar resonances results in the need to adopt a quantification algorithm in which several assumptions are made (see below), (3) an additional step or internal standard is required to obtain absolute quantification of sugar concentration for hydrolyzates and, (4) no linkage information is derived.

Experimental Section Wood or other lignocellulosic biomass samples are typically ground to a fine powder using a Wiley Mill with 20 mesh screens followed by drying at 110°C for 8-16 hours. Samples of microcrystalline cellulose, guar gum, larch arabinogalactan, and gellan gum were obtained as powders from the following sources, respectively: F M C Corp., Halliburton Oil Services, St. Regis Paper Company, and Kelco. Wood samples were obtained from a collection maintained at SUNY-ESF. For N M R analysis, 0.2ml of 72% H S 0 was added to 0.040 g of dried biomass. After stirring, the dispersion was allowed to digest at 4 0 ° C for 1 hour in a bath or oven with additional stirring every 15 min. Following this digestion, 5.4 ml of D 0 (NMR solvent) was added to the dispersion which was then autoclaved in a high pressure sealed glass tube at 121 °C for 1 hour. After cooling, an additional 0.42 ml of 96% H S 0 is added for reasons discussed below. Hydrolyzates prepared in this fashion were then 2

4

2

2

4


126 filtered into NMR tubes without neutralization of the H S 0 . For "model" sugar compounds, NMR analysis was performed at 3.3 wt % concentration in a solvent containing H S0 and D 0 in same ratio as used for the hydrolysis procedure although these samples were not autoclaved. Since the H S0 employed in the digestion contains 28% H 0 , the samples tested by NMR contained approximately 1% water (in D 0) which can interfere with the observation of *H signals resulting from the sugars. However, the low pH of the acidic hydrolysis medium shifts the "water" NMR resonance (due to H 0) awayfromthe region of CI anomeric proton resonances which are used to quantify each sugar. The magnitude of this water peak shift is illustrated in Table I. Specific NMR details and conditions include: Bruker AVANCE 600 MHz NMR system (proton frequency = 600.13 MHz), Broadband Observe probe type (BBO), 30°C, 90° Pulse = lip. sec, delay between pulses = 10 sec, acquisition time = 2.73 sec, sweep width =10 ppm, center of spectrum = 4.5 ppm, reference = acetone at 2.2 ppm (lfil added to sample tube). Baseline corrections were made using a polynomial procedure. 2

2

4

4

2

2

4

2

2


2

Table I. Influence of Acid Concentration on Water Peak Chemical Shift H S 0 (%) 0 4 8 15 2

4

H 0 Chemical Shift (ppm) 4.6-4.7 5.2-5.4 5.7-5.9 6.1-6.3 2

Results and Discussion Sugar Quantification Algorithm In this analysis, both the cellulose and hemicellulosefractionsof woody biomass are ideally hydrolyzed to monomeric sugars in aqueous acidic solution. Under these circumstances, sugars undergo a mutarotation process whereby an equilibrium is established between the a and P CI anomer of the five and sixmember ring forms of the sugars and the open chain form (13). As shown in Table II, for most common sugars, the 6-membered a and p ring form of the sugar predominates, suggesting that quantification of only the pyranose forms will provide an adequate estimate of sugar concentration. (This assumption is less appropriate for galactose and arabinose where the furanose form approaches 5-6%). Figure 1 shown below is the proton NMR spectrum of an acid hydrolyzed sample of a fast-growing species of willow (Salix spp.). Although the spectral region from 3.2-4.0 ppm is very complex, the well resolved resonances centered near 5.2 ppm and 4.6 ppm can be assigned to the a and p anomeric CI protons of D-glucose and D-xylose. The higher intensity resonance of each pair is D-


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Table II. Conformation of Sugars in Aqueous Solution at 30-40°C (13) Open Chain Sugar a and 3 Pyranose a and P Furanose %

