Anal. Chem. 1997, 69, 3329-3332
Gas Chromatographic Tuning of the Uk′37 Paleothermometer Joan Villanueva and Joan O. Grimalt*
Department of Environmental Chemistry (C.I.D.-C.S.I.C.), Jordi Girona, 18, 08034-Barcelona, Catalonia, Spain
The extensive use of the C37 di- and triunsaturated alkenones for the estimation of sea surface temperatures in ancient oceans prompts an investigation of the analytical constraints on reliable gas chromatographic measurements. Several tests reproducing the alkenone amounts currently encountered in high-resolution stratigraphy studies show that no significant errors are specifically related to differences between on-column, splitless, and septum-programmable injection. However, irreversible adsorption on the chromatographic column is one major source of error that has significant effects (temperature deviations higher than 0.5 °C) when the total amounts of alkenones introduced into the system are lower than 5-10 ng. These deviations are more important for lower relative proportions of the triunsaturated species (higher temperature). The observed adsorption evidences that, in the practical operating conditions for analysis of large data sets (average of three replicate injections for 1 g of sample), a concentration of 50 ng/g is the lowest limit for reliable paleotemperature estimation. One achievement of molecular marker studies during the 1980s1 was the discovery of the link between the proportion of di-, tri-, and tetraunsaturated methyl and ethyl C37-C39 ketones (usually referred as the alkenones) and the temperature of the waters in which they were biosynthesized. This relationship is substantiated by studies of precursor organisms within the Haptophyceae, notably Emiliania huxleyi,2 and the global distributions of these alkenones in the marine sediments, particularly their latitudinal trends.3,4 As a result, the relative abundance of these alkenones has been proposed as a paleoclimatic proxy indicator of sea surface temperature (SST),3,5 a fundamental parameter for the understanding of the interactions between atmosphere and hydrosphere in the past. The SST method is based on the measurement of the concentrations of heptatriaconta-15E,22E-dien-2-one (C37:2) and heptatriaconta-8E,15E,22E-trien-2-one (C37:3) to determine a relative composition index [Uk′37 ) C37:2/(C37:2 + C37:3].3,6 Diverse relationships between Uk′37 and temperature have been established by means of E. huxleyi cultures under temperature-controlled condi(1) Philp, R.; Oung, J.-N. Anal. Chem. 1988, 60, 887A-896A. (2) Conte, M. H.; Eglinton, G. Deep-Sea Res. 1993, 40, 1935-1961. (3) Brassell, S. C.; Eglinton, G.; Marlowe, I. T.; Pflaumann, U.; Sarnthein, M. Nature 1986, 320, 129-133. (4) Rosell-Mele´, A.; Eglinton, G.; Pflaumann, U.; Sarnthein, M. Geochim. Cosmochim. Acta 1995, 59, 3099-3107. (5) Brassell, S. C. In Organic Geochemistry. Principles and Applications; Plenum Press: New York, 1993; pp 699-753. (6) Prahl, F. G.; Muehlhausen, L. A.; Zahnle, D. L. Geochim. Cosmochim. Acta 1988, 52, 2303-2310. S0003-2700(97)00038-3 CCC: $14.00
© 1997 American Chemical Society
tions6,7 and by core top sediment analyses and correlation with SST.4,8 There is a general agreement on the slope and range of application of the resulting straight lines.5 The first equation published was reported by Prahl and Wakeham (Uk′37 ) 0.033T + 0.043).7 From a geochemical perspective, these alkenones possess several characteristics that permit their widespread use in paleoclimatic studies: (a) they are major lipid compounds in Quaternary ocean sediments, and they are present in many ancient sediments (as old as Cretaceous),5 (b) they are specifically produced by a few members of the Haptophyceae algae,9 (c) their principal precursor species are widespread in the global ocean,2 (d) they constitute one of the lipid groups more resistant to biodegradation and diagenesis,10 and (e) their features of composition related to temperature are not modified