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Method for Batch Processing Small Wood Samples to Holocellulose for Stable-Carbon Isotope Analysis Steven W. Leavitt’ and Shelley R. Danzer Laboratory of Tree-Ring Research, University of Arizona, Tucson, Arizona 85721
INTRODUCTION Cellulose and holocellulose have become the preferred material for isotopic analysis of tree rings because of the singular composition of cellulose relativeto whole tissue which has many compounds of different isotopic composition’ and because cellulosic compounds are relatively immobile and remain confined to the growth rings in which they were actually formed, whereas whole tissue contains some mobile compounds which may move across ring boundaries.2 Holocellulose is a combination of cellulose (a glucan polymer) and hemicellulose (mixtures of polysaccharides).3 Standard methods for extraction of holocellulose from wood with chlorine dioxide or acid sodium chlorite have the effect of removing most of the lignin, but prolonged treatment to remove the final few percent of lignin results in some loss of hemicelluloses.4 For the purpose of streamlining the processing of wood samples to holocellulose, and especially to increase ease of handling and to limit loss of small samples with which we commonly work, we have devised a modified method of processing built around the basic ‘Jayme-Wise” technique of acid sodium chlorite delignification summarized by Green.4 Traditionally, samples are processed in large thimbles through various extractions, at times requiring removal and transfer to other separate vessels. In our method, a batch of 26-30 samples is collectively processed using one large beaker and one flask after initial solvent extraction of the batch in a single soxhlet. We describe here a test of the method in which both yield and stable-carbon isotope composition were measured on samples progressively taken through processing to various degrees of acid chlorite reaction.
EXPERIMENTAL SECTION Apparatus. Small samples of air-dried, ground (30-40-mesh) plant matter (usually 0.1-0.2 g if abundant and much smaller in the case of samples of limited supply) are placed into individual pouches made of glass-fiber filter paper. We have found the Schleicher & Schuell (Keene, NH 03431) no. 30 glass fiber paper as 5.5-cm circles or as 6-cm squares cut from large &in. X 10-in. sheets performs quite well, whereas other types such as Whatman GF/A 5.5-cm glass micropore filter circles disaggregate too much during processing. The filter paper is shaped into a pouch by forming it around the round end of a 14-mm-diameter test tube and placing it into the round 18-mm top opening of a plastic test tube holder. With a 10-hole test tube holder, 10 pouches can be lined up at a time to receive ground plant samples. For routine preparation of holocellulose where mass measurements are not needed during processing, a small plastic or glass tab (ca. 5-mm diameter) with a number inscribed is placed into the pouch along with the sample, and the sample name and corresponding tag number are recorded. The plastic tags degrade with time so they must be inspected prior to each use and replaced if necessary. The pouches are cinched up with waxed lacing string or waxed ribbon flossing tape (dental). Because of the corrosive nature of (1)Deines, Peter Handbook of Environmental Isotope Geochemistry: Elsevier: Amsterdam, 1980;Vol. 1, Chapter 9. (2)Long, Austin; Arnold, Larry D.; Damon, Paul E.; Lerman, Juan C.; Wilson, Alex T. Radiocarbon Dating; University of California Press: Berkeley, 1979; pp 532-537. (3)Pettersen, Roger C. The Chemistry of Solid Wood; American Chemical Society: Washington, DC, 1984;pp 57-126. (4)Green, John W. Methods of Carbohydrate Chemistry; Academic: New York, 1963;pp 9-21. 0003-2700/93/0365-0087$04.00/0
the treatment, we tie the pouch with two separate lengths of string in case one fails. An alternate method of securing the pouch is with 4-in. nylon cable ties (typically available from electronic supply stores). When weight determinations are to be made at various stages during processing, it is important to be able to identify the pouches from the outside, so a number or letter is inscribed directly onto the cable-tie ‘buckle” of each sample. Excess flossing tape or cable tie, and excess pouch flap material above the tie is then trimmed away. Inscription and reading of these small numbers is aided with a binocular microscope. The results described herein employ the latter method of labeling samples. Procedure. In the first processing step, extractives such as waxes, oils, and resins are removed. A set of 25-30 samples can usually fit into a soxhlet extractor (50-mm i.d., 200-mL capacity to siphon top). The reservoir flask is filled with a 2:l toluene/ ethanol mixture and