Anal. Chem. 1995, 67, 1283-1289
Determination of the Samples
UK37
Bndex in Geological
Antoni Rosell=Mele,*James F. Carter, Adrian T. Paw, and Geoffrey Eglintont
Organic Geochemistry Unit, Environmental and Analytical Section, School of Chemistry, University of Bristol, Cantocks Close, Bristol BS8 1 TS, United Kingdom
The U%, index is a tool for sea surface paleotemperature (SST) reconstruction and is defined by the relative abundance of three long-chain alkenones (C37) commonly found in Quaternary marine sediments. A simple, fast, and reliable analytical technique is required for the analysis of these compounds in deep-sea sediment cores in order to provide time series of SST and hence contribute to the understanding of past climates. The method described here employs a robotic workstation for sample preparation and gas chromatography (GC) coupled to mass spectrometrywith ammonia chemical ionization (CIMS) to quantify the targeted analytes. This semiautomated procedure provides enhanced sensitivity, selectivity, and speed of an&sis when compared to conventional, manual sample preparation and GC flame ionization detector analysis.
Alkenones
Long-chain alkenones are a group of organic compounds that occur ubiquitously in marine sediments' (Figure 1),their presence being first observed in sediments from the Walvis Ridge, off southwestern Africa.2 Subsequently, their structure was determined (tri-and diunsaturated methyl and ethyl C37-C39 alkenones; see Table 1 and Figure 1)13 and their double bond configuration was found to be the biologically unusual E (trans).4 Long-chain alkenones and the related alkyl alkenoates Vable 1, Figure 1) are biosynthesized by several species of phytoplankton and hence, in organic geochemical terms, can be considered as biomarkers3s5 for algae of the class Prymne~iophyceae.~,~ These algae are widespread in the present day oceans, Emiliania huxleyi being the predominant species.*-1° In the early 1980s, a relationship between the relative abundances of alkenones and the
Alkyl alkenoates
t Current address: Biochemistry Centre, Department of Geology, University of Bristol. (1) Brassell, S. C. In Organic Geochemisty: Principles and Applications; Engel, M. H., Macko, S. A, Eds.; Plenum Press: New York, 1993; pp 699-738. (2) Boon, J. J.; van de Meer, F. W.; Schuyl, P.; de Leeuw, J. W.; Schenck, P. A; Burliigame, A L. In Initial Rep. Deep Sea Dn'lling Proj. 1 9 7 8 , 4 0 , pp 627637. (3) de Leeuw, J. W.; van de Meer, F. W.; Rijpstra, W. I. C.; Schenck, P. A In Advances in Organic Geochemisty 1 9 7 9 Douglas, A G., Maxwell, J. R, Eds.; Pergamon: Oxford, 1980; pp 211-217. (4) Rechka, J. A; Maxwell, J. R Org. Geochem. 1988, 13, 727-734. (5) Volkman, J. IC; Eglinton, G.; Comer, E. D. S.; Sargent, J. R In Advances in Ovganic Geochemistry 1 9 7 9 Douglas, A G., Maxwell, J. R, Eds.; Pergamon: Oxford, 1980; pp 219-227. (6) Volkman, J. IC; Eglinton, G.; Comer, E. D. S.; Forsberg, T. E. V. Phytochemistry 1980, 19, 2619-2622. (7) Marlowe, I. T.; Green, J. C.; Neal, A C.; Brassell, S. C.; Eglinton, G.; Course, P. A BY.PhycOl. 1984, 19, 203-216. (8) McIntyre, A; BC, A W. H. Deep-sea Res. 1967, 14, 561-597. (9) Okada, H.; Honjo, S. Deep-sea Res. 1973, 20, 355-374. (10) Okada, H.; McIntyre, A Microflaleontologv 1977, 23, 1-55.
