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Anal. Chem. 1991, 63, 2456-2459
DSC resulta, and for the many discussions concerning polymer blends. I also thank David Weiss (Eastman Kodak Co.) for producing the coatings and for the numerous discussions. In addition, I thank Kim Goppert-Berarducci (Eastman Kodak Co.) for measuring the surface energies of these polymers and the discussions concerning contact-angle measurements. Registry NO.PS (homopolymer),9003-53-6; BPAPC (SRU), 24936-68-3; BPAPC (copolymer), 25037-45-0; TMPC (SRU), 38797-88-5;TMPC (copolymer), 136132-65-5.
LITERATURE CITED (1) Bletsos, I.V.; Hercules, D. M.; Fowler, D.; van Leyen, D.; Bennlnghoven, A. Anal. Chem. 1990, 62, 1275-1284. (2) Brlggs, D. 61.Powm. J . 1989, 21, 3-15. (3) Hook, K. J.: Qardella, J. A. J . Vac. Scl. Techno/. A 1989, 7 ,
1795-1800. (4) Lub, J.; van Leyen, D.; Bennlnghoven, A. Polym. Commun. 1989, 30, 74-77. ( 5 ) van Velzen, P. N. T.; Wierenga, P. E.: Schaake, R. C. F.; van Leyen, D.; Bennlnghoven, A. Trlbol. Trans. 1988, 37,489-496. (6) Zhao, C. L.: Dobler, F.; Pith, T.; Holl, Y.; Lambla, M. J . Colldd Interfece Scl. 1989, 728, 437-449. (7) Hearn, M. J.; Ratner, 8. D.; Brlggs, D. Mecromolecules 1988, 21,
2950-2959. (6) Hearn, M. J.; Briggs, D.;Yoon, S. C.; Ratner, 8 . D. Surf. Interface Anal. 1987, IO, 384-391. (9) Brlggs, D.; Ratner, B. D. Polym. Commun. 1988, 29, 6-8.
(IO) Bhatla. 0. S.; Bwrell, M. C. Suf. Interface Anel. lOW, 75, 388-391. (11) Bt'lggs. D.;Munro. H. S. POJLm. Commun. 1087, 28(Part lo), 307. (12) Hennequln. J. F. J. phys. 1008, 29, 855. (13) Kelner, L.; Patel, T. C. secondery Ion Mss Spectromeby, SIMS V ; Bennlnghoven. A., Colton, R. J., Simons,
D. S., Werner, H. W., Eds.;
Sprlnger-Verlag, Berlin, I986 pp 494-496. (14) Mamyrln, B. A.; Kartaev, V. I.; Schmlkk, D. V.; Zagulln, V. A. Sov. Fhys.-JETP (Engl. Trans/.) 1973, 37, 45. (15) Poschenrieder, W. P. Int. J. Mass Specfrom. Ion Phys. 1972, 9 , 357. (16) Hagenhoff, B.; van Leyen, D.;Nlehuis, E.; Benninghoven, A. Secondary Ion Mess Specfromeby, SIMS VI; Bennlnghoven, A,, Huber, A. M., Werner, H. W.. Eds.; John Wlley & Sons: New Yak, 1988 pp 235-238. (17) Brlggs, D.;Brown, A,; Vickerman, J. C. Mendbook of stcltlc Secondery Ion Mess Specfromeby (SIMS);John Wlley & Sons: Chlchester, U. K., 1989. (16) Waugh, A. R.; Klngham, D. R.; Hearn, M. J.; Brlggs, D. Secondery Ion Mess Specfrometry,SIMS VI; Bennlnghoven. A., Huber, A. M., Werner, H. W., Eds.; John Wlley 8 Sons: New York, 1988; pp 231-234. (19) Brlggs, D.; Heam, M. J.; Fletcher, I.W.; Waugh. A. R.; McIntosh, 6. J. Surf. Interface Anal. 1990. 75, 62-85. (20) Fadley, C. S.Prog. SOlM Sfate Chem. 1876, 2, 265-343. (21) Dann, J. R. J . ColEOM Interface Scl. 1970, 32, 302. (22) Brown, A.; Vickerman, J. C. Surf. Interface Anal. 1988, 8 , 75-81. (23) Briggs, D. Org. Mess Specfrom. 1987, 22, 91-97.