run overnight (ca. 16-18 h). After shutdown and cooling, samples are poured from the soxhlet and allowed to dry for 1-2 h before returning them to a soxhlet in which the reservoir flask is filled with 100% ethanol and then extracting them for another 16-18 h. Samples are poured from the soxhlet to dry for 1-2 h before boiling in a 1OOO-mL beaker filled with deionized water for 6 h. Some inorganic salts and low molecular weight polysaccharides including gums and starches are soluble in hot water.3 The pouches (still wet) are then transferred to a 1500-mL Erlenmeyer flask containing ca. 700 mL of deionized water with an addition of sodium chlorite (NaC103, technical grade) in the amount of 6-8 g along with 1 mL of glacial acetic acid. The flask is placed on a hot plate at 70 OC with a 50-mL Erlenmeyer flask inverted over the top of the large flask and allowed to react overnight. The following day, three more additions of sodium chlorite and acetic acid are made at 2-h intervals, and the reaction is continued over the second night. The following morning, the samples are rinsed by decanting the solution and adding deionized water. After allowing to sit for 0.5-1 h, the process is repeated until the conductivity of the supernatant is less than 0.5 Mmhos cm-’ (SI, 0.5 pS cm-l). We find this usually takes 5-7 such rinsings. Samples are then ovendried overnight, and the pouches are opened by cutting the string or cable tie to recover the cellulose. Care must be taken in removing the cellulose because it is usually white like the glassfiber paper. Because the holocellulose tends to clump together, it is farily e p y to remove but sometimes a binocular microscope or hand lens may be used to facilitate the process. For our test of the method, we first started out with 26 pouches fiied with wood (sampleno. UA-ALTP-4),amodern spruce (Picea glauca) sample from Alaska. Samples were progressively taken out at various steps of the bleaching process on a schedule outlined in Table I. Two additional groups of four modern spruce samples were each treated together with separate batches of 24-26 other samples that were being routinely processed for other projects in order to compare our uniform processing with the test results. A third additional group of four spruce samples was treated with a batch of 16 other samples. Finally, a group of four large splits of spruce wood (ca.0.4-0.5 g each) were extracted in large,separate glass thimbles, poured from their thimbles, and boiled and bleached in separate beakers and flasks. The holocellulose yield was calculated as the percent of ovendried (70 “C) holocellulosemass scraped from the pouches relative to the original mass of air-dried, ground wood. Samples were combusted in a recirculation microcombustion line at 800 OC in the presence of excess oxygen. The COz product was cryogenically purified and analyzed mass spectrometrically. The W / W ratios are expressed as 613C values with respect to the PDB standard6 ( 5 ) Craig, Harmon Geochim. Cosmochim. Acta 1967,12, 133-149. 0 1993 American Chemical Society
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Table I. Bleaching Schedule after Initial Solvent and Hot-Water Extraction of 26 S D l i t s of Test Samde action date time, h 6March 1630 hot plate on at 70 OC, 8.03 g of NaC103 and 1 mL of glacial acetic acid added 7March 0815 four samples removed from solution (batch 1) 0820 7.99 g of NaC103 and 1mL of glacial acetic 1025 four samples removed (batch 2) 1030 7.90 g of NaC103 and 1mL of glacial acetic 1225 four samples removed (batch 3) 1230 8.05 g of NaC103 and 1mL of glacial acetic 1435 four samples removed (batch 4) 1440 6.10 g of NaC103 and 1mL of glacial acetic 8March 0815 four samples removed (batch 5) 0820 6.35 g of NaC103 and 1mL of glacial acetic 9March 0805 last five samples removed (batch 6) ~~
~
through the use of secondary standards. To judge the accuracy, one split of NBS-21graphite standard combusted to COz following the experiment gave P3C = -28.46% compared to the reported value of -28.13700.~Additionally, NBS-19 marble and NBS-20 limestone standards analyzed after reaction with 100% phosphoric acid to produce COz gave P3C values of +1.91700and -0.48%, respectively, whereas their reported true values are +1.93% and-l.OG%, respectively. A "softwoodstandard"ground wood material obtained from the Laboratory of Isotope Geochemistry, University of Arizona, was processed to holocellulose using our method. The 613Cresult was -23.65% (s = 0.32, n = 2) which may be compared to results previously obtained using standard sodium chlorite methods which yielded P3C of -23.55% (s = 0.07, n = 2).' Repeated combustion and analysis (n = 8) of a holocellulose lab standard to monitor precision during the course of the experiment produced a standard deviation of 0.23%.