0003-2700/95/0367-1283$9.00/0 0 1995 American Chemical Society
0
a
37:4Me
b
373Me
0
L
L
37:2Me
c 0
38:2Me
g 0
38:3Me
e 0
I 38:2Et 0
38:3Et
d
0
36:30Me
h
L
L
36:20Me
I
0
J
36:20Et
Figure 1. Structures and shorthand notation of the alkenones and alkyl alkenoates discussed in the text. The systematic names for these compounds are given in Table 1.
temperature of the media in which they were biosynthesized was demonstrated, both in laboratory cultures and in the open sea." Further studies gave a numerical expression to this relationship with the definition of the U57 index:l2'l3
UK37
[37:2Me] - [37:4Me] [37:3Mel [37:4Mel
= [37:2Mel
+
+
where U% stands for unsaturated ketones with 37 carbon atoms. This equation was later modified by removing the term corresponding to the 37:4Me alkenone (U57') because this compound was, at the time, rarely found in open sea marine (11) Marlowe, I. T. Lipids as palaeoclimatic indicators. Ph.D. Thesis, University of Bristol, 1984. (12) Brassell, S. C.; Eglinton, G.; Marlowe, I. T.; Waumann, U.; Sarnthein, M. Nature 1986, 320, 129-133. (13) Brassell, S. C.; Brereton, R G.; Eglinton, G.; Grimalt, J.; Liebezeit, G.; Marlowe, I. T.; Waumann, U.; Sarnthein, M. Ovg. Geochem. 1 9 8 6 , 1 0 , 6 4 9 660.
Analytical Chemistry, Vol. 67, No. 7, April 7, 7995 1283
Table I.Long-chain Alkenones and Alkyl Alkenoates in Marine Sediments Considered in rhis Work*
designation alkenones a b
shorthand notation
name
g
37:4Me 37:3Me 37:2Me 383Et 383Me 38:2Et 382Me
octatriaconta-9E,16E,23E-trien-2-one octatriaconta-16E,23E-dien-3-one octatriaconta-16E.23E-dien-2-one
526 528 530 542 542 544 544
j
3630Me 36:20Me 36:20Et
methyl hexatriaconta-7E,14E,21E-trienoate methyl hexatriaconta-14E,21E-dienoate ethyl hexatriaconta-14E,21E-dienoate
544 546 560
C
d e f
alkyl alkenoates h i
heptatriaconta-8E,15E,22E,29E-tetraen-2-one heptatriaconta-8E,15E,22E-trien-2-one heptatriaconta-15E,22E-dien-2-one
octatriaconta-9E,16E,23E-trien-3-one
544
546 548 560 560 562 562
*
* *
* *
562
564 578
E1 stands for electron impact ionization. CI-MS stands for chemical ionization mass spectrometry. Those compounds used to derive the U%7 or U57' index are indicated with an asterisk.
sediments or water column particulate~.~~J* The U%7 index is with a high organic content. Hence, they are easy to study by currently being used as a means of reconstructing sea surface the methods normally employed by organic geochemists. Howpaleotemperature~."-~~J~-~~ The stratigraphic distribution of longever, there are many locations where the organic content of the chain alkenones represented by the U%7 index was first detersediment is low and these methods prove to be inadequate. One mined for a core from the Kane Gap, off northwestern A f r i ~ a . * ~ - ~ ~of the simplest procedures for the determination of a U157 The results revealed a covariance between the U% index and stratigraphy is to ultrasonically extract the long-chain alkenones the oxygen isotope record from the same core, across several from freeze-dried sediments with organic solvents and to analyze glacial/interglacial transitions, showing that the UK37index was trimethylsilylated total extracts by gas chromatography employing effective in preserving climatic information. The use of the index a flame ionization detector (GC-FID). This method has been as a climatic proxy has been further proven by showing, for reliably applied to sediment cores from upwelling areas being instance, the coincidence between a yearly laminated 20th century studied in our laboratory (off Namibia and M a ~ r i t a n i a ) . ~ ~ ~ ~ ~ ~ ~ UK37 record (Santa Barbara basin, California) and historical However, it has proved unsuitable for the analysis of cores from measurements of El Niiio events.23 The spatial reproducibility of 50" N and 72" N in the North Atlantic, as an increase in the relative the VI57 data has also been demonstrated through the similarity abundance of long-chain alkyl alkenoates (see Table 1,Figures 1 found in the UK37records obtained from two cores drilled 20 m and 2), on moving to colder water site^,^^-^^ complicates the chromatogram, requiring longer analysis times to achieve the apart from each other off northwestern Africa.25 Whereas the widespread occurrence of long-chain alkenones in marine surficial