Microwave-Supported Preparation of r-Cellulosefor Analysis of δ13C in Tree Rings

Marika Haupt* and Tatjana Boettger

Department of Isotope Hydrology, UFZ Centre for Environmental Research Leipzig-Halle, Theodor-Lieser-Strasse 4,D-06120 Halle, Germany

The microwave technique is found to be very applicablefor the preparation of r-cellulose from wood samples andcan be recommended for analyzing the stable carbonisotopes in tree rings. At a reaction temperature of 80 °C,the extraction time can be decreased from 36 h to 15 min.Microwave-supported prepared cellulose contains moreamorphous constituents, resulting in a relatively higherreactivity and amenability for a following nitration withregard to determination of nonexchangeable hydrogen.The δ18O values of microwave-enhanced extracted cel-lulose remain significantly lighter than reference values,possibly as a result of an increased oxygen isotopeexchange between bleaching solution and cellulose underconditions of high energy input. Therefore, this techniquecannot be recommended for oxygen isotope analyses inwood cellulose.

Tree rings are known as a natural, highly resolved palaeocli-matic archive, because the fixation of the light elements, such ascarbon, oxygen and hydrogen, in these annual wood incrementsdepends on conditions for growth. Wood consists predominantlyof cellulose, hemicelluloses, lignin, and some minor constituents,such as resins and tanning agents. The composition variesconsiderably according to tree species; site conditions; and, infossil woods, the storage conditions and time of storage.

Traditionally, cellulose is used for palaeoclimatic investigationsbecause it’s a very stable, natural biopolymer. Different nomen-clatures of cellulose modifications can be found. R-Cellulose isdefined as the 17.5% sodium hydroxide-insoluble cellulose fractionwith a degree of polymerization above 200.1,2 Other sourcesdeclare cellulose with a special latter configuration as R-cellulose3,whereas 13C NMR spectra of algal and bacterial cellulose underliethis definition.4

The isolation of cellulose from tree-ring wood is commonlycarried out with several modifications of the sodium chloritepulping process described by Sohn and Reiff.5-8 Building of stable

isotope chronologies requires the cellulose extraction of a greatmany individual tree-ring samples, consuming time and materialon a big scale. Therefore, it is necessary to search for method-ological modifications to improve the economical efficiency.

The microwave technique is well-established in sample prepa-ration, mainly for the determination of pollutants in soils, sedi-ments, sludges, etc,9 but Pensado et al.10 also describe theextraction of polycyclic aromatic hydrocarbons from wood.Microwave-enhanced production of cellulose derivatives is alreadywell-known, too; for example for esterification11,12 and phospho-rylation.13 Microwave-supported bleaching of pulps with hydrogenperoxide and magnesium carbonate for increasing the brightnessof fibers is presented by Wan et al.14

Microwave heating allows the drastic reduction of extractiontime because temperatures above the boiling point of the solventcan be used, thereby accelerating the process and minimizing theconsumption of chemicals. The aim of this work is to check theapplicability of the microwave technique for cellulose extraction,provided that the produced cellulose is identical in structure andproperties to cellulose prepared with conventionally used methods.Of particular importance for palaeoclimatic investigations are thecarbon and oxygen stable isotope ratios in cellulose and, withregard to analysis of nonexchangeable hydrogen, their crystallinitydecisively influencing the process of nitration.

EXPERIMENTAL SECTIONMaterials. For the described tests, incorporated in the 5th

EC Framework Program “Energy, Environment and SustainableDevelopment” funded project ISONET, “400 Years of AnnualReconstructions of European Climate Variability using a High-Resolution ISOtopic NETwork” (2002-2006), well homogenizedwood standards of three recent oaks (Quercus I, II, III) and pines(Pinus I, II, III), a laboratory standard (RH; homogenized pine

* Corresponding author. E-mail: marika.haupt@ufz.de.(1) Falbe, J., Regitz, M., Eds. Rompp Chemielexikon; Georg Thieme: Stuttgart,

1989.(2) Gaudinski, J. B.; Dawson, T. E.; Quideau, S.; Schuur, E. A. G.; Roden, J. S.;

Trumbore, S. E.; Sandquist, D. R.; Oh, S.; Wasylishen R. E. Anal. Chem.2005, 77, 7212-7224.