RECEIVED for review January 28, 1991. Accepted August 9, 1991.
Determination of the Concentration and Stable Isotopic Composition of Nonexchangeable Hydrogen in Organic Matter Arndt Schimmelmann University o f California at San Diego, Scripps Institution o f Oceanography, La Jolla, California 92093-0215
The hydrogen stable isotope ratb of organic matter containing hydrogen that is not solely conservative, nonexchangeable, carbon-bound hydrogen depends on sample preparation, because organic hydrogen bound to oxygen and nitrogen may exchange isotopically with ambient-water hydrogen. The method described here permits the determination of the concentration and stable isotopic composition of the nonexchangeable hydrogen in complex organic matter, such as geochemicais and biological and archaeiogicai organic materials. Aiiquots of organic substrates were independently equilibrated with water vapors of different hydrogen isotopic compositions, followed by determinations of the bulk D/H ratios. Mass-balance calculations permit eliminating or minlmizlng the interference of exchangeable hydrogen. The precision of the calculated stable isotope ratio of nonexchangeable hydrogen can be better than k3 per mil, depending on the precislon of the measured values for bulk hydrogen. The accuracy of D/H ratios of nonexchangeable hydrogen was improved over that of previously available methods, as shown for ceiiuiose/celiuiose nitrate.
INTRODUCTION Stable hydrogen isotope ratios of modern and fossil organic matter bear potentially valuable geochemical, environmental, dietary, and climatic information (1-4). The problem of uncontrolled isotopic exchange between organic hydrogen bound to oxygen (O-H) and nitrogen (N-H)with ambient-water 0003-2700/91/0363-2456$02.50/0
hydrogen limits the usefulness of measuring total D/H ratios in most organic compounds. Notable exceptions are hydrocarbons and lipids, nitrated cellulose, and chemical derivatives of chitin (5-9), all of which ideally contain only isotopically conservative, nonexchangeable, carbon-bound hydrogen (C-
HI. A method is described for the determination of the concentration and stable isotopic composition of the nonexchangeable hydrogen in organic matter, based on controlled isotopic equilibrations of O-H and N-H with isotopically distinct water vapors. Standard techniques are then employed for the determination of the stable isotope ratios of the resulting bulk hydrogen (10, 11). After isotopic equilibration of an organic substance with water hydrogen, the deuterium in total organic hydrogen is the sum of the deuterium in the nonexchangeable and the exchangeable hydrogen pools, or 6~ = (1 - fe)Sn
+ f e ( 6 w + e)
(1) 6~ = 6, - f e s n + f e e + f e b (2) where 6T = isotopic composition of total organic hydrogen, f e = fraction of total hydrogen which is exchangeable, 6 , = isotopic composition of nonexchangeable hydrogen, 6 w = isotopic composition of water, and e = isotope fractionation effect between exchangeable organic hydrogen and water hydrogen, in per mil. Equation 2 is of the form y = a bx, where y is analogous to 6T,a to (6, - f,6, + f e e ) , b to f , , and x to 6 w . A calculation of 6, is possible based on the basis of two isotopic equilibration experiments, with knowledge of (1) the 6w values of two different water vapors used for equili-
+
0 1991 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 63, NO. 21, NOVEMBER 1, 1991
_.. _..