baseline separation needed to obtain accurate peak area integrasediments has been documented for most areas of the ocean^,^^^^ tions. In addition, coelution of other compounds with the targeted alkenones further complicates the chromatogram. For instance, their stratigraphic occurrence has not been fully evaluated. The sediment cores analyzed are mainly from low latitudes and plant wax esters carried over from earlier separations and other, upwelling areas.11-13,15-26These locations are usually highly unknown compounds are potential sources of coelution. Varying sample composition presents a number of difEculties. A single productive, with high sedimentation rates producing sediments sediment core may span 1 million years, and the sediment (14) Prahl, F. G.; Wakeham, S . G. Nature 1987,320, 367-369. composition (e.g., chemical, lithological) may exhibit extreme (15) Jasper, J. P.; Gagosian, R B. Paleoceanography 1989,4, 603-614. changes due to changing climatic and sedimentary conditions. (16)Poynter, J. G. Molecular straqraphy. Ph.D. Thesis, University of Bristol, 1989. Sample quality is also highly variable. Marine sediment cores (17) Poynter, J. G.; Fammond, P.; Brassell, S. C.; Eglinton, G. In Proceedings of are often contaminated with organic material (e.g., lubricant oils the Ocean Drilling Program Leg 108; ODP College Station, TX,1989 pp from drilling devices, plasticisers from containers) due to sample 387-394. (1% Farrimond, P.; Poynter, J. G.; Egliiton, G. In Proceedings ofthe Ocean Drilling recovery and handling procedures. Unfortunately, this is true of Program Leg 112; O D P College Station, TX,1990; pp 547-553. many of the cores already collected for geological studies. Finally, (19) McCaffrey, M. A; Farrington, J. W.; Repeta, D. J. Geochim. Cosmochim. Acta in order to obtain sufficient data for climatic iiterpretation, large 1990,54, 1671-1682. (20)ten Haven, H. L.; Baas, M.; Kroot, M.; de Leeuw, J. W.; Schenck, P. A.; numbers of samples need to be analyzed, which emphasizes the Ebbing, J. Geochim. Cosmochim. Acta 1987,51, 803-810. need for a rapid and reliable analytical procedure to obtain U57 (21) ten Haven, H. L.; Kroon, D. In Proceedings ofthe Ocean Drilling Program data. .Scientzj?c results, Leg 117; O D P College Station, TX 1991; pp 445-452. (22) Lyle, M. W.; Prahl, F. G.; Sparrow, M. A.Nature 1992,355, 812-815. In this paper we describe the use of GC/MS using positive (23) Kencedy, J. A; Brassell, S. C. Nature 1992,357, 62-64. ion ammonia chemical ionization (GC-NH3CI-MS) for the rapid (24) Eglinton, G.; Bradshaw, S. A,; Rosell, A; Sarnthein, M.; Waumann, U.; characterization and quantitication of the long-chain akenones and Tiedemann, R Nature 1992,356, 423-426. (25) Zhao, M.; Rosell, A; Eglinton. G. Palaeogeogr. Palaeoclimatol. Palaeoecol. alkyl alkenoates present in extracts of marine sediments. This 1993,103, 57-65. method combines the characteristic high sensitivity and selectivity (26) Rostek, F.; Ruhland, G.; Bassinot, F. C.; Muller, P. J.; Labeyrie, L. D.; Lancelot, Y.; Bard, E. Nature 1993,364, 319-321. (27) Rosell-Mele, A Lungchain alkenones, alkyl akenoates and total pigment abundances as climatic proxy-indicators in the Northeastern Atlantic. Ph.D. Thesis, University of Bristol, 1994.
1284 Analytical Chemistty, Vol. 67, No. 7, April 1, 1995
(28) Conte, M. H.; Eglinton, G.; Madweird, L. A. S. Org. Geochem. 1992,19, 287-298. (29) Conte, M. H.; Eglinton, G. DeepSea Res. 1993,40, 1935-1961.
of selected ion monitoring (SIM) mass spectrometry and the specific reaction of ammonia with the targeted compounds in the sample. GC-NHQ-MS has already been used to quantify a range of analytes, e.g., c l e m b ~ t e r o l ,l~i ~d ~ c a i n e ,isoniazid ~~ and its trichothecene haloperidol, 34 sarin and s ~ m a n ?and ~ alkylcarbazole and benzocarbazole It has been used similarly for the characterization of triacylglyce r o l ~alkaloid , ~ ~ mixtures,38volatile terpenoid esters and phenylpropan0ids,3~cholesteryl esters:O and sulfur ~esicants.4~ The use of NH3CI-MS to measure the U%i ratio was originally suggested by Rechka and Maxwell: who analyzed synthetic alkenone standards by probe MS. However, the applicability of the technique to the determination of the U%j index in real samples was not demonstrated.