(3) Ebert, G. Biopolymere; Teubner Studienbucher Chemie: Stuttgart, 1993.(4) Kunze, J.; Fink, H.-P. Papier 1999, 12, 753-764.(5) Wiesberg, J. Ph.D. Thesis; Rheinisch Westfalische Technische Hochschule,

Aachen, 1974.(6) Gray, J.; Song, S. J. Earth Planet. Sci. Lett. 1984, 70, 129-138.

(7) Borella, S.; Leuenberger, M.; Saurer, M.; Siegwolf, R. J. Geophys. Res. 1998,103 (D16), 19519-19526.

(8) Sohn, A. W.; Reiff, F. Papierfabrikant 1942, 40, 1-7.(9) Gerhardt, U.; Romer, H.-P. Intern. Z. Lebensm. Technol. Verfahrenstech. 1985,

36, 309-316.(10) Pensado, L.; Casais, C.; Mejuto, C.; Cela, R. J. Chromatogr., A 2000, 869,

505-513.(11) Satge, C.; Verneuil, B.; Branland, P.; Granet, P.; Krausz, P.; Rozier, J.; Petit,

C. Carbohydr. Polym. 2002, 49, 373-376.(12) Antova, G.; Vasvasova, P.; Zlatanov, M. Carbohydr. Polym. 2004, 57, 131-

134.(13) Gospodinova, N.; Grelard, A.; Jeannin, M.; Chitanu, G. C.; Carpov, A.; Thiery,

V.; Besson, T. Green Chem. 2002, 4, 220-222.(14) Wan, J. K. S.; Radoiu, M.; Kalatchev, I.; Depew, M.C. Res. Chem. Intermediat.

2000, 26, 931-939.

Anal. Chem. 2006, 78, 7248-7252

7248 Analytical Chemistry, Vol. 78, No. 20, October 15, 2006 10.1021/ac060523a CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 09/12/2006

wood from the Leipzig region), and 31 subfossil pine woodsamples (Pinus sylvestris L., ∼1000-1300 cal AD) from the centralKola Peninsula, a region of the Chibiny Low Mountains,15 wereused.

The stable carbon and oxygen isotope ratios, obtained in woodcellulose extracted with our standard preparation method, areregarded as carbon reference (CR) and oxygen reference (OR)values for comparison with the microwave-assisted (MW) ex-tracted cellulose.

Standard Preparation Method. R-Cellulose is prepared witha multistage procedure according to Sohn and Reiff,8 whereas atfirst, an extraction with methanol/benzene (1:1; room temperature;36 h) is carried out to remove resins and other soluble ingredients.After flushing with acetone, bleaching with a 7% sodium chloritesolution (60 °C; 36 h) follows, whereby lignin is broken down anddissolved together with a part of the hemicelluloses. The residualmixture of R-cellulose and hemicelluloses is treated with dilutedsodium hydroxide solutions (5%; 60 °C; 4 h and 17%; roomtemperature; 40 min) to separate R-cellulose. The final product,rinsed with 10% acetic acid as well as distilled water, is dried at50 °C overnight.

Microwave-Assisted Preparation Method. According toRinne et al.,16 a solvent extraction is nonessential for analysis of

stable isotope ratios in cellulose of recent and well-preservedwoods, which is the case with all standard woods and subfossilpine wood samples from Kola Peninsula that were used. Therefore,samples are treated directly with 7% sodium chlorite solution usingthe microwave system Microwave 3000 (Anton Paar). This systemcan be used in temperature- or power-controlled mode. Thatmeans that, in contrast to common household appliances, tem-perature or power can be adjusted and the other parameter isfound automatically. We used the temperature-controlled modebecause only reaction temperature and reaction time are selectedas influencing variables for dissolving process. Alkaline treatmentand cleaning of final cellulose samples are similar to the standardmethod described above.