-1
dcb a
cm
113 'mI U
I'
Figure 1. Apparatus for the isotopic equilibration of exchangeable organic hydrogen with water vapor. Quartz or Vycor ampules are filled with (a) quartz wool, (b) copper(I1) oxide and silver foil, (c) organic substrate, and (d) copper wool or foil and are additionally fumished with a segment of Teflon shrink tubing (e). The arrangement within the oven consists of several filled ampules that are connected via shrink tubing (e), Teflon capillary tubing (9, Teflon manifold (g), stainless steel capillary (h) coiled within an aluminum heating block, and a Teflon capillary (i) to a port outside of the oven. The port receives either dry nitrogen gas or deionized water.
bration, (2) the resulting iiT values of the equilibrated organic substrates, and (3) the isotope fractionation effect E for exchangeable hydrogen present in the substrate. Isotope effects E are temperature-dependent and can be determined experimentally (shown below for cellulose)or can be calculated for different types of bonds (12-1 7). Chemically complex substrates with no precise stoichiometry do not permit a precise calculation of E. Each molecule of kerogen, for example, would have a unique set of hydroxyl, carboxyl, amino, etc. groups with exchangeable hydrogen. A value E for all exchangeable hydrogen can be estimated in the form of a "mean fractionation effect", by first calculating the various fractionation factors for individual functional groups and then using an overall weighted average according to the relative abundances of the functional groups in the substrate. The presented method is applicable to organic comounds that are chemically stable in water vapor at a chosen equilibration temperature, regardless of the ratio of C-H versus 0-H and N-H. Caution should be exercised with compounds containing high concentrations of sulfur, because sulfur has been shown to decrease the hydrogen recovered after the combustion and may thus introduce isotopic artifacts (18).
EXPERIMENTAL SECTION Apparatus. Samples (3-12 mg) are loaded together with quartz wool, approximately 1 g of copper(I1) oxide wire, a few square millimetersof 0.025 mm thick silver foil, and approximately 300 mg of copper foil or wool in a Vycor or quartz ampule (0.d. 9 mm) that has an open tip (0.d. approximately 2 mm, length 6 cm) at one end (Figure 1). The organic sample can be wrapped in quartz wool or another gas-permeable,inert material to avoid contact with CuO, if necessary. All inorganic materials, except Cu foil, are precombusted at 550 "C. The tip of the ampule is fitted with a 1cm long segment of Teflon shrink tubing (0.d. 3 mm; Cole-Parmer;shrunk 5 mm onto tip), and the ampule receives a constriction at the other end. The shrink tubing is then connectecd to Teflon capillary tubing within an oven. Several ampules are connected to a Teflon manifold. The temperaturecontrolled oven with fan-forced air convection holds an aluminum heating block kept at a temperature of 150-200 "C, with a stainless steel capillary (i.d. 0.8 mm) coiled within. The Teflon manifold connects via stainless steel and Teflon capillaries to a port outside of the oven. The port can be linked either to a supply line of dry nitrogen gas or to a Teflon capillary leading to a raised reservoir
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of deionized water (with known isotopic composition; head space and evaporative isotope fractionation are avoided by employing an air-free, collapsible, medical-type infusion container). Reagents. Distilled deuterium-depleted water (from Athabasca Glacier, Canada) and various additions of D20to distilled San Diego tap water yielded five waters for the exchange experiments, with 6D values of -157, -87, +112, +286, and +704 per mil. Cellulose and the corresponding cellulose nitrate were prepared according to standard techniques by Sternberg (19). Purified cotton was obtained from medical supplies. Humic acid and protokerogen from Recent marine sediment from the Santa Barbara Basin (20) and kerogen from the Miocene Monterey Formation were exhaustively demineralized (21,22). Chitin from a lobster (Homarus americanus) was prepared via decalcification and deproteinization of the carapace (23). Reagent grade oxalic acid diammoniun salt was obtained from Sigma. Procedure. Figure 1 describes the preparation of ampules containing organic samples. The connected ampules are continuously flushed with dry N2,while the oven temperature is raised to the desired degree, in this study between 104 and 156 "C. After switchingfrom nitrogen to distilled water, the steam pressure and overall flow rate are regulated by adjusting the height