A
C
B
EXPERIMENTAL SECTION Solvents. Dichloromethane, methanol, and 2,2,4trimethylpentane @isto1 grade) were obtained from Fisons (Loughborough, Leicestershire, U.K.).Solvents were specified as suitable for geochemical hydrocarbon analysis and were assayed before use. The silylating reagent, BSTFA (bis(trimethylsily1)trifluoroacetamide), was obtained from Sigma (St. Louis, MO). Standards. The alkenone standards heptatriaconta-l5E,22Edien-2ane (37:2Me) and heptatriaconta-SE,15E,22E-trien-2ane(37: 3Me) were provided by Prof. Maxwell at Bristo14 and dissolved in 2,2,4trimethylpentane (isooctane). Sample Preparation. Samples were processed partly with a robotic workstation (BenchMate, Zymark Corp., Hopkinton, MA) before GC injection, and the different tasks are summarized below: 1. Grind freeze-dried sediment (analyst). 2. Tare tubes (workstation). The workstation tared each tube (up to 100) and remained in standby mode until instructed by the analyst to continue with the next operation. 3. Addition of sediment to the tubes (analyst). Approximately 1-2 cm3 of sediment was manually added to each tube. 4. Weigh tubes (workstation). The weight of each sample was saved in ASCII code to a floppy disk. The weights of the samples varied between 0.3 and 1.5 g. 5. Extract sediment (workstation). To each tube were added 3 mL of dichloromethane and 1 mL of methanol, and then the tube was placed in a vortex mixer at a predetermined speed for 250 s. 6. Centrifuge tubes (analyst). Twenty tubes at a time were centrifuged to facilitate the removal of the extract.
C
b c
D
b
E
d c
~
F
25
26
27
28
mins
Figure 2. Representative GC-FID traces of the eluting region of the long-chain alkenones and alkyl alkenoates at different oceanic locations. Refer to Table 1 for identification of peak designations. (A) core MD900948, 35 cmbsf, Indian Ocean 2'08' N, 76"22' E; (B) M 16789, surface sediment, Atlantic Ocean 4'19 N, 7 5 8 W; (C) M 17045, 35 cmbsf, Atlantic Ocean 52" N, 16'39' W; (D) M 17048-4, surface sediment, Atlantic Ocean 54"18 N, 1 8 " l l ' W; (E) HM 796.2,41 cmbsf, Norwegian Sea 62'58 N, 2"42 E; (F) M23262, surface sediment, Norwegian Sea 72'13' N, 14'26 E.
~~~~~~
(30) Forster, H. J.; Rominger, K. L.; Ecker, E.; Peil, H.; Wittrock, A Biomed. Environ. Mass Spectrom. 1988, 17, 417-420. (31) Karlaganis, G.;Bircher, J. Biomed. Environ. Mass Spectrom. 1987, 14,513516. (32) Karlaganis, G.; Peretti, E.; Lauterburg, B . j Chromatogr. 1987,420,171177. (33) D'Agostino, P. A; Provost, L. R; Drover, D.J Chromatogr. 1 9 8 6 , 3 6 7 , 7 7 86. (34) Szczepanik-Van Leeuwen, P. j. Chromatogr. 1985, 339, 321-330. (35) D'Agostino, P. A; Provost, L. R; Brooks, P.J Chromatogr. 1991,541,121130. (36) Dzidic, I.; Balicki, M.; Hart,H. Fuel 1 9 8 8 , 67, 1155-1159. (37) Evershed, R P.; Prescott, M. C.; Goad, L. J. Raprd Commun. Mass Spectrom. 1990,4,345-347. (38) McCoy, J. W.; Roby, M. R; Stermitz, F. R J. Nut. Prod. 1983, 46, 894900. (39) Lange, G.; Schultze, W. 0%.Mass Spectrom. 1 9 9 2 , 2 7 , 481-488. (40) Evershed, R P.; Goad, L. J. Biomed. Environ. Mass Spectrom. 1987, 14, 131-140. (41) D'Agostino, P. A; Provost, L. R J. Chromatogr. 1992, 600, 267-272.