by diagenesis, even in the case of extensive oxidation of organic matter.11,12 Accordingly, no sources other than a few Haptophyceae contribute to the alkenones found in marine sediments, and their climatic information is well preserved. These properties have encouraged the extensive use of the sedimentary alkenones to obtain estimations of paleo-SST in Quaternary studies.13-17 However, temperature changes involved in paleoclimatic models (excluding the North Atlantic) are only a few degrees Centigrade, 1-3 °C.18 Thus, SST estimates need to be performed with a maximum error of 0.5 °C to be reliable for such studies. This corresponds to a maximum error in the Uk′37 measurements of only 0.0165 if the above-mentioned equation is used. In this respect, there is a major gap between the wealth of Uk′37 data reported in the literature and the very limited number of studies concerned with the reliability of the determinations. Unfortunately, strong analytical requirements in terms of precision and accuracy have to be fulfilled to achieve correct SST estimates and credible paleoclimatic information. (7) Prahl, F. G.; Wakeham, S. G. Nature 1987, 320, 367-369. (8) Sikes, E. L.; Farrington, J. W.; Keigwin, L. D. Earth Planet Sci. 1991, 104, 36-47. (9) Marlowe, I. T.; Brassell, S. C.; Eglinton, G.; Green, J. C. Chem. Geol. 1990, 88, 349-375. (10) Sun, M.-Y.; Wakeham, S. G. Geochim. Cosmochim. Acta 1994, 58, 33953406. (11) Prahl, F. G.; de Lange, G. J.; Lyle M.; Sparrow M. A. Nature 1989, 341, 434-437. (12) Poynter, J.; Eglinton, G. Fresenius, J. Anal. Chem. 1991, 339, 725-731. (13) Jasper, J. P.; Hayes, J. M. Nature 1990, 347, 462-464. (14) Eglinton, G.; Bradshaw, S. A.; Rosell, A.; Sarnthein, M.; Pflaumann, U.; Tiedemann, R. Nature 1992, 356, 423-426. (15) Kennedy, J. A.; Brassell, S. C. Nature 1992, 357, 62-64. (16) Rostek, F.; Ruhland, G.; Bassinot, F. C.; Mu ¨ ller, P. J.; Labeyrie, L. D.; Lancelot, Y.; Bard, E. Nature 1993, 364, 319-321. (17) Schneider, R. R.; Mu ¨ ller, P. J.; Ruhland, G. Paleoceanography 1995, 10, 197-219. (18) CLIMAP Project Members. Geol. Soc. Am. Map. Chart. Ser. 1981, 36, 1-50.
Analytical Chemistry, Vol. 69, No. 16, August 15, 1997 3329
Gas chromatography coupled to flame ionization detection is the technique most widely used for the alkenone analyses.13-17 Its use requires specific analytical constraints to avoid systematic and random Uk′37 errors. Some of them, namely the problems of coelution, have been considered elsewhere.19 The present study is focused on discrimination at injection and irreversible adsorption, which constitute the most usual error sources. They are particularly important in studies involving high stratigraphic resolution due to the limited amounts of sample available. Major errors may be introduced in the alkenone paleothermometer as a consequence of inadequate consideration of these problems. EXPERIMENTAL SECTION Materials. Residue-analysis isooctane was from Merck (Darmstadt, FRG). The synthetic alkenone standards, heptatriaconta15E,22E-dien-2-one and heptatriaconta-8E,15E,22E-trien-2-one, were kindly provided by Prof. Maxwell (Organic Geochemistry Group, University of Bristol).20 The alkenone mixture used for the tests at the University of British Columbia (Vancouver, Canada) were obtained from a pure culture of E. huxleyi. The algal biomass was extracted with dichloromethane/methanol (2:1 v/v), hydrolyzed with 6% KOH in methanol, and the alkenone fraction was separated from the neutral lipids by column chromatography. Gas Chromatographic Instruments. The chromatograms used for the analyses encompass different injection facilities: (A) Carlo Erba Model HRGC5300 equipped with a flame ionization detector, splitless and on-column injection. Carrier gas, hydrogen (50 cm/s). Oven temperature program, from 80 to 200 °C at 20 °C/min, then to 295 °C at 6 °C/min, holding the final temperature for 20 min. Detector temperature, 320 °C. Injection (solvent, toluene) either in splitless mode (300 °C, split valve closed for 55 s, hot needle technique) or on-column (70 °C). (B) Varian Model 3400 equipped with a septum-programmable injector (SPI) and a flame ionization detector. Carrier gas, oven and detector temperatures as described above. Injection temperature program, from 80 °C (held 1 min) to 300 °C at 200 °C/min, holding the final temperature for 15 min. The injector was cooled with a gas stream to reach 80 °C. (C) The instrument used at the University of British Columbia was a Hewlett-Packard Model 5880A equipped with a splitless injector and a flame ionization detector. Carrier gas, helium (50 cm/s). Oven temperature program, from 90 to 200 °C at 15 °C/min, then to 320 °C at 6 °C/min, holding the final temperature for 25 min. Detector temperature, 320 °C. Injection (solvent, toluene) in splitless mode (310 °C, split valve closed for 3 min, hot neddle technique). A 50 m × 0.32 mm i.d. CPSIL-8CB column (Chrompack, Middleburg, The Netherlands) coated with 100% dimethylsiloxane (0.12 mm film thickness) was used in all cases. RESULTS AND DISCUSSION Injection. The influence of the different injection systems on the Uk′37 measurements has been examined. Standard alkenone mixtures providing commonly encountered values in warm and cold waters have been used for this purpose. Diluted solutions representing current marine sedimentary concentrations have been prepared with these mixtures and repeatedly analyzed (three (19) Villanueva, J.; Grimalt, J. O. J. Chromatogr. 1996, 723, 285-291. (20) Rechka, J. A.; Maxwell, J. R. Org. Geochem. 1988, 13, 727-734.
3330 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
Figure 1. Uk′37 indexes of two series of standard mixtures of C37:2 and C37:3 alkenones diluted at various ratios. The two mixtures constitute commonly encountered values of warm and cold waters. Injection systems: (A) SPI, (B) splitless, and (C) on column.
replicates). All results (SPI, on-column injection, and splitless injection) show comparable trends toward higher Uk′37 values at lower alkenone amounts (Figure 1). These trends are observed irrespective of the composition of the mixture and the dominance of the C37:2 or C37:3 alkenones. In all three cases, the total alkenone amount of 5-10 ng appears to be a threshold, below which significant errors in accuracy are produced (Uk37 drifts > 0.0165). The errors below this threshold are higher at warm temperatures (0.08, 0.035, and 0.035 for SPI, splitless, and on-column, respectively) than at cold temperatures (0.03, 0.03, and 0.03 for SPI, splitless, and on-column, respectively), which implies a greater dependence on the relative amounts of the more unsaturated species (C37:3). Uk′37 drifts are also observed above this threshold of 10 ng, but they are below the 0.0165 value. They are lower for the on-column (0.006-0.008) than for the splitless (0.011-0.012) and the SPI injectors (0.0100.010). The standard deviation values resulting from the replicate chromatograms obtained with the SPI, on-column, and splitless injectors show, in general, lower dispersion at higher concentrations (Figure 2). Like in the previous case, these values are higher for the warm temperatures (maximum standard deviation 0.007, 0.01, and 0.006 for SPI, splitless, and on-column, respectively) than for the cold temperatures (0.006, 0.003, and 0.0045, respectively). The maximum standard deviation that can be accepted in order to ensure a random error lower than the 0.0165 threshold is 0.0098 (calculated from the Student test assuming three replicate injections, standard deviation ) 0.0165(3-1/2)/t, where t ) 2.92, the tabulated statistics for two degrees of freedom). The measured standard values are always below this threshold, irrespective of the injection systems used or the relative proportion of the C37:2 and C37:3 homologues. Accordingly, dispersion effects associated with the different injection systems are not significant sources of error in paleotemperature determinations.