%

%

D-Glucose D-Mannose D-Galactose

99

0.002

99 94

1 1 6

0.02

0.005

D-Xylose

99.4

0.6

0.002

D-Rhamnose

99

1

0.005

L - Arabinose

95.5

4.5

0.03

glucose. These doublet signals are shifted away from the "complex" region of the spectrum since the CI protons are adjacent to two electron withdrawing oxygen atoms (the pyranose ring oxygen and the CI hydroxy 1). By summing up the integrated intensity of the a and P doublets, the relative molar concentration of xylose and glucose can be easily determined. Figure 2 contains the proton N M R spectra, recorded under acidic conditions, for a series of "model" sugar compounds commonly found in woody plant polysaccharides. For all sugars, the observed a and P peaks are "doublets" because the anomeric proton attached at CI is coupled to a single proton at C2 of the pyranose ring. Figure 3 superimposes these sugar spectra onto one axis in a fashion that simulates the mixture of sugars resulting when an actual cellulose and hemicellulose-containing biomass sample is hydrolyzed by aqueous acid. Although excellent resolution of sugar types is achievable in most cases, peak overlaps do exist. The relative concentration of each sugar is determined by summing up the total integrated intensity from its respective a and P anomeric proton doublets (the a doublet occurs above 5.00 ppm and the P doublet occurs below 4.95 ppm) and dividing by the total spectral intensity observed for the a and P doublets of all sugars in the mixture. In our current quantification algorithm, a and P signal intensities for D-glucose (Glu), D-mannose (Man) and D-rhamnose (Rha) are summed directly since they are well resolved. In normal practice for woody materials, the arabinose (Ara) a peak is significantly lower in intensity compared to the nearby glucose a peak and it is not resolved (See Figure 3). To determine the total N M R intensity associated with arabinose, the p doublet between 4.5 and 4.55 ppm is integrated while the a intensity is calculated from the measured a / p ratio determined for this compound (from data in Figure 2). The observed P signal is then added to the calculated a intensity to yield the total arabinose contribution. The calculated a intensity for arabinose is also subtracted from the a intensity of the glucose peak since these peaks overlap. As shown in Figure 3, p peak overlaps for D-xylose (Xyl) and D galactose (Gal) are also observed at 4.57 ppm. To alleviate this conflict, the well resolved "right" P resonance of xylose is doubled to yield a total P intensity which is added to the a doublet intensity. Similarly, for galactose, the "left " P resonance intensity is doubled and it is added to the a doublet. In order to determine the exact chemical shift of the a and p peaks associated with Dglucuronic acid (GluA), a 2D HSQC experiment was performed as shown in Figure 4. The spectral region between 4.3 and 5.4 ppm is significantly more In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


A

1

D-Glucose

D-Galactose

A

Cellobiose

D-Mannose

L-Rhamnose

D-Xylose

1


1

00


5.3

L - Arabinose

5.2 5.0

4.9

4.8

4.7

Figure 2. H NMR spectra of common sugars in the CI anomeric proton region, a resonances are > 5.00 ppm and /3 resonances are < 4.95 ppm (reversedfor L-arabinose).

5.1

r-r-p-


4.6

J 4.5

ppm


R

M

11

GL

5.30

5.10

(ppm)

4.90

4.80

4.70

4.60

*H NMR spectra of common sugars simulating a complex mixture.

5.00

Figure 3. Superimposed

5.20

4.50

\ \ i x \ i i \ i i \ i i i i i i i i t ; » i i i i i i i i i i \ \ \ i i i i i ; r i \ i | i i i i | i i i i } i i i » { \ t i i { r i r i | i i i i { i i t i r i i i i | i" i t » \ r i » t { i i i

GL

L-Arabinose (A) D-Galactose (GL) D-GIucose (G) D-Mannose (M) L-Rhamnose (R) D-Xylose (X)