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
1285
7. Transfer extract (workstation). A fixed amount of the supernatant was transferred into a second tube. The cannula employed in this operation was rinsed with dichloromethane/ methanol after each transfer. Steps 5-7 were repeated twice and the three extracts combined. 8. Concentrate to dryness (analyst). The tubes containing the total solvent extracts were placed in the concentrator Uurbo Vap LV, Zymark). The trays containing the tubes in the workstation were placed in a thermostatic water bath at 25 “C, and solvent was removed using a gentle nitrogen stream. 9. SPE (workstation). To the dry extract was added 1mL of dichloromethane, and the tube was vortexed for 60 s at maximum speed. The adsorbent (3 cm3 silica Bond Elut SPE column, Analytichem International, Varian, Harbour City, CA) was successively conditioned with dichloromethane (1 mL), and dichloromethane containing 1%methanol (1 mL). The sample was loaded onto the column and eluted with dichloromethane containing 1%methanol (4 mL), collecting the eluant in an empty tube. The cannula used in transferring both the solvent and the sample was rinsed with dichloromethane. 10. Step 8 was repeated. 11. Silylate total extract (analyst). To each tube was added 40 pL of BSTFA, and the tubes were then sealed and placed in a desiccator overnight. 12. Concentrate to dryness (analyst). The extracts were transferred with a Pasteur pipete to GC autosampler vials and dried (concentrator SF50 and Cole vacuum pump CVPlOO MK4, Genevac Ltd., Ipswich, England). 13. Redissolve extract and inject into GC. The dry extracts were diluted with 30-150 pL of 2,2,4-trimethylpentane, and aliquots were taken for analysis by GC-NH3CI-MS. Instrumental Analysis. Analyses were performed using a Varian 3400 gas chromatograph fitted with a septum-equipped programmable injector (SPI) directly coupled to a Finnigan MAT TSQ 70 triple stage quadrupole mass spectrometer. Suitable separation of the analytes was achieved using a 50 m, 0.32 mm i.d. column coated with 0.12 pm CPSIWCB (Chrompack, Netherlands). Hydrogen was employed as the camer gas with a head pressure of 8 psi. Aliquots of 1pL of the samples were injected by a CTC MOOS autosampler. The SPI was operated in “high performance” nonvaporizing mode, whereby the injector was held at 80°C during injection and then immediately temperature programmed from 80 to 300 “C at 200 “C min-l. The oven was programmed from 200 to 300 “C at 6 “C min-l with no initial hold time and final isothermal period of 10 min, during which the compounds of interest eluted. This gave a total analysis time of 27 min. Operating conditions for the mass spectrometer were optimized for sensitivity with respect to the pseudomolecular ions, [M NH4]+, of the C37 methyl alkenones (Table 1). The conditions used were as follows: ion source temperature, 170 “C; electron energy, 70 eV; electron current, 400 p& and electron multiplier voltage, 1500 V, with an electrometer gain of lo8. Chemical ionization was performed with high-purity ammonia (BOC, micrographic grade, London, U.K) introduced to the ion source through the conventional CI gas inlet. The pressure inside the ion source was regulated to -0.85 Torr, equivalent to a pressure of 4.5 x Torr in the vacuum manifold. Ten ions, corresponding to the [M NHJ+ adducts of the analytes (Table
+
+
1286 Analytical Chemisfry, Vol. 67, No. 7, April I , I995
l ) , were monitored by scanning the third quadrupole at a scan rate of 0.1 Da/s. The overall procedure was controlled through the TSQ70 instrument control language OCL) which controlled the autosampler, the gas chromatograph, and the mass spectrometer. This ensured that the analytical conditions were highly reproducible and allowed multiple injections for large number of samples. Once acquired, the data were processed using the mass spectrometer data system Finnigan MAT ICIS I). Integrated peak areas for selected ion chromatograms were written to an ASCII file, which was then transferred to a PC for further processing. RESULTS AND DISCUSSION