Stable Isotope Analysis. The carbon and oxygen isotoperatios of cellulose samples are analyzed simultaneously17 with themass spectrometer Delta plus XL (ThermoFinnigan) coupled witha high-temperature (1450 °C) pyrolysis reactor (HEKAtech, HT-O). The results are given as δ-values: δ ) (Rsample - Rstandard)/Rstandard × 1000 (R ) 13C/12C, 18O/16O, respectively) in per mil(‰) versus the IAEA (International Atomic Energy Association)standard materials VPDB (Belemnitella Americana, Peedee For-mation, Cretaceous, South Carolina) and VSMOW (Vienna Stan-

(15) Kremenetski, K.; Boettger, T.; MacDonald, G.; Vaschalova, T.; Sulerzhitsky,T.; Hiller, A. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2004, 209, 113-125.

(16) Rinne, K. T.; Boettger, T.; Loader, N. J.; Robertson, I.; Switsur, V. R.;Waterhouse, J. S. Chem. Geol. 2005, 222, 75-82.

(17) Knoller, K.; Gehre, M.; Boettger, T.; Weise, S. M. Rapid Commun. MassSpectrom. 2005, 19, 343-348.

Figure 1. Influence of the microwave reaction temperature on theδ13C (a) and δ18O (b) values of standard wood celluloses (tconst ) 30min).

Figure 2. Influence of the microwave reaction time on the δ13C (a)and δ18O (b) values of standard wood celluloses (Tconst ) 80 °C).

Analytical Chemistry, Vol. 78, No. 20, October 15, 2006 7249

dard Mean Ocean Water). The methodical uncertainty of themethod amounts to (0.2‰ for δ13C and (0.3‰ for δ18O.

Structural Analysis. The structure of the prepared cellulosesis investigated by means of high-resolution, solid-state CP/MAS13C NMR spectroscopy (nuclear magnetic resonance spectroscopyin the cross-polarization magic angle spinning mode) (Avance 400;Bruker) according to Kunze and Fink4 and NIR FT Ramanspectroscopy (near infrared Fourier transform Raman spectros-copy, RFS 100; Bruker) according to Schenzel and Fischer.18

RESULTS AND DISCUSSIONAccording to Sparr-Eskilsson and Bjorklund,19 both solvent

(kind, volume, concentration) and reaction conditions (time, tem-perature) are relevant parameters for a microwave-enhanced extrac-tion method, whereas according to Kornilova and Rosell-Mele,20

the temperature mostly influences the results. On the basis of ourstandard preparation method, where a treatment with 7% sodiumchlorite solution at 60 °C (36 h) is specified, we increase thetemperature for an initial microwave system test to 120 °C. The

reaction time is fixed at 15 min. Under these conditions, thecellulose is completely dissolved, and the sodium chlorite solutionbecomes colorless. That means it is not possible to heat thethermally instable cellulose above the boiling point of the bleach-ing solvent.

To determine the critical temperature at which cellulosedecomposes, three recent wood standards (Quercus I, Pinus I, RH)were treated at temperatures increasing stepwise from 60 to 110°C (tconst ) 30 min). For pine celluloses, the δ13C value (Pinus I,RH) apparently reached a maximum at 70 °C (Figure 1a), whereasthe deviation from the reference (∆δ13C ) δ13CMW - δ13CCR) alsois the lowest at this temperature (∆δ13CPinusI ) -0.5‰; ∆δ13CRH

) -0.2‰). The cellulose of oak (Quercus I) shows no trend ,andthe lowest deviation from the reference is given at 80 °C(∆δ13CQuercusI ) -0.5‰). On the other hand, the δ18O valuesgenerally attain their maximum at 80 °C (Figure 1b), whereasthe deviation from the reference values (∆δ18O ) δ18OMW -δ18OOR) is likewise the lowest at this temperature (∆δ18OQuercusI )-0.7‰, ∆δ18OPinusI ) -0.7‰; ∆δ18ORH ) 0.1‰). At 90 °C, the lossof cellulose is already observable; at 100 °C, the cellulose iscompletely dissolved. Therefore, 80 °C is determined as the upperlimit for the reaction temperature when the microwave system isused for cellulose preparation.

