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ISOTOPE GEOSCIENCE

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Chemical Geology

136 (1997) 313-317

Technical Note

An improved technique for the batch processing of small wholewood samples to a-cellulose N.J. Loader ‘I, I. Robertson a, A.C. Barker b, V.R. Switsur a, J.S. Waterhouse

b

a The Godwin Laboratory, Godwin Institute for Quaternary Research, Department of Earth Sciences, University of Cambridge, Free School b Environmental

Lane, Cambridge, CB2 3RS, UK Science Research Centre, Anglia Polytechnic University, East Road, Cambridge, CBI IPT, UK Received

15 February

1996; accepted

1 September

1996

Abstract We present details of a modified technique for the extraction of cl-cellulose from wood samples. The revised technique, based upon the sodium chlorite oxidation method of Green (1963) utilises an ultrasonic bath and small Soxhlet thimbles to prepare a-cellulose from slivers of wholewood. These modifications facilitate the rapid batch processing of small wholewood samples to a-cellulose and yield a material with sufficient homogeneity as required for palaeoclimatic reconstruction. Keywords:

a-Cellulose;

Stable carbon isotopes; Palaeoclimate

reconsfruction;

1. Introduction The analysis of stable isotope ratios in organic materials has provided valuable information in the fields of carbohydrate research, plant physiology and palaeoclimatology (Hill et al., 1995; Switsur et al., 1995). In the case of stable isotope dendroclimatology, analyses of wholewood shavings produce a more ambiguous signal, both temporally and chemically, than may be derived from the analysis of a single component such as the ol-cellulose (Wilson and Grinsted, 1977; IBurk and Stuiver, 1981). Currently ol-cellulose is the favoured component for isotope analyses of wood for palaeoclimate research (Ramesh et al., 1985; Loader, 1995; Switsur et al., 1995). 0009-2541/97/$17.00 Copyright PII SOOOS-2541(96)00133-7

Soxhlet thimble

The techniques available for the extraction of cu-cellulose from wholewood are well established and have been described fully by Green (1963). The method adopted for this study was based upon the acidified sodium chlorite extraction of Jayme and Wise (Green, 1963) as modified by Mullane et al. (1988) for the delignification and extraction of OLcellulose from oak (Quercus sp.>. These processes may be scaled either up or down according to the amount of wood shavings available. In situations where sample sizes are small, accurate measurement of the reagents becomes more prone to error and the smaller volumes of liquids involved means that the wood slivers are not fully immersed in the acidified sodium chlorite solution leading to an incomplete extraction. As a consequence of these concerns it

0 1997 Elsevier Science B.V. All rights reserved.

314

NJ. Loader et ul. /Chemical

was decided to make modifications to the technique to enable a more effective extraction of small samples ( < 100 mg) such as are encountered in practice.

Geology

136 (1997) 313-317 EtdxdTukNlrmba

t

2. Development

of technique

Whilst the essential chemistry remained unchanged, the general technique was modified through the development of the sample containers, processing times and filtering apparatus. Reagents were standard laboratory grade with the exception of sodium chlorite (technical grade) and were used without further purification in the ratios shown in Table 1. The wholewood shavings for this study were obtained from 12 mm cores collected from Sandringham Park, Norfolk, UK. The latewood from each ring cut into slivers ( N 40 p,m thick) using a razor blade, weighed and placed into labelled borosilicate Soxhlet extraction thimbles (Fig. 1). Each thimble was then placed into a beaker containing the deionised water, acetic acid and sodium chlorite in the ratios outlined in Table 1, made up to a quantity sufficient for the total mass of wood shavings being processed. The volume of reagents was such that it did not exceed the height of the open Soxhlet thimble. The beaker containing the thimbles was then loosely covered with a watch glass and supported in an ultrasonic bath at N 70°C. The ultrasonic bath (Ney Dental International Model 57X, Output frequency 43-47 kHz) was set to maximum power and maximum degas for 4 hr. Three further additions of the acetic acid and sodium chlorite reagents were made to the beaker containing the sample thimbles, one after each hour (Table 1). At the end of this time Table 1 Quantities of reagents used per gram of wood shavings extraction of a-cellulose from small wholewood samples Reagent

Quantity of reagent

Wood shavings De-ionised water Sodium chlorite (per addition) Acetic acid (per addition) 10% (w/v) sodium hydroxide 17% (w/v) sodium hydroxide

1.0 g 175 ml 2.5 g 1.7 ml 75 ml 67 ml

Sodium chlorite and acetic acid quantities for each hourly addition.

in the

reflect those required

7omm

+

w

_)

2ontul Fig. 1. The modified u-cellulose.