Figure 3. Uk′37 indexes corresponding to a purified E. huxleyi extract diluted at various ratios.
Figure 2. Uk′37 standard deviations (three replicates) of the series reported in Figure 1.
Some of these tests were also performed at the Department of Oceanography at the University of British Columbia (splitless injection). In this case, the alkenone solutions were obtained by several dilutions of a purified extract of E. huxleyi cultured at 13.9 °C. These solutions were quantified with an internal standard of n-hexatriacontane. The results also show that the Uk37 values are deviated toward warm values when the total alkenone amounts injected are lower than 5-10 ng (Figure 3). Irreversible Adsorption. The uniformity of the trend observed in all concentration plots (Figures 1 and 3) suggests that the Uk′37 increase at lower alkenone amounts can be attributed to a common phenomenon, namely irreversible adsorption in the chromatographic column. Thus, curve-fitting of the following equation,
Uk′37 ) (C37:2 - A2)/(C37:2 - A2 + C37:3 - A3)
(1)
to the average replicate values obtained in these tests gives values of 0.1 and 0.25 for A2 and A3, respectively (Figure 4). These constants are fully consistent with the curves shown in Figure 1. Thus, a portion of both ketones is irreversibly adsorbed on the capillary column, and the adsorption of the triunsaturated species is 2.5 times higher than that of the diunsaturated counterpart, leading to Uk′37 deviations toward warmer temperatures. Accordingly, these deviations should be quantitatively more important at warm temperatures, when the relative amount of C37:3 is lower. The curves found in Figures 1 and 4 define a threshold, below which the paleotemperature estimations using the Uk′37 index are unreliable. In the chromatographic system used for this test, alkenone amounts below 5-10 ng give rise to systematic
Figure 4. Changes in the Uk′37 index at decreasing total alkenone amount. Injection on column. (s) Average index of the experimental determinations. (- - -) Curve-fitted line as described in eq 1.
deviations of more than 0.5 °C. Taking 10 ng as a safe threshold and assuming a dilution ratio of 1:5 and the processing of 1 g/sample, this threshold implies that samples containing less than 50 ng/g of C37 alkenones will not be reliable for paleotemperature determination, a case that is not unusual in paleoceanographic studies. CONCLUSIONS Irreversible adsorption is a major potential source of error in the gas chromatographic determination of paleotemperatures from the analysis of the C37 alkenones. It affects both the di- and triunsaturated species and has significant effects (deviations of the temperature measurements higher than 0.5 °C) when the total amounts of ketones introduced into the system are lower than 5-10 ng. The higher adsorption of the C37:3 species gives rise to deviations of the Uk37 measurements toward higher temperatures that become more important with lower proportions of this triunsaturated species in the mixture. Adsorption also increases the dispersion of the results. Again, higher standard deviation values are observed as both the total alkenone amount and the proportion of C37:3 decrease. The different modes of injection, on-column, splitless, and SPI, do not introduce significant dispersion in comparison to the adsorption effects in the capillary column. On-column is the injection technique that provides the lowest standard deviation and drift at high concentration. Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
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ACKNOWLEDGMENT We thank Francesc Mocholi (Varian Iberica) for access to a Varian Model 3400 chromatograph equipped with an SPI system. Financial support from EEC, ENVIRONMENT Program EV5V-CT92-00117, is acknowledged. J.V. is a grateful for a Ph.D. grant from the Spanish Ministry of Education. We are thankful to the Department of Oceanography of the University
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Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
of British Columbia for their analyses of an extract of E. huxleyi diluted at various ratios. Received for review January 22, 1997. Accepted April 17, 1997.X AC9700383 X
Abstract published in Advance ACS Abstracts, July 15, 1997.