131

Figure 4. A 2D HSQC spectrum of Glue A. Horizontal axis is the proton spectrum dimension and the vertical axis is the C dimension. Off axis peaks represent protons attached to specific carbon atoms in the sugar structure. The C peaks at > 90 ppm correspond to CI while the C2-C6 peaks are located between 70 and 80 ppm. 13

13


132


complicated for GluA compared to other sugars, perhaps due to lactone formation. Figure 4 reveals that the proton resonances observed near 4.7 and 5.3 ppm are attributable to CI anomeric protons. In the quantification algorithm, these intensities are summed to provide an estimate of GluA concentration. Once all of the a and (3 doublets in a complex mixture are resolved and / or estimated, the algorithm sums these to yield a total sugar intensity from which the contribution of individual sugars can be calculated as a relative mol % or wt % since the molecular weight of each sugar is known. To test the quantification procedure, solutions of sugar mixtures were prepared by weighing out a known amount of several sugars followed by proton NMR analysis under acidic conditions. Results shown in Table HI compare the estimates of sugar concentration provided by NMR to the actual concentrations of two separate mixtures. It appears that the NMR method provides a reasonably good estimate of sugar concentration over the range of concentrations that might be expected for woody biomass hydrolyzates. To test the repeatability of the biomass hydrolysis / sugar quantification procedure, a sample of wood pulp was subjected to acid hydrolysis followed by NMR analysis a total of 5 times. Results presented in Table IV illustrate that the repeatability of the combined hydrolysis / analysis protocol is excellent.

Table III. Sugar Mixture Quantification by NMR (wt%) Sugar Glucose Xylose Mannose Galactose Arabinose Rhamnose

Actual % Mixl 49.5 24.9 9.9 10.6 2.5 2.5

NMR % Mixl 51.5 23.7 9.3 10.1 2.5 2.9

Actual % Mix 2 50.5 23.9 10.6 7.5 3.3 4.2

NMR % Mix 2 51.4 24.2 10.1 7.2 3.1 4.0

Table IV. Repeatability of Hydrolysis / NMR Analysis for Wood Pulp Sugar Glucose Xylose Mannose Galactose Arabinose

Avg. Mole %-5Runs 90.0 3.11 5.89 0.38 0.64

Standard Deviation 0.24 0.14 0.12 0.07 0.05



133 One source of potential variability in the overall analysis protocol is the degree to which each cellulose or hemicellulose molecule in the original woody biomass is degraded by acid to oligomers, monomeric sugars, and then to degradation products such as 2-fiiraldehyde (furfural; derived from xylose) or hydroxymethylfurfural (from glucose). Typically, hydrolysis conditions such as the duration of each temperature stage, are optimized for each type of sample (ground wood, isolated polysaccharides, plant stalks, pulp, etc) by monitoring N M R signals associated with "under-converted" cellobiose (cellulose dimer), specifically, the resonances near 4.65 - 4.67 and 4.45 - 4.48 (See Figure 2). At the same time, we attempt to avoid the over-conversion of xylose to furfural, which is a much faster reaction compared to glucose conversion (14). Furfural exhibits four well-resolved resonances from 6.7 to 9.5 ppm, as shown in Figure 5, that can be used to monitor its presence in the hydrolyzate. Although our algorithm is designed to compute relative concentrations based on monomeric sugars, it is possible to integrate both the cellobiose and furfural resonances and to "correct" the relative concentrations of glucose and xylose, respectively, by adding in these under- and over-converted species. The hydrolysis conditions summarized in the Experimental Section are typical for ground wood samples.

Analysis of Model Polysaccharides and Wood Samples To test the N M R quantification protocol, a number of "model" polysaccharides were studied for which the monomeric sugar composition has been determined by other techniques. Table V shows that the N M R method agrees well with the known structures of microcrystalline cellulose, guar gum and the arabinogalactan polysaccharide from western larch. However, the GluA content observed for the microbial polysaccharide gellan, as well as xylan from birch, is consistently lower than reported by others. Since it is well known that the linkage between GluA and other sugars is very resistant to acid hydrolysis (J), it is likely that the underestimation of this sugar by N M R occurs because it is not hydrolyzed to a monomeric form but remains linked in higher molecular weight oligomers that are not adequately quantified by our procedure.