Soxhlet thimble

used in the extraction

of

the thimbles were retrieved and the solution removed by vacuum filtration. The holocellulose was washed once with hot deionised water (_ 50 ml) and then once with cold deionised water (N 50 ml). At this stage the holocellulose should be quite white and not tinged with yellow or orange. Should these colours persist, the sample needs to be oxidised further and the purification repeated. On completion of the filtration the individual Soxhlet thimbles were placed in a clean beaker and 10% (w/v) sodium hydroxide was added to each of the Soxhlet thimbles. The beaker was placed in the ultrasonic bath and agitated for 45 min at N 80°C. The sample thimbles were next removed, filtered and washed once more with cold deionised water (N 50 ml> before being placed in a third beaker containing 17% (w/v) sodium hydroxide which was subjected to ultrasound for a further 45 min at room temperature. This procedure leached carbohydrates such as mannan and xylan from the holocellulose. The extraction thimbles were then removed from the beaker and the residual sodium hydroxide solution removed by filtration under vacuum. The cll-cellulose was washed with 17% (w/v) sodium hydroxide solution (- 20 ml) followed by copious amounts of deionised water and then 1% (w/v) hydrochloric

NJ. Loader et al. /Chemical

acid (N 20 ml). Finally, a large volume of cold deionised water was flushed through until the washings were neutral. The a-cellulose was then dried in a vacuum oven at 40°C for at least 4 hr. The dry a-cellulose was retrieved from the Soxhlet thimbles and stored prior to isotopic analysis. Each Soxhlet thimble was washed in deionised water and cleaned further in chromium(V1) oxide solution in 80% (w/v> sulphuric acid for at least 12 hr. After this time the thimbles were removed from the solution and washed in copious amounts of deionised water prior to re-use. For softwoods, resin must first be extracted in the Soxhlet apparatus with a 2:l toluene-methylated spirit azeotrope. As noted by Green (1963), extraction times will vary according to the nature of the sample being analysed. In the work currently in progress at the Godwin Laboratory samples of Pinus syhestris are processed in the azeotrope for 6 hr and then the wood shavings washed in methylated spirit and then deionised water prior to extraction of a-cellulose. The nature of the extraction thimbles described are such that they will fit the Soxhlet apparatus making it possible to process small softwood samples from wholewood to a-cellulose in the same vessel (Loader, 1995; D.L. Hemming, pers. commun.). Three tests were performed in order to assess the efficiency of the modified technique. The first was a comparison between the technique described by Green ( 19631, and our modified (ultrasound) version. In this instance samples of a homogeneous wood powder laboratory standard derived from a sample of pre-industrial Quercus robur from Ickworth Park, Suffolk, were delignified and the resulting o-cellulose converted to carbon dioxide for stable isotope analyses using a technique modified from Sofer (1980). The carbon isotope ratios were analysed

Table 2 A comparison Method

Green (1963) is study.

between the two techniques

of cellulose extraction

,

6’V-PDBC 3 (%o)

Number of analyses

a, _ (%o)

(o/o)

-24.27 -24.26

12 12

0.11 0.10

29+4 30*5

Yield

The results of both yield and stable carbon isotope ratios demonstrate that the two extraction procedures are indistinguishable within the statistical limits.

Geology 136 (1997) 313-317

315

Table 3 Carbon isotope ratios obtained from analyses of a-cellulose extracted from wholewood slivers of a single ring using the moditied technique Sample year

6’3C V-PDB

(AD)

(%,I

Number of analyses

o,,-I (%o)

1920 1921 1922 1923 1924 1925 1926 1927 1928 1929

- 24.28 - 22.97 - 24.62 - 23.47 - 24.66 - 24.91 - 24.38 - 24.87 - 23.52 -24.10

4 4 4 4 4 4 4 4 4 4

0.06 0.08 0.04 0.08 0.14 0.03 0.08 0.15 0.06 0.08

Results demonstrate the ability of the technique to yield an homogenous a-cellulose from originally inhomogeneous wholewood slivers

using a SIRA II isotope ratio mass spectrometer. The results are expressed as per mil (o/00)deviations from the Vienna Pee Dee Belemnite (V-PDB) standard. The two techniques compare favourably; both yield N 30% o-cellulose and demonstrate a high degree of isotopic homogeneity (Table 2). Whilst these initial results demonstrated the effectiveness of the extraction process using a powdered wholewood standard of high initial homogeneity it is likely that under normal circumstances samples will be in the form of wood shavings and rarely in such a quantity as to enable sample homogeneity to be obtained by milling without sample loss. Recent high-resolution analyses of stable isotope ratios in tree-rings have demonstrated the potential for significant isotopic variation across a single ring (Ogle and McCormac, 1994; Loader et al., 1995; Robertson et al., 1996). For this reason it was decided to assess the ability of the modified technique to produce a homogeneous a-cellulose from wholewood shavings of oak (Quercus robur) collected from the Sandringham Estate, Norfolk, and to test the homogeneity of the o-cellulose extracted from slivers rather than homogenised wholewood. Ten individual tree-rings were sampled. The latewood from each ring was isolated and cut into slivers ( _ 40 pm> using a razor blade and the a-cellulose extracted. Four stable carbon isotope