at which the water reservoir bag is positioned above the heating block (=vaporizer) level. Average water consumption over 20 h of operation was approximately 10 mL at 0.1 bar water vapor pressure. At the end of the equilibration period, the samples are again flushed with dry N2for at least 3 h while the oven is allowed to cool during the last 2 h. While a positive pressure is still maintained with dry N2,the samples are then sequentially sealed off with a torch, close to the Teflon connections. The 9 mm 0.d. openings are temporarily sealed with parafilm to exclude moist air. Angular break seals are formed at the sealed tips (Figure l), using a fine torch. After removal of the parafilm, the samples are immediately evacuated on a vacuum line and finally sealed under vacuum. The subsequent processing of the samples and the conversion of water to H2 follow routine methods (10,11). Engel and Maynard's (24) observation regarding the possibility of carbon isotope fractionation within combusted ampules after cooling is likely to be valid for hydrogen as well, via formation of hydrated sulfates (18)and hydrated copper carbonates (24). All samples need to be processed on a vacuum line within a few hours following combustion. Gas yields are measured manometrically. Isotopic results are expressed in GDsMownotation in per mil referring to standard mean ocean water (SMOW) isotope standard
The mass spectrometric precision of the measurements is f2 per mil for FDsMow values.
RESULTS AND DISCUSSION The isotopic responses of various organic comounds to equilibrations with five isotopically different water vapors at 114 "C and 20 h are shown in Figure 2. The slope of a linear regression line connecting data from a given compound reflects a measure of the substance's overall hydrogen isotopic exchangeability [in percent of total hydrogen; this study uses functional GM linear regressions which take into account that both variates are subject to error of measurement (25)].For example, a slope of 45" would indicate that all hydrogen in a sample is exchangeable. A slope of zero would identify the y intercecpt as the 6D value of the conservative D/H ratio of a compound with no exchangeable hydrogen. Case Study of Celluloseversus Cellulose Nitrate. The slope of the cellulose regression line in Figure 2 translates into an exchangeability of 26.7% at 114 "C, or f, = 0.267 in eqs 1 and 2 (linear regression coefficient R = 0.999, n = 8). In theory, 30% of the hydrogen in the biopolymer [C6H702(OH3], is 0-H and thus potentially exchangeable, but in practice, cellulose is a fibrous, closely knit agglomerate of biopolymers in which not all 0-H are physically accessible (26). This cellulose can be compared with its nitrated counterpart,
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 21, NOVEMBER 1, 1991
-
J
-50
.d
7
ammonium oxalate
400
200
400
800
800
6Dmow (per mil) of water Figure 2. ,6 D , values of isotopically equilibrated organic materials versus ,,D ,6 values of the waters used for the equilibration at 114 “C over 20 h.
cellulose nitrate, in Figure 2. Even exhaustively nitrated cellulose still contains some hydroxyl hydrogen (27), resulting here in a 3.9% exchangeability, or f, = 0.039 (R = 0.991, n = 7). The algebraically calculated intersection 2 (Figure 2) of the regression lines for cellulose and cellulose nitrate falls on -157 per mil on the x axis (6D of water, aWz) and -77 per mil on the y axis (6D of equilibrated substrate, 6Tz). Using eq 2, we can fill in the known bT, f,, and 6w values for both cellulose and cellulose nitrate, yielding two equations with two unknowns (a, E ) and permitting the calculation of 6, = -77 per mil and E = 80 per mil. As expected for higher temperatures, the E value falls in the lower range of hydrogen isotopic fractionation for organic 0-H bonds [for example, ethanol 110 per mil, benzyl alcohol 100 per mil, phenol 70 per mil; all calculated for 25 “C (12)]. The x and y coordinates of the intersection 2 of the regression lines describe unique equilibration conditions when water of -157 per mil is used: The isotopic composition of the exchangeable hydrogen, after equilibration with water of -157 per mil, is identical with the isotopic composition of the nonexchangeable hydrogen (-77 per mil). The isotope fractionation effect E between water hydrogen and cellulose hydroxyl hydrogen accounts for 157 - 77 = 80 per mil. By rearranging eq 2 to