(18) Schenzel, K.; Fischer, S. Cellulose 2001, 8, 49-57.(19) Sparr-Eskilsson, C.; Bjorklund, E. J. Chromatogr., A 2000, 902, 227-250.(20) Kornilova, O.; Rosell-Mele, A. Org. Geochem. 2003, 34, 1517-1523.

Figure 3. Combined influence of microwave reaction temperatureand reaction time to the δ13C (a) and δ18O (b) values of standardwood celluloses as a deviation from reference values (∆δ13C )δ13CMW - δ13CCR and ∆δ18O ) δ18OMW - δ18OOR).

Figure 4. Microwave-assisted cellulose preparation (T ) 80 °C; t) 15 min) of subfossil wood samples (Pinus sylvestris L.) - δ13CMW

(a) and δ18OMW (b) values in comparison to reference δ13CCR (a) andδ18OOR (b) values.

7250 Analytical Chemistry, Vol. 78, No. 20, October 15, 2006

The influence of the reaction time, now at a constant temper-ature of 80 °C, on the carbon and oxygen isotope ratios is shownin Figure 2. The δ13C values decrease for times lower than 30min and remain relatively constant up to 60 min (Figure 2a), butthe least deviation from the reference values is observable after15 min for Quercus I and Pinus I (∆δ13CQuercusI ) -0.3‰; ∆δ13CPinusI

) -0.1‰) and not until 60 min for RH (∆δ13CRH ) -0.1‰). The

δ18O values also show differences between various tree species(Figure 2b). The deviation from the reference values is alreadythe slightest after 30 min for pine wood cellulose (δ18OPinusI )-0.7‰; ∆δ18ORH ) 0.1‰), but not until after 60 min for oak woodcellulose (∆δ18OQuercusI ) -0.6‰).

The combined importance of reaction temperature and timewas appraised by treating the seven recent standard materialsunder varying conditions as follows: (a) 60 °C, 30 min; (b) 70 °C,60 min; and (c) 80 °C, 15 min. The δ13C reference values areattained approximately with method c (Figure 3a). In this case,the deviation lies within the mass spectrometric error range (σ(0.2‰) for Pinus I and for the other six wood standards, in arange of 2σ. In all cases, no statistically significant difference at p) 0.01 (|t| ) 2.6 < t6;0.01 ) 3.7) between δ13C values of cellulosesextracted with microwave support and celluloses extracted usingthe standard preparation method can be stated. But it should benoted that the δ13C ratios of all microwave-enhanced preparedcelluloses, except RH, are measured marginally lighter than inreference celluloses.

The δ18O values converge to the reference values when thereaction time increases (Figure 3b) but persist generally lighterusing all tested conditions. In contrast to the carbon results andto Kornilova and Rosell-Mele,20 the δ18O values are apparentlyinfluenced more strongly by extraction time than by extractiontemperature.

Analogue results (Figure 4) were obtained for microwave-supported treatment of 31 subfossil pine samples at the “opti-mized“conditions. The deviation of δ13C values from referencevalues remains within the mass spectrometric error range (σ(0.2‰) for 56% of the samples, in the range of 2σ ((0.4‰) for78%; the residual 22% diverge even more. The δ13C values ofmicrowave-assisted-prepared cellulose compared with referencevalues are statistically identical at p ) 0.01 (|t| ) 0.9 < t30;0.01 )2.8), whereas the δ18O values also remain significantly lighter thanthe reference values.

Impurities, caused by adherent solvents after rinsing, can bespaced out, because the samples prepared by both methods,

Figure 5. NIR/FT Raman spectra of cellulose (Pinus III) extracted with (a) microwave-assisted preparation method (T ) 70 °C; t ) 60min) and(b) standard preparation method (T ) 60 °C; t ) 36 h).

Figure 6. CP/MAS 13C NMR spectra of cellulose (RH) extractedwith (a) standard preparation method (T ) 60 °C; t ) 36 h) and (b)microwave-assisted preparation method (T ) 80 °C; t ) 15 min).

Analytical Chemistry, Vol. 78, No. 20, October 15, 2006 7251

microwave-supported and conventional, are cleaned in the sameway.