RESULTS AND DISCUSSION The results of this study are contained in Table 11,including the characteristics of the resulting "holocellulose", the condition of the cable tie with which the pouch was secured, the percent holocellulose yield, and the stable-carbon isotopic composition (613C). There is a progressive decrease in yield with length of processing, with batches 5 and 6 representing a particularly dramatic, highly statistically significant decrease in yields associated with their extended treatment. For comparison, Hiigglunds reported the residue after acid sodium chlorite (or chlorine dioxide) treatment to be in the range of 60-63 % for spruce and pine samples, and 62-74% for a variety of hardwood samples. Pettersen3 found the "Cross and Bevan cellulose" content of 8 white spruce and 19 black spruce as 61 % and 60 % ,respectively. Cross and Bevan cellulose is the product after chlorine dioxide bleaching followed by extraction with hot aqueous sodium sulfite solution; this "cellulose'! retains a large quantity of the original hemicelluloses.4 At the extended level of delignification represented in batches 5 and 6, even the plastic cable ties were beginning to decompose. The 613C of the original whole wood is -25.92% (n = 4, s = 0.12). The isotopic composition of the holocellulose shows a shift toward increased enrichment in 13C (less negative P C ) with continued treatment. This is consistent with loss of lignins which tend to be isotopically more W-depleted than cellulose and especially hemicellulose.' The increasingly 13Cenriched composition of batches 5 and 6 is puzzling because hemicelluloses are more 13C-enrichedthan cellulose, so that as hemicellulose is decomposed one would expect the re(6) Coplen, Tyler B.;Kendall, Carol;Hopple, Jessica Nature 1983, 302,236-238. (7) Siegel, Randall D. Paleoclimatic Significance of DIH and 13C/22C Ratios in Pleistocene and Holocene Wood; M.S. Thesis, University of Arizona: Tucson, 1983. (8)HEgglund, Erik Chemistry of Wood; Academic: New York, 1951.