(3) we see that the expression in the bracket is always zero (80 - 157 + 77 = 0 in the numerical example). It follows that JTZ is dependent only on 6,. Therefore, fe can be chosen freely between 1and 0 (for non-nitrated cellulose and completely nitrated cellulose, respectively) without effect on the locus of 2. In other words, we can substitute any number of hydroxyl groups (equilibrated to -77 per mil) with nitrate groups without changing the overall isotopic composition of -77 per mil. The extent of nitration determines the slope of the cellulose nitrate line, but it does not influence the location of the intersection 2 of the cellulose and cellulose nitrate lines. Moreover, all celluloses, regardless of their C-H isotopic compositions ,a have their 2 intersections of respective lines of their non-nitrated and nitrated forms falling on a 45O line marked “C(H20),”in Figure 2. Note that “C(H20),”,following the formula y = x + a, or 8T = 6w E , is valid for a specific
+
temperature only, here 114 “C, because t is responsible for the position of the line along the x axis. A “C(H,O),” line is valid for all celluloses,and possibly for all carbohydrates as well, as long as they express the same t value, for a given equilibration temperature. For example, with knowledge of E the determination of the isotopic composition of C-H of an organic compound is possible by opting for one of the following strategies: If the relative amount of exchangeable hydrogen and the slope of the equilibration line are well-known, it may be sufficient to equilibrate one aliquot of a sample, determine its overall 6D value, and construct the intersection of two lines as in the cellulose example. Higher precision could be achieved by multiple equilibrations with various water vapors in combination with regression analyses. If the isotopic exchangeability of a compound is not known, one needs at least two D/H exchange reactions to construct such a line. For cotton, which represents almost pure carbohydrate, the y coordinate of the intersection with “C(H20)n” in Figure 2 suggests a 6D value of C-H of -31 per mil (exchangeability 16.1%; R = 0.997, n = 17). In terms of accuracy of D/H ratios of C-H in cellulose, the equilibration method outperforms the use of nonequilibrated nitrated cellulose, because the isotopic influence of even small amounts of remaining exchangeable hydrogen in nitrated cellulose is taken into account. Implications for Non-Carbohydrate Substrates. A suitable line parallel to the “C(H20),” line is valid for all organic comopunds with the same t value, for a given equilibration temperature. The “C(H20),” line would need to be shifted along the x axis to account for differences in the isotope factionation effect t. The displacement can either be determined experimentally by chemical replacement of exchangeable hydrogen with inert groups (e.g., nitration, if chemically possible), or can be calculated on the basis of the knowledge of stretching and bending force constants (12-17). For example, Bigeleisen and Ishida’s simple first-order calculation of isotope effects, particularly applicableto deuterium substitution, reflects the relative importance of bending and stretching forces to the isotope effect as a function of temperature (14). Unlike all other theoretical methods, Polyakov (17)uses a! and p factors in an innovative approach based on Galimov’s (28) theory of thermodynamically controlled intramolecular isotopic ordering. One may also choose compounds for comparison that contain only one kind of hydrogen bond, such as N-H, for example in ammonium oxalate (NH4)2(C00)2(Figure 2). In crystalline ammonium oxalate, 98.1% of the hydrogen was found to be exchangeable. A graphical evaluation, similar to the one discussed for cellulose 0-H, yields a N-H fractionation of 17 per mil, at 114 “C.For comparison, Bigeleisen’s (12) calculated values for N-H at 25 “C vary between -4.5 (ammonia) and +110 per mil (aniline). Chemically complex kerogen, humic compounds, bulk organic tissues, etc. do not permit chemical replacement of 0-H and N-H and would require the use of a calculated 0-H and N-H mean isotope effect. The calculation could be aided by lH NMR and IR data on the distribution and abundance of different types of functional groups bearing exchangeable hydrogen. Figure 2 does not attempt the determination of 6, for complex compounds, but it documents different degrees of exchangeability. The decline of exchangeability from Recent humic acid (18.8%;R = 0.999, n = 7) over Recent protokerogen (11.4%; R = 0.979, n = 6) to Miocene kerogen (6.3%; n = 2; see also Table I) is consistent with decreased abundances of functional 0-H and N-H containing groups in that order (29). Effect of Temperature on Isotopic Equilibration. Table I illustrates the effect of temperature on the observed hydrogen exchangeability in various organic compounds.
