Technica1 Notes Anal. Chem. 1994,66, 2409-241 1
Improved Method for the Determination of the Oxygen Isotopic Composition of Cellulose Peter E. Sauer INSTAAR and Department of Geological Sciences, University of Colorado, Boulder, Colorado 80309-0450 Leone1 d. S. L. 0. Sternberg' Biology Department, University of Miami, Coral Gables, Florida 33 124
A modification of a chemical preparation procedure for oxygen isotope analysis of cellulose has been developed which yields greater precision and requiresless time for sample preparation. In the place of isoquinoline, zinc is used to remove HCI from the reaction products of the sample and HgCh. The two methods give systematically different results because isoquinoline biases measurements by contaminating the sample with additional C02. For the zinc method, precision is *0.2460 (lu standard deviation) compared with *0.9460 for the isoquinoline method. The stable isotopes of oxygen in organic material are of interest to plant physiologists' and paleoclimatologists2 who use isotopic ratios to constrain water relations and climate models. Because changes of only 1-2460 may be of biological or climatic importance, it is desirable to improve analytical precision better than the *0.9% variability of existing techniques. Natural changes in 180/160 ratios are small, and oxygen isotope ratios are expressed in 6 notation as per mil (460) variation from a standard:
in which R is the 180/160 ratio in the sample or standard, respectively. In this paper, the standard used for all measurements is standard mean ocean water (SMOW). Because different plant components have different isotopic compositions,a singlecompound is usually isolated for analysis. Cellulose (C6H1005)~is traditionally chosen because it is relatively easily isolated, it is a major component of plant material, and its resistance to chemical alteration subsequent to synthesis allows it to preserve its original isotopic composition. The method presented here was developed and tested for cellulose, but in principle it could be used for oxygen isotopic analysis of any carbohydrate. To measure the oxygen isotopic composition of cellulose, it is necessary to convert all the oxygen in the sample to C02 (1) Sternberg, L. d. S. L. Stable Isotopes in Ecologiccrl Research: Springer-
Vcrlag: New York, 1989; pp 124-141. (2) Burk, R. L.; Stuiver, M. Science 1981, 211, 1417-1419. 0003-2700/94/03662409$04.50/0 0 1994 American Chemical Society
without introducing any additional oxygen. Combusting cellulose with an excess of mercuric chloride in vacuum provides insufficient oxygen to oxidize all the carbon to C02, and a significant portion of the oxygen combines to form CO. The CO is converted to C02 by using a 5000-Vspark chamber partially submerged in liquid nitrogen; immediate condensation of the newly formed C02 shifts the reaction equilibrium toward the formation of additional C02. Hydrogen also poses a problem during pyrolysis because it reacts with oxygen to form H2O. This source of error is eliminated by adding HgCL to the combustion a m p ~ l e .The ~ chlorine gas evolved when the ampule is heated reacts with hydrogen to form HCl and prevents the formation of H2O. The HCl must be removed from the sample before it is loaded into the mass spectrometer. In earlier methods, HCl was removed using either a nickel reaction vessel4 or organic bases such as quinoline and 5,6benz~quinoline.~ Isoquinoline is commonly used today.5 An amalgalm of zinc has also been used for the determination of the 6I8Oof inorganic compounds, but precision was poor by modern standards (*30%0).~No test of the zinc amalgalm method has been performed on organic compounds to determine whether it can distinguish the small isotopicchanges of interest to plant physiologists or paleoclimatologists. To minimize the handling and generation of isoquinoline waste (which is toxic), a single batch of isoquinoline (1 5-20 mL) is normally used to process several dozen samples.5 Unfortunately, gases dissolvein isoquinoline,and the procedure for removing C02 from the reaction vessel is time-consuming, involving a number of heating and cooling steps. A small amount of C02 always remains in solution trapped within the isoquinoline. Thus, the analysis is very sensitive to the condition of the isoquinoline, which changes as samples are processed. A drift of 2.2% in the determination of standards was observed over a period of several weeks of heavy use of the preparation system (Table 1). (3) Rittenbcrg, D.; Ponticorvo. L. Int. J. Appl. Radiat. hot. 1956, I , 208-214. (4) Thompson,P.; Gray, J. In?. J. Appl. Radiat. hot. 1977, 28, 411-415. ( 5 ) Sternbcrg, L. d. S. L. In Plan? Fi6es; Linskens. H. F., Jackson, J. F., Eds.;
Modern Methods of Plant Analysis, New Series 10; Springer-Verlag: New York, 1989; pp 89-99. (6) Anbar, M; Guttmann, S. In?. J . Appl. Rodiat. hot. 1959, 5, 233-235.