maining residue to become more W-depleted. This result may represent degradation of specific,more W-enriched than average, hemicelluloses under extreme chlorite delignification conditions. Alternatively, under these harsh conditions perhaps even some of the cellulose is degrading. The 613C averages of batches 1,2,3, and 4 were not statistically different from one another, but batches 5 and 6 were significantly heavier than the first four. The three groups of four splits of the modern spruce wood which were processed along with separate batches of 24,25, and 16 other unrelated wood samples, respectively, using our standard acid chlorite procedure provide an interesting comparison. The average yield for the first group of four was 62.1 f 1.3% and the 613C was -24.67% (s = 0.22%). The second group of four had an average yield of 59.7 1.3 5% and a 613C of -24.66% (s = 0.33960); all samples were fully white except one. The third group of four had a yield of 50.8 f 1.6% andaPCof-24.94% (s = 0.17%). Theseyieldssuggest the treatment of 25-30 samples produces a holocellulose product approximately consistent with that reported in the literature and intermediate in extent between that received by samples in batch 4 and that received from the extended bleaching in batches 5 and 6 of the test run. Complete treatment of 20 samples resulted in lower yields, but the apparently lower 6l3C of the third group was not significantly different from the first two groups. The 613C values from runs with 25-30 samples are not statistically different from those of batches 5 and 6 of the test run. It thus appears that our routine with 25-30 samples effectively represents the holocellulose content, but at the same time produces an isotopic composition suggesting a greater degree of lignin removal. Yields were not calculated for the final group of four samples which were treated in thimbles and separate glassware, but their average 613C was -24.66% (s = 0.21%). This is consistent with our results using the pouch method. The seemingly high standard deviations of the 613C means, even for the whole wood tissue splits, is comparable to the standard deviation for combustions of the lab standard over the period of the experiment. However, sample heterogeneity may also contribute to the scatter in 6%' beyond that simply attributable to the actual delignification treatment effects. Sample no. UA-ALTP-4 was from a full disk of wood from the trunk of a tree that was ground and mixed. There is actually much inhomogeneity among and within tree rings of an individual tree? and the ultimate combustion to COZof only ca. 3 mg of holocellulose from a sample originally in excess of 150 g in size may also contribute to some of the variability.
*
CONCLUSIONS With appropriate caution, the processing of small samples of plant matter in glass-fiber pouches provides a reliable method of treatment that eliminates much of the extra glassware needed in traditional methods, and it eliminates material losses which might otherwise occur from transferring samples from one vessel to the next and in filtering. Our standard processing (25-30 samples bleached from late afternoon one day to morning of the day after, with a total of four additions of NaC103 and acetic acid) appears to produce a good holocellulose product, similar in yield to that reported in the literature using the standard techniques for large samples, and approximating a treatment intermediate between that of batch 4 and batch 5 in the bleaching test conducted herein. Further treatment and treatment of fewer samples runs the risk of major loss of mass which includes (9) Leavitt, Steven W.;Long, Austin Ecology 1986, 1002-1010.
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Table 11. Characteristics and Yields of Cellulose Batches batch character of cellulose 1 yellow; powdery 2 yellow, but less so on outside; less powdery 3 light yellow 4 mostly white, slight yellow inside 5 white, hard packed 6 white, very hard packed
hemicellulose and perhaps even cellulose in the extreme. Small significant differences in yield also occur during insufficient treatment. The stable-carbon isotopic composition of the residue generally shows a pattern of increasing 613C with treatment, a t least initially consistent with the loss of isotopically "light" lignin. The continued 13C enrichment of samples under prolonged delignification conditions suggests that selective loss of certain hemicelluloses and/or attack on cellulose is enriching the residue further in l3C. Because of the variation of both yield and 613Cassociated with the extent of delignification processing, a protocol for constant and uniform processing of samples is essential. However, if precise yield determination is not needed, it
cable tie good condition good condition good condition good condition broke on 2 of 4 broke on 5 of 5
mean yield, % (1s) 73.1 (0.3) 72.3 (0.6) 70.5 (1.8) 68.8(0.8) 53.1 (1.9) 48.9 (2.2)
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mean P C , 9bo (Is) -25.27 (0.15) -25.32 (0.17) -25.13 (0.20) -25.09 (0.11) -24.64 (0.15) -24.61 (0.18)
appears that overbleaching is not as critical to accurate measurement of holocellulose 613C.
ACKNOWLEDGMENT
Thiswork was supported by NSF Grant No. ATM-9096319. Kyle DeWitte carefully prepared and weighed samples and Teresa Newberry analyzed COS from the samples a t the Laboratory of Isotope Geochemistry, Department of Geosciences, University of Arizona.
RECEIVED for review June 8, 1992. Accepted October 20, 1992.