Chemical ionization can allow selective ionization of certain compound classes in a sample by the use of specific reagent gases. For marine sediment extracts, we have observed that ammonia selectively ionizes organic compounds containing heteroatoms or unsaturation. Although many gases may be employed for CI, ammonia has several advantages; for example, it has been reported that lower detection limits are achieved with ammonia as a reagent gas than when other gases are used.42 However, there are also some problems associated with ammonia CI. Both the reagent ion plasma and the sample ion spectra are very sensitive to functions in ion source conditions,42 and consequently, the reproducibility of the spectra is sometimes poor. In addition, the mechanism of sample ionization is not iully understood, and there can be marked differences in the ionization efficiency for different compound classes.42 However, this did not present a significant problem for this analysis, as the group of compounds considered are chemically similar and only their relative abundances were calculated. Figure 3 shows typical ammonia CI spectra of the three C37 alkenones (Table 1) obtained from natural samples. Figure 4 shows the mass chromatograms corresponding to the pseudomolecular ions [M NHJ+ for the compounds listed in Table 1. Identification was confirmed by relative retention time and comparison of electron ionization spectra to those reported for synthetic standards4 or for well characterized natural products.3,5.6J1,16 The characteristic CI-MS ions of the 37:3Me and 37: 2Me alkenones are identical to those reported by Reckka and Maxwell4 for synthetic standards. Ketones containing isolated carbonyl groups have lower proton affinities than ammonia,42and proton transfer from NHd+ to form [M HI+ is, therefore, not energetically favorable. However, the ammonium ion adduct is readily formed, giving rise to an intense pseudomolecular ion ([M NH*]+),42 which is the base peak for all the spectra. There are also significant ions corresponding to m/z [M NH4 11’ and m/z [M NH4 2l+. The former is probably due to the isotopic contribution of 13C and the latter to hydrogenation of the intact molecule or pseudomolecular ion in addition to the 13C2isotope peak. Attempts to identify these species by accurate mass spectrometry proved ineffective as insufficient resolution could be obtained due to the high pressure of ammonia. Weak ions corresponding to protonated [M + HI+ and nonprotonated M+ molecular ions are also evident in all the spectra (Figure 3). CI vs EI. Ammonia chemical ionization is often described as a “soft”ionization technique when compared to EI. The technique generally produces intense pseudomolecular ions with little fragmentation, as observed in the spectra of the alkenones (Figure 3). This has the effect of increasing both the sensitivity and the
+
+
+
+
+
+
+
(42) Westmore, J. E.;Alauddin, M. M. Mass Spectvom. Rev. 1986.5, 381-465.
I
b
"1
A' 37:4 Me
"1
B, 37:3 Me
lli
'I
Total ion current
I t
37:2 Me
Figure 3. Representative ammonia chemical ionization mass spectra of the C37 alkenones. Refer to Table 1 for complete compound identities.
selectivity of the analysis due to the high abundance of specilic pseudomolecular ions and the nonionization of potentially interfering components. In contrast, the electron ionization spectra of the alkenones, as described in the literature,3s5s6J1J6 show a weak molecular ion (Table 1) and numerous fragments, none of which are sufficiently specific to be useful for quantitative work. Although highly specific, the molecular ions are too weak to be monitored at the concentration range of interest (units of nanograms per microliter). EI, however, has proved useful in the characterizationof alkenones in sediments, as the spectra provide sufficient information for structural confirmation. In order to further increase sensitivity, only the pseudomolecular ions of the targeted analytes were recorded by selectedion monitoring (SIM) (Table 1). Further, a fast scan rate (0.1 Da/s) was employed in order to obtain sufficient data points to minimize errors due to peak area i n t e g r a t i ~ n . ~ ~ GC vs Probe. The sample extracts could be introduced to the mass spectrometer via a capillary column or a probe inlet.
20.0
21 .o
22.0
min.
Figure 4. Ammonia chemical ionization mass chromatograms of the total ion current and pseudomolecular ions of the alkenones and alkyl alkenoates quantified in a lipid extract from a surficial marine sediment in the Norwegian Sea. See Table 1 for compound identities.
The latter technique was briefly evaluated and subsequently discarded because of the high background signal and, especially, because of overlap in the masses of the pseudomolecular ions, e.g., the [M NH41+ ion of the 37:2Me alkenone (m/z 548) and the [M NHI 21+ ion of the 37:3Me alkenone (m/z 548). Automatic GC injection can be easily performed and the chromatographic condition optimized to achieve a quick analysis. However, it is still necessary for the alkenones to be completely resolved (Figure 4) in order to distinguish between the contributions from molecules yielding ions of the same mass. Satisfactory resolution was achieved for alkenones with dif[erent degrees of unsaturation and for the alkyl alkenoates, but not for the methyl and ethyl isomers of the C38 alkenones. However, these compounds are not included in the U57 index and hence are not strictly necessary, at present, for paleoclimatic work.