Ana&ticaiChemistty, Vol. 66, No. 14, Ju& 15, 1994 2409
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Long-term drift of the determinations m a y result from the changing condition of the Isafurpoline. It is reasonable to expect t h p changes during normal sam e proceasing because (1) the amount of ~soquinohe dccrcam with eacg sample r d , 88 it is converted to the chloride salt, (2) isoquinoline degrates through time, and (3) the mount a d isotopic composition of the COz retained from each sample changes as different samples are processed. a
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METHOOS Only the new aspects of this procedure are described here in detail. Additional details including a description of the preparation vacuum line were described earlier by Sternberg.' Cellulose is first isolated from plant material according to the Jayme and Wise method, described by Green.' An aliquot of 6-1 0 mg of cellulose is sealed in a Vycor breakseal ampule with0.375gofHgClzandis bakedfor6 hat 550°C,producing a mixture of C02, CO, and HCl. The breakseal ampule is loaded in a vacuum line fitted with a spark chamber. Carbon monoxide is converted to C02 in a 5OOO-V spark chamberas Two grams of zinc is loaded into a 9-mms.d., 10-cm-long Vycor ampule and evacuated. While still under vacuum, the zinc is heated for approximately 1 min with a blue flame from a glass-blowingtorch. The flame is applied only to the bottom portion of the ampule where the zinc is located. A portion of the zinc sublimes and condenses as a purified coating higher on the ampule wall. As the vapor pressure of the zinc rises, the zinc pellet suddenly jumps several centimeters up into the ampule and sticks to the ampule wall. At this point, the flame is removed and the ampule is allowed to cool. This process removes oxides and provides a clean zinc surface which will react with the HCI. The mixture of HCl and C02 from the spark chamber is then frozen into the ampule for a period of 5 min, then scaled, and baked for 2 h a t 200 OC. This ampule is reloaded onto the extraction line. Carbon dioxide is frozen in a liquid nitrogen trap, and H2 and other residual gasses are pumped away. The volume of C02 is measured manometrically to determineyield. Finally, the pure CO2 is cryogenically transferred to a Pyrex ampule for transfer to the mass spectrometer. A mass scan for HC1 within the C 0 2 being transferred indicated that zinc completely removed HCI from the combustion products. RESULTS Replicates of three standards (Table 2) and 22 other samples (Figure 1) were measured with the old and the new (7) Green, J. W.Methods of carbohydrate Chemhtry;Academic Res:New YorL 1963; Vol. 3, pp 9-21.
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improved method. The repeatability of the measurement is substantially improved: standard deviations of replicate analyses are 0.2% for the improved method using zinc as opposed to 0.2-0.9960 using the isoquinoline method. The two methods for preparing samplesdo not always yield the same results (Table 2, Figure 1). Although both methods produced identical results for LS9 (6I8O= 24.3%) and Sigma (29.3%), the isoquinolinemethod gave higher 6 l 8 0 values for samples having lower 6l80values (RLIO and field samples). This suggests the isoquinoline may be providing additional oxygen, which biases the analysis. To test this possibility, we measured the P O of C02 gas evolved from the isoquinoline as it sat under vacuum overnight. Although the amount of gas evolved was small, its composition (6l80 = 19%) could account for the tendency of the isoquinoline to produce misleading values (Figure 1). This test did not replicate the conditions of normal sample processing, however, in which substantial amounts of HCI come into contact with the isoquinoline. To test the effect of HCI on the gas derived from the isoquinoline, we introduced 200 pmol of pure HCl into the isoquinolineand again measured the 6 l 8 0 of the evolved C02. This test yielded 9 Mmol of C02 (from an isoquinoline volume of 15-20 mL) with a S1So of +349&. Such an amount of C02 could seriously contaminate samples, which typically produce 100-150 pmol of C02, especially sampleswith 6'80 values very different from +34% Because many factors may affect the amount and the isotopic composition of COz gas given off by the isoquinoline during routjne sample preparation, the impact of the C02 released from isoquinoline will have both a memory effect and a systematic bias which cannot be eliminated. Instead, it is
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better to remove HCl gas without the use of organic compounds. A comparison of preparations using zinc from two sources showed no differences in measured 6 l 8 0 values. The 6 l 8 0 value of the Sigma standard using zinc from Mallinckrodt was determined at 29.4 f 0.2 (n = 4) and using zinc from Aldrich at 29.3 f 0.2 (n = 12). In conclusion, using zinc to remove the HCl from the HClC02 gas mixture has several advantages over the isoquinoline method. The new method is more precise, removing a significant source of COZcontamination. Differences between analyses performed by different workers and long-term variation, two problems with the use of isoquinoline, are eliminated. The use of zinc takes less time per sample, primarily because this approach avoids the multiple heating and cooling steps required to get the dissolved C02 gas out of the isoquinoline. Finally, this new method is cleaner,
preventing the introduction of organic reactants into the vacuum line and generating less toxic waste.
ACKNOWLEDGMENT We thank Professor Carl Hoff, Department of Chemistry, University of Miami, for suggesting the use of zinc. This research was funded by NSF Grant ATM-9122974 and a Student Research Grant from the Geological Society of America. This is publication 42 1from the program in Ecology, Behavior and Evolution of the Department of Biology, University of Miami, Coral Gables, FL, and contribution 9 from the Paleoclimateof Arctic Lakes and Estuaries (PALE) Program. Recetved for revlew December 2, 1993. Accepted Aprll 11. 1994.@ Abstract published in Aduance ACS Abstracu. May 15, 1994.
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