Table V. Sugar Composition of "Model" Polysaccharides (Dry wt. %) Ara

Observed N M R Ratio 1.69/1

Reference Ratio 1.66/ 1

87%

13%

6.41 /1

6.14/1 (16)

Rha

Xyl

GluA

96%

12% 3%

100% Glu 2.25 / 1 / 0.44 32/1

100% 2 / 1 / l (77) 7 / 1 (18)

Polysaccharide

Man

Gal

Guar Gum

61%

36%

(15)

Larch Arabinogalactan MCC Gellan Xylan

Glu 100% 61%

27%



r r m f T

9.0

Figure 5. 'HNMR

8.0

6.0

(ppm)

2

5.0

4.0

3.0

_

2.0

i i i | i |-rr-j rT-| i | i I T I | I I I T-J

spectrum of acid hydrolyzed willow wood. Peak at 6 ppm is due to H 0.

7.0

T T T I I I I I I I I M p r i F T "] * T Y T T~[~T "T T T r i ^ T T j i ~ r i - r T T - - r T r | i i i i | i i T T j r r r r ] - !


135


To resolve this limitation of the current protocol, enzymatic digestion (79) or an acid methanolysis procedure could be employed to more completely liberate GluA (20) from polysaccharides. Since this N M R method has been developed to rapidly characterize biomass samples with minimal sample treatment, the added complexity of additional treatment or derivatization is not consistent with the objectives of the present work. It should also be noted that we have not resolved the effect of 4-O-methylation on the chemical shift of the GluA anomeric protons. Typically wood xylans contain 4-O-methyl-GluA in their structure and not unsubstituted GluA which generally occurs in microbial polysaccharides. It is anticipated that a substituent on 04 in the GlucA structure will have only a minor effect on the chemical shift of the CI protons. Having successfully demonstrated the utility of the N M R technique for model sugar compounds and polysaccharides, attention was focused on the characterization of wood samples. A *H N M R spectrum recorded for balsam fir hydrolyzate is provided in Figure 6. A summary of results for several wood samples is provided in Table VI along with "reference" values published for the same species. Good agreement exists between the N M R method and published results for the major sugars (glucose, xylose and mannose for softwoods only) while deviations may exist for sugars present in lower concentrations. Based on the repeatability data shown in Table IV, it is possible that the results determined by other techniques are not as sensitive to low sugar concentrations as N M R . For wood samples, we also observe a systematic underestimation of GlucA by our acid hydrolysis / N M R procedure (except balsam fir) as seen for isolated polysaccharides (See Table V). Since GluA underestimation is most likely the result of incomplete acid hydrolysis, it should also be noted that "observed" GluA would be linked to other sugars in an oligomer so the quantification of these other sugars would also be adversely influenced. It also appears that the N M R estimate of galactose is consistently high compared to other methods. Based on the results in Table III, however, we do not believe that this an "artifact" of the N M R method itself.

Table VI. Sugar Composition of Acid Hydrolyzed Wood Samples (% of Carbohydrate Fraction) Sample Glu Xyl Sugar Maple (SM) 70.9 20.9 70.4 SM-Ref(l8) 20.3 Paper Birch (PB) 56.4 35.1 PB- Ref. (18) 56.2 34.0 Balsam Fir (BF);1 63.5 8.1 BF-Ref (18) 66.4 9.2 Loblolly Pine (LP) 66.6 10.6 LP-Ref (18) 63.5 9.9 1. Also includes 1.9% rhamnose

Man 2.9 3.1 1.1 2.4 15.4 17.3 14.1 15.5

Gal 0.8

Proton NMR Methods in the Compositional Characterization of

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