+
+
+
(43) Pettit, B.R Biomed. Mass Spectrom. 1986, 13, 473-475.
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
1287
UKv' . . . . . . . .
0.80 0.78 0.76 0.74
+
.../..................................................
. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I
0.4
0.5
0.6
0.7
0.8
0.9 100
120
Pressure (torr) Q
548dZ
140
160
180
200
Temperature ('C)
*
546dZ
*
UK,'
Figure 5. (a) Abundance of the pseudomolecular ions (left y-axis; relative units) of synthetic standards of the 37:3Me (m/z 546) and 37:2Me (m/z 548) alkenones produced in the ion source and changes in the UK37' values (right y-axis). (b) Changes in the dispersion of the results (coefficient of variation, CV) at different ammonia pressures and temperatures of the ion source.
Operational Conditions. The mass spectrometer operating conditions were optimized using 37:3Me and 37:2Me alkenone standards to give the highest yield of pseudomolecular ions, compromising with the reproducibility of the measurement of the m/z abundances and instrumental constraints. Figure 5 shows the effects of ion source temperature and pressure upon both the yields of ions and their ratio (Ufs;). In agreement with Rudewicz and Munson,u it was found that the production of ammonium adduct ions increased with increasing ammonia pressure, with a corresponding decrease in the reproducibility of the response. However, the U%' values remained approximately constant throughout the range of pressure considered (-0-1 Torr). The temperature of the ion source was found to have a critical effect on ion production (Figure 5 ) . At temperatures below 120 "C, no ions were produced. Increasing the temperature above this threshold produced an increase in ions which maximized at approximately 150 "C. As the ion source temperature was further increased to 160 "C, the ion production decreased slightly and remained constant for any further temperature increment. Across the range of temperatures examined, there was no apparent relationship between the ion source temperature and the reproducibility of the measurement of ion abundances. Using a solution (3:l) of the 37:3Me and 37:2Me alkenone standards, the method described was found to give linear quantitation over a range of concentration from 70 to 4 x lo5 pg/pL. Natural sample concentrations lie within this range. By injecting a number of increasingly diluted alkenone standards until the response was not statistically different (95%confidence) from the background noise, the detection limit for this method was (44) Rudewicz, P.; Munson, B. Anal. Chem. 1986,58,2903-2907.
1288 Analytical Chemistty, Vol. 67,No. 7,April 7, 7995
calculated to be -10 pg/pL. However, in order to achieve reproducible peak area determinations, the minimum analyte concentration should be at least 100 pg/pL. Replicate analyis of 119 samples produced an average standard variation of 0.01 for the U%' index. This reproducibility could be improved by an order of magnitude if replicate analyses were carried out sequentially as long-term instrument stability was affected by the continuous presence of ammonia in the vacuum system. The selectivity of the method was ensured as each compound was characterized by both its retention time and the mass of its pseudomolecular ion, the abundance of which was used for quantification. Effect of Alkenone Concentration on the Uy&' Index. The effect of analyte concentration was studied using standard solutions of synthetic 37:3Me and 37:2Me alkenones at different concentrations and in two ratios (2:l and 1:2 w/w 37:2Me/37: 3Me). It was observed that the value of the U%' index changed as a function of the alkenone concentration, but not the standard deviation, except at extreme concentrations (Figure 6), with a general trend that the U%; value decreased with increasing concentration of alkenone injected. However, this trend was not found to be reproducible, and the reason for these fluctuations may be irreversible adsorption of the alkenones in the chromatographic system, the 37:3Me alkenone being preferentially adsorbed in comparison to the 37:2Me. Adsorption may occur at active sites of the injector or the column or in deposits of noneluted polar compounds accumulated in the system. The triunsaturated alkenone is slightly more polar than the diunsaturated alkenone and hence more prone to being adsorbed. Therefore, if a significantly wide range of sample concentrations
0.76
k
0.74 0.72
- 2 0.70
a1 st.
f\
4
Table 2. Results of the comparison between the Two Methods (GC-FID and GC=CI-MS)Evaluated To Determine the UKsr' Index
t
FID
3 0.68 0.66 0.64 0.62
UK3j
0
UK371
0
0.789 0.692 0.468 0.408 0.241
0.002 0.001 0.001 0.002 0.001
0.800 0.665 0.456 0.375 0.231
0.010 0.011 0.009 0.013 0.011
a A series of alkenone standards (37:3Me and 37:2Me) with different relative proportions (-1:4, 1:2, 1:1, 2:1, 4:l w/w) and approximately the same concentration (-20 ng/,uL of both compounds) were used. In each case, three aliquots of 1 L were injected. The results showed that both techniques provided &e same results with a 99%confidence limit.