Possibly, these effects are caused by an incomplete microwave-supported extraction of R-cellulose and, consequently, highercontent of residual lignin. Borella et al.7 and Loader et al.21 detectedδ13C values of lignin up to 4‰ lighter in comparison to R-cellulose.According to Barbour et al.,22 the δ18O values of lignin are,depending on species, up to 9‰ lighter than those of R-cellulose.During previous experiments, we also observed lighter δ13C andδ18O values in the case of the residual lignin. Nevertheless, it couldnot be reasoned that the microwave-enhanced extracted R-cel-lulose is clearly more impure than that conventionally prepared,because the NIR/FT Raman spectra obtained from Pinus IIIcellulose (Figure 5) indeed offers a higher peak at 1600 cm-1 formicrowave-assisted prepared cellulose in comparison to standardprepared cellulose, indicating more lignin residues.16 But this verysmall difference could be related only to marginal variations inthe case of δ13C values. In contrast, the strong deviations in δ18Oresults cannot be explained by traces of lignin. Maybe they arecaused by microwave-generated oxygen isotope exchange be-tween the acidified sodium hypochlorite solution and cellulose.

The CP/MAS 13C NMR spectra of recent pine standardcellulose (RH) reveals the structural similarity of microwave-supported-prepared cellulose and cellulose prepared using thestandard preparation method, but there are some differences inthe crystallinity of the produced celluloses (Figure 6). Themicrowave-enhanced-prepared cellulose contains more amorphouscomponents, which are quite visible due to an increasing side linein the shared peak of the C-4 position. The lines for the C-1 andC-2, -3, -5 positions become more indistinct, which is alsoattributable to a rise in amorphous components.4 The enhance-ment of amorphous components has consequences for cellulosereactivity in paleoclimatic applications, especially for the nitrationof cellulose, because cellulose nitrate is commonly used for isotope

analysis of nonexchangeable hydrogen. More amorphous com-ponents implicate more susceptible regions for acid attacks.23

Furthermore, according to our own experience with commerciallyavailable microcrystalline cellulose, the nitration of predominantlycrystalline cellulose is quite impossible.

CONCLUSIONSGenerally, the microwave technique is applicable for the

preparation of R-cellulose from wood samples and can be recom-mended for analyzing the stable carbon isotopes in tree rings.Under optimized conditions, reaction temperature 80 °C andreaction time 15 min, the δ13C reference values will be achievedapproximately within analytical uncertainty of the mass spectrom-eter.

The microwave-enhanced-prepared cellulose contains moreamorphous components than that conventionally prepared, result-ing in a relatively higher reactivity with regard to nitration anddetermination of nonexchangeable hydrogen.

Even past 60 min at 80 °C, the δ18O values of microwave-supported prepared cellulose still remain significantly lighter thanreference values, which is possibly caused by increased oxygenisotope exchange between the bleaching solution and the celluloseunder conditions of high energy input. Therefore, this techniquecannot be recommended for oxygen isotope analyses in woodcellulose.

ACKNOWLEDGMENTThis work was made possible by EC Grant EVK2-CT-2002-

00147 ISONET. We thank Mr. Kainrath from S-prep for providingthe microwave system Microwave 3000 (Anton Paar) and technicalsupport. Special thanks goes to E. Brendler, TU BergakademieFreiberg, for CP/MAS 13C NMR spectroscopy; K. Schenzel, MLUHalle-Wittenberg, for NIR FT Raman spectroscopy; and U.Helmstedt, UFZ Centre of Environmental Research Leipzig-HalleGmbH, for analysis of stable isotopes.

Received for review March 22, 2006. Accepted August 16,2006.

AC060523A

(21) Loader, N. J.; Robertson, I.; McCarroll, D. Palaeogeogr. Palaeoclimatol.Palaeoecol. 2003, 196, 395-407.

(22) Barbour, M. M.; Andrews, T. J.; Farquhar, G. D. Aust. J. Plant Physiol. 2001,28, 335-348.

(23) De Souza Lima, M. M.; Borsali, R. Macromol. Rapid Commun. 2004, 25,771-787.

7252 Analytical Chemistry, Vol. 78, No. 20, October 15, 2006