ANALYTICAL CHEMISTRY, VOL. 03, NO. 21, NOVEMBER 1, 1991 T a b l e I. H y d r o g e n I s o t o p i c E x c h a n g e a b i l i t y (in P e r c e n t of T o t a l H y d r o g e n ) in V a r i o u s O r g a n i c Compounds That Were E q u i l i b r a t e d f o r 20 h at 104, 114, 140,143, and 166 O c a exchangeability, % compd
104 OC
114OC
cellulose chitin cotton humic acid kerogen
23.1 (2)
nd 15.7 (3)
nd nd
14OOC
143 OC
156 OC
26.7 (8) 15.8 (2) 16.1 (17)
nd
nd nd
26.8 (2) 17.2 (2)
nd
19.2 (6)
18.8 (7)
20.7 (2) 8.4 (2)
nd nd
nd nd
6.3 (2)
OThe number of bD values used entheses; nd = not determined.
16.4 (2)
strates, for example phyllosilicates and zeolites.
ACKNOWLEDGMENT I sincerely thank John Hayes, Max Coleman, and Michael J. DeNiro for their helpful criticism. Leonel Sternberg provided cellulose and cellulose nitrate samples. I am grateful to Dave Winter (UCLA) and to the Stable Isotope Laboratory at the Southern Methodist University (Dallas) for their support and their mastery over stable isotope ratio mass spectrometry.
9.1 (2)
for calculation is given in par-
Isotopic equilibrations were performed over 20 h at 104,114, and 143 "C in water vapors with 6D values of -157, -87, and +704 per mil. At higher temperatures, exchangeability is larger, because isotopic exchange is kinetically controlled by diffusion of hydrogen species through the substrate. The exchange rate may widely vary depending on chemical composition and structure. Future studies of specific classes of compounds (e.g., crystalline or amorphous substrates, biopolymers, geopolymers, etc.) would be required to establish a temperature dependence and time regime for isotopic equilibration that achieves reproducibleexchangeability while not compromising the chemical stability. For the isotopic equilibration of temperature-sensitive substrates the oven temperature could be set below 100 "C if a regulated vacuum oven is used that can avoid having a liquid water phase in contact with the samples. One should bear in mind that even temperatures of a few degrees above boiling point, below critical temperature, may permit the existence of liquid water in porous samples, due to reduced vapor pressure within narrow pore spaces. Soluble constituents in the sample have a similar effect by forming solutions with lower vapor pressure. Any hydrous liquid phase in contact with a sample would be enriched in deuterium and would thus cause increased D/H ratios compared to properly equilibrated samples.