0.48
-,.
ammonia CI-MS
0.46
Y *
3 0.44 0.42 0.40
c
0.01
:s -0 a,
E o 0.1
1
10
100
w
lo00
obtained with the CI method. However, this is not significant for paleoclimatic reconstruction, as a change of 0.01 unit in the U%{ index corresponds to a change of only -0.3 OC in sea surface temperature.14 In addition, the calibration equation of the index with water temperature, using laboratory cultures or field samples, has a larger error, providing values with a dispersion of 1-2 oC.14,27-29,45-47
lg ng/pI (total of both alkenones)
* UK3; * STD -
a
expected UK3,'
Figure 6. UK37' values and standard deviations when standards of the synthetic alkenones at 2:l and 1 :2 (37:2Me/37:3Me) proportions are injected at different concentrations into the gas chromatograph coupled to the CI-MS system. The x-axis is logarithmic, the left y-axis is the mean of the UK3+ values, and the right y-axis is the standard deviation of UK37'.
is analyzed, systematic errors may be introduced into the U%7 index when results are compared among laboratories or transformed into temperature values using a calibration. This problem may be rectified in the future by distributing standard samples among laboratories and undertaking intercalibration exercises. GC-CI-MS Compared to GC-FID. A series of alkenone standards (37:3Me and 37:2Me) were prepared with different relative proportions (-1:4, 1:2, 1:1, 2:1, and 4 1 w/w) and approximately the same concentration (-20 ng/pL of both compounds) to compare the value of U%{ obtained by two methods (GC-FID and GC-CI-MS). In each case, three aliquots of 1pL were injected. The results showed that both techniques provided the same results with a 99%confidence limit (Table 2). Besides the higher selectivity of the CI method, the main difference between the techniques is the higher reproducibility obtained by GC-FID for the U%7) index, with an average standard deviation (obtained for more than 500 real samples, three replicates) of 0.001, an order of magnitude smaller than that (45) Prahl, F. G.; Muehlhausen, L,A; Zahnle, D. L. Geochim. Cosmochim. Acta
CONCLUSIONS
The GC-CI-MS method described has proved to be a viable alternative to conventional GGFID analysis when undertaking U%7 stratigraphicstudies. Both procedures provided comparable data, but the GC-CI-MS method offers improved selectivity, sensitivity, and speed of analysis when compared to GC analysis with FID. The GC-CI-MS technique did, however, give poorer reproducibility, but always within acceptable limits. Since alkenone concentration has been shown to affect the Uy37) index, it is advisable to work within a restricted range of alkenone concentrations, in order to have consistent U57) values for a given set of samples. A suggested optimum range of concentration is between 3 and 30 ng/component during GC analysis. The greater sensitivity of the GC-CI-MS method means that smaller samples may be extracted and that less organic rich sediments can be now analyzed. Hence, this new technique has increased the range of oceanic sitedcores which may now be studied and widened the potential of molecular stratigraphyas a tool for paleoceanographic research. ACKNOWLEDGMENT
We thank the EEC-EPOCH project @POCH-O004@DB)) for financial support and the Natural Environmental Research Council for providing mass spectrometry facilities (GR3-6619and F60-G636-10). We thank J. R Maxwell for providing alkenone standards. Received for review September 14, 1994. January 16, 1995."
Accepted
1988,52,2303-2310. (46) Sikes, E. L.; Volkman,J. K. Geochim. Cosmochim. Acta 1993,57,1883-
AC940917E
1889. (47) Sikes, E.L.;Farrington, J. W.; Keigwin, L. D.
EPSL 1991, 104, 36-47.
@
Abstract published in Advance ACS Abstracts, February 15, 1995.
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
1289