NJ. Loader et al./Chemical

316

analyses were determined for each o-cellulose sample. The reproducibility of the isotope values for each ring indicates the ability of the ultrasonic method to produce a homogeneous a-cellulose from an initially inhomogeneous wholewood sample (Table 3). In the third test, a sample of the homogeneous wood powder was subjected to an increasing period of acid delignification. After each hour an aliquot was removed from the mixture, washed with water and converted to o-cellulose. This process was repeated for 10 hr to investigate whether the processing time might be critical under such harsh chemical conditions, as suggested by Leavitt and Danzer (1993). The results obtained from the carbon isotope analysis fail to show that extraction time is a factor. Indeed, it is apparent, using this wholewood standard, that there is no significant difference between the isotope ratios of carbon dioxide derived from o-cellulose extracted after 1 or 10 hr of sodium chlorite delignification (Table 4). However, as outlined by Wise et al. (19461, the length of the oxidation period is largely a matter of experience. Indeed we suggest that the period of the oxidation should be modified for the species and physical nature of the sample (shavings or powder), being processed. Based upon analyses carried out at the Godwin Laboratory a 4-hr sodium chlorite extraction for wholewood Oak (Quercus sp.> (40 pm> slithers and a 6-hr sodium chlorite extraction for Scats pine (Pinus syluestris) prior to sodium hydroxide treatment is sufficient to yield a-cellulose of high purity. Table 4 Carbon isotope results obtained from a-cellulose extracted from the powdered wbolewood standard subjected to various delignification times Processing

time

6’3C

V-PDB

old

(%*I

1 2 3 4 5 6 7 8 9 10

-24.15 - 24.18 - 24.20 - 24.19 - 24.24 -24.18 - 24.19 -24.19 - 24.29 -24.12

Mean

-24.19f0.05

Geology 136 (1997) 313-317

Since the reaction vessel remains the same throughout the extraction process problems associated with the loss of sample during the mechanical transfer between filtrations as used in other techniques are minimised. The batch process also reduces the amounts of reagents required for the complete extraction of small samples and in doing so reduces the amount of chlorine(IV) oxide gas that is produced per sample. Ultrasonic treatment promotes a rapid and more complete penetration of the reagents into the wood particles and assists greatly in the disaggregation of the individual cellulose fibres. The adoption of the re-usable sample vessels (themselves not infinitely durable), reduces dramatically the amount of glassware required for processing, and enables a superior extraction of small samples to a-cellulose. At present the authors are aware of only one other batch technique for wood processing. This, described by Leavitt and Danzer (19931, involved the construction of an individual small glass fibre filter parcel for each sample, tied together with waxed flossing tape. A batch of these were loaded into a large Soxhlet apparatus and extracted for several hours prior to the oxidation of lignins using acidified sodium chlorite solution. The total processing time was > 2 days and produced holocellulose, rather than o-cellulose. The extraction efficiency showed some degree of variation that appeared to depend on the length of processing time. The reproducibility of that technique was also at the margins of those acceptable for palaeoclimatic reconstruction. The greater spread of results may be a product of the initial inhomogeneity of the sample being processed or that it was holocellulose, which contains small amounts of sugars, rather than the homogeneous o-cellulose, that was being analysed. The additional risk of loss of sample through rupture of tbe filter papers during processing was also noted by Leavitt and Danzer (1993).

3. Conclusions We have shown that the modifications described here to the standard technique (Green, 1963) y&l significant improvements for the extraction of a-cellulose. In 6 13Canalysis of samples of Quercus sp. a reproducibility was obtained which was sufficiently

N.J. Loader et al./ Chemical Geology 136 (1997) 313-317

high for palaeoclimate reconstruction ( < 0.1%0). Neither the recent reviews of cellulose processing techniques by Sheu and Chiu (1995) nor the batch analyses of Leavitt and Danzer (1993) and van de Water et al. (1994) obtain a better overall reproducibility than the data presented here. The number of samples processed per batch is typically between 25 and 50, yet this is limited primarily by the number of extraction thimbles available and the specification of the ultrasonic bath used. This method is now in regular use at the Godwin Laboratory where large numbers of low-mass samples are being prepared for isotopic analysis.