CONCLUSIONS The determination of the D/H ratio of nonexchangeable hydrogen in organic substrates via controlled isotopic equilibration of the exchangeable hydrogen pool is applicable to a large variety of materials. The equilibration method is the only approach feasible for those oxygen- and nitrogen-containing compounds that do not permit a chemical replacement of all their hydroxyl, amino, etc. groups by nitrate or other inert groups. Paleodietary, paleoclimatic, and paleoenvironmental stable isotope studies can utilize the hydrogen isotopic composition of chemically complex substrates, for example collagen from bone, amino acids, lignin, and various solid fossil fuels and biogeochemical isolates. Further use of the isotopic equilibration method might be extended to inorganic sub-
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LITERATURE CITED (1) Buchardt. E.; Frk, Peter, Haandbook of E~vkonmentalIsofope Qeochemistry; Elsevier: Amsterdam, 1980; Vol. 1, Chapter 12. (2) Estep, Marilyn, F.; Dabrowski. Halina. science 1980, 209. 1537-1538. (3) Smith, J. W.; Rigby, D.; Schmidt, P. W.; Clark, D. A. Nefure 1983, 302, 322-323. (4) Schldlowski, Manfred; Hayes, John M.; Kaplan, Isaac R. Eerih's €adest Biosphere; Princeton University Press: Princeton, NJ, 1983; pp 149-186. (5) EDStdn. Samuel; YBDD. . . c. J.; Hall J. E 8 h PletWf. SCl. Lett. 1978, 30, 241-251. Schoell, Martin, Org. Oeochem. 1984, 6, 645-663. Schimmeimann, A r m DeNiro. Michael J. oeochlm. Cosmochm. Acte 1988, 50, 1485-1496. Miller, Randail F.; Fritz, Peter; Morgan, Alan V. Pakreogeogr., PaleeoCllmafOl., Pelaeoecol. 1988, 66, 277-288. Sternberg, Leonel. &We 1988. 333, 59-61. Stump, R. K.; Frazer. J. W. Nwi. Sei. Absfr. 1973. 28, 746. Northfeit, Donald W.; DeNiro, Michael J.; Epstein, Samuel, M m . Cosmochim. Acta 1981, 45. 1895-1898. Bigeleisen, Jacob. Science 1985. 147, 463-471. Stern, M. J.; Wolfsberg, M. J. Chem. Phys. 1988, 45. 4105-4124. Blgeieisen. Jacob; Ishida, Takanobu, J. Am. Chem. SOC. 1973, 95, 8155. Bigeleisen, Jacob; Ishida, Takanobu, J. Chem. Phys. 1975. 62, 80-88. Bigeleisen, Jacob; Ishida. Takanobu, J. Chem. M y s . 1975, 63,
1702.
Polyakov, V. E. Geochem. Inf. 1988. 25, 117-121. Krishnamurthy, R. V.; DeNko. Michael J. Anal. Chem. 1982, 54,
153- 154.
Sternberg, Leonel. plent Fibers; Modern Methods of Plant Analysis, New Series; Springer: Berln, 1989; Vol. 10, pp 89-99. Schimmelmann, Arne Lange, Carina B.; Berger, Wolf H. Limnd. OCsenOgr. 1990, 35,165-173. Wedeking, K. W.; Hayes, John M.; Matzigkeit, Udo. Earth's €ar/ksf Biosphere; Princeton University Press: Princeton, NJ, 1983 pp
428-441.
Idir. Erdem, F. Ph.D. Dissertation, University Callfornla Los Angeles,
1987.
Schimmelmann, Amdt; DeNiro, Michael J. Chifin /n Netwe and Tech-; Plenum, Publishing Corp.: New York. 1986; pp 357-364. Engei, M. H.; Maynard, R. J. Anal. Chem. 1989, 61, 1996-1998. Ricker, W. E. J. Flsh. Res. Boardcan. 1973, 30, 409-434. Young, Raymond, A.; Roweii. Roger M. Cellulose: Strucfufe, W i t & cathm, andHydrdLsls; Wiley: New York, 1986; 379 pp. DeNiro, Michael J. &hPletWt. SCi. Lett. 1981, 54, 177-185. Galimov. Erlk Mikhaliovlch. The Biobglcal Fractlonetion of Isotopes; Academic Press: Orlando, FL, 1985. Tissot, Bernard, P.; Wehe, Dietrich, H. Pefr&um Fwmafbn and Occurrence, 2nd ed.;Springer: Berlin, 1984; 899 pp.
RECEIVED for review February 11,1991. Accepted July 8,1991. This work was supported by the Universitywide Energy Research Group Account No. 6-506290 to M. Kastner. Additional support was provided by Scripps Industrial Associates and by a Biomedical Research Support Grant to A.S.