Acknowledgements The authors thank M.A. Hall for his helpful advice and the use of the Godwin Laboratory’s mass spectrometers; J. Rolfe for his efficient wood sample preparation; A.H.C!. Carter, D.L. Hemming and A.C. Gerrard of the Goclwin Institute for their constructive criticism and assistance with this paper. We thank also the Sandringham Estate and Ickworth Park for allowing sample collection, and A. Kershaw of the Department of Chemistry and Chemical Technology, University of Bradford, for making the modified Soxhlet extraction thimbles. This research was funded by a studentship to N.J.L. from the Natural Environment Research Council, and a grant awarded to V.R.S. and J.S.W. from the European Commission (EV5V-CT94-0500), for which we express our thanks. The authors are also grateful to Professor S.W. Leavitt and Dr. F.G. McCormac for their helpful comments on the manuscript.

References Burk, R.L. and Stuiver, M., 1981. Oxygen isotope ratios in tree cellulose reflect mean annual temperature and humidity. Science, 211: 1417-1419.

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Green, J.W., 1963. Methods of Carbohydrate Chemistry, III (Ed. R.L. Whistler). Academic Press, New York, NY, pp. 9-21. Hill, S.A., Waterhouse, J.S., Field, E.M., Switsur, V.R. and ap Rees, T., 1995. Rapid recycling of triose phosphates in oak stem tissue. Plant, Cell Environ., 18: 931-936. Leavitt, SW. and Danzer, S.R., 1993. A method for the batch processing of small wood samples to holocellulose for stable carbon isotope analysis. Anal. Chem., 65: 87-89. Loader, N.J., 1995. The stable isotope dendroclimatology of Pinus syluestris from Northern Britain. Ph.D. Thesis, University of Cambridge, Cambridge (unpublished). Loader, N.J., Switsur, V.R. and Field, E.M., 1995. High resolution stable isotope analysis: implications of “micro-dendroclimatology” for palaecenvironmental research. Holocene, 5(4): 457-460. Mullane, M.V., Waterhouse, J.S. and Switsur, V.R., 1988. On the development of a novel method for the determination of stable oxygen isotope ratios in cellulose. Appl. Radiat. Hot., 39: 1028-1035 Ogle, N. and McCormac, F.G., 1994. High resolution 6°C measurements of oak show a previously unobserved spring depletion. Geophys. Res. Lett., 21(22): 2373-2375. Ramesh, R., Bhattacharya, S.K. and Gopalan, K., 1985. Dendroclimatological implications of isotope coherence in trees from the Kashmir Valley, India. Nature (London), 317: 802-804. Robertson, I., Pollard, A.M., Heaton, T.H.E. and Pilcher, J.R., 1996 Seasonal changes in the isotopic composition of oak cellulose. In: J.S. Dean, D.M. Meko and T.W. Swetnam (Editors), Tree-rings, Environment and Humanity. Radiocarbon, Tucson, AZ, pp. 617-628. Sheu, D.D. and Chiu, C.H., 1995. Evaluation of cellulose extraction procedures for stable isotope measurements in tree ring cellulose. Int. J. Environ. Anal. Chem., 59: 59-67. Sofer, Z., 1980. Preparation of carbon dioxide for stable carbon isotope analysis. Anal. Chem., 52: 1389-1391. Switsur, V.R., Waterhouse, J.S., Field, E.M., Carter, A.H.C. and Loader N.J., 1995. Stable isotope studies in tree rings of oak from East Anglia. In: B. Frenzel, B. Stauffer and M.M. Weiss (Editors), Problems of Stable Isotopes in Tree-rings Lake Sediments and Peat Bogs as Climatic Evidence for the Holocene. Palaoklimaforsch./Palaeoclimate Res. 15, ESF (Eur. Sci. Found.), Strasbourg, pp. 129-140. van de Water, P.K., Leavitt, S.W. and Betancourt, J.L., 1994. Trends in stomata1 density and ‘3C/‘2C ratios of Pinus jlexilis needles during the last glacial-interglacial cycle, Science, 264: 239-243 Wilson, A.T. and Grinsted, M.J., 1977. 13C/ ‘*C in cellulose and lignin as palaeothermometers. Nature (London), 265: 636-639. Wise, L.E, Murphy, M. and D’Addieco, A.A., 1946. Paper Trade J. (TAPPI), pp. 11-19.