Anal. Chem. 2005, 77, 6026-6031
Development of a Proxy for Past Surface UV-B Irradiation: A Thermally Assisted Hydrolysis and Methylation py-GC/MS Method for the Analysis of Pollen and Spores Peter Blokker,*,† Dan Yeloff,‡ Peter Boelen,† Rob A. Broekman,† and Jelte Rozema†
Faculty of Earth and Life Sciences, Department of Systems Ecology, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, and Faculty of Science, Research Group Palaeoecology and Landscape Ecology, Universiteit van Amsterdam, Kruislaan 318, 1090 GB Amsterdam
A method was developed for the analysis of the UVabsorbing sporopollenin monomers p-coumaric acid and ferulic acid in very low numbers of pollen. This enables the analysis of pollen or spores from cultured plants, from herbarium collections, and from sediment, soil, and peat cores. The method involves thermally assisted hydrolysis and methylation using tetramethylammonium hydroxide combined with gas chromatography and mass spectrometry. Pyrolysis, gas chromatographic, and mass spectrometric conditions were optimized for the analysis of minimal amounts of pollen. The method has a detection limit of ∼60 fresh pollen of Alnus glutinosa and a relative standard deviation of ∼10% between 100 and 600 pollen. Knowledge of past fluctuations in stratospheric ozone concentrations and surface UV-B irradiance is absent before 1920 when instrumental monitoring began at Arosa (Switzerland).1 The Antarctic ozone hole was first detected in 1974 based on Dobson spectrometer ozone monitoring at the Halley Research Station at the Antarctic.2 A decrease in stratospheric ozone will result in an increase in the flux of solar UV-B radiation to the lower atmosphere. A recently reported method for the reconstruction of past solar UV-B flux showed that the chemical information recorded in pollen and spores may be used to reconstruct UV-B irradiance prior to modern times.3,4 Plants have an active defense against UV radiation to protect vulnerable tissues against damage.5 Spores and pollen are protected by the biomacromolecule sporopollenin in their * To whom correspondence should be addressed. E-mail: peter.blokker@ ecology.falw.vu.nl. † Vrije Universiteit Amsterdam. ‡ Universiteit van Amsterdam. (1) Staehelin, J.; Mader, J.; Weiss, A. K.; Appenzeller, C. Phys. Chem. Earth, Parts A/B/C 2002, 27, 461-469. (2) Farman, J. C.; Gardiner, B. G.; Shanklin, J. D. Nature 1985, 315, 207-210. (3) Blokker, P.; Boelen, P.; Broekman, R. A.; Rozema, J. Plant Ecol. In press. (4) Rozema, J.; Broekman, R. A.; Blokker, P.; Meijkamp, B. B.; de Bakker, N. V.; van de Staaij, J.; van Beem, A.; Ariese, F.; Kars, S. M. J. Photochem. Photobiol., B 2001, 62, 108-117. (5) Rozema, J.; Bjo ¨rn, L. O.; Bornman, J. F.; Gaberscik, A.; Hader, D.-P.; Trost, T.; Germ, M.; Klisch, M.; Gro ¨niger, A.; Sinha, R. P. J. Photochem. Photobiol., B 2002, 66, 2-12.
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outer exine layer.5 As sporopollenin is a structure that is very resistant against chemical and microbial degradation; it preserves well in sediments and soils, and thus, a historic record of UV-B radiation is preserved. Plants from herbarium collections form an alternative source for historic UV information. By sampling small numbers of pollen for analysis, the original plants remain undamaged. A historic record of a wide variety of species covering periods of a few hundred years at high resolution may be unveiled, providing a record for the reconstruction of UV-B concentrations of the recent past. One drawback of analyzing the chemistry of fossil spores and pollen is that sufficient organic material is required for analysis. As fossil pollen and spores compose part of a heterogeneous sediment/peat matrix, laborious manual isolation under a microscope is required. Pollen and spores of taxa of intent are often in low abundance, requiring a sensitive analytical method capable of managing low amounts of analyte without the need for additional sample pretreatment. The chemical nature of sporopollenin has been the subject of debate for many years. Recent evidence suggests that the monomers p-coumaric acid (pCA) and ferulic acid (FA) compose an important part of this polymer, combined with an aliphatic constituent.3,6,7 These moieties form an intricate network of a nonhydrolyzable, and in most cases, insoluble biomacromolecular material. The latter properties greatly limit the possible techniques that can be used for the analysis of this material, since it would require laborious chemical steps prior to analysis, which is difficult when dealing with extremely small samples (e.g., Alnus pollen of diameter ∼20 µm). Bulk analytical techniques such as infrared spectroscopy, nuclear magnetic resonance, and UV/visible spectroscopy generally also require too much material to make them feasible option. Pyrolysis (py) techniques are particularly useful for the analysis of small amounts of organic macromolecular material, which is (6) Wierman, R.; Ahlers, F.; Schmitz-Thom, I. In Biopolymers; Hofrichter, M., Steinbu ¨ chel, A., Eds.; Wiley-VCH: Weinheim, 2001; pp 209-227. (7) van Bergen, P.; Blokker, P.; Collinson, M. E.; Sinninghe Damste´, J. S.; de Leeuw, J. W. In Evolution in Plant Physiology; Hemsley, A. R., Poole, I., Eds.; Elsevier: Amsterdam, 2004; Vol. 21, pp 133-154. 10.1021/ac050696k CCC: $30.25
© 2005 American Chemical Society Published on Web 08/19/2005
Figure 1. (a) SEM of fresh A. glutinosa pollen. (b) 50 Alnus pollen in pyrolysis quartz liner. Photographs used for quantification of pollen.
heated in an inert environment generating small volatile fragments.8 These fragments can be introduced into a gas chromatograph coupled to a detector of choice. Since the pyrolysis of natural material often generates a myriad of compounds resulting in coelution, a mass spectrometer is often preferred as a detector, enabling deconvolution, selectivity, and sensitivity. Pyrolytic heating of organic samples results in a wide variety of thermal reactions. In the case of the compounds of interest for UV-B research, pCA and FA, this will result in decarboxylation. Decarboxylated fragments can originate from more sources than pCA and FA.3 Furthermore, secondary reactions will generate more fragments, resulting in a decrease in sensitivity due to chemical noise. The use of thermally assisted hydrolysis and methylation (TMH) reagents will decrease secondary pyrolysis reactions and increase the yield of the analyte. There is evidence to suggest that tetramethylammonium hydroxide (TMAH) is the best reagent of choice for this purpose.9, 10 In this paper, we describe the issues involved in the development of a method for the analysis of low amounts of pollen and spores using THM-py-GC/MS, for the purpose of obtaining past information on UV-B fluctuations recorded in fossil pollen and spores and in other small organic remains. RESULTS AND DISCUSSION Placement of the Sample in the Pyrolysis Unit. For the setup of the analysis method, a pyrolysis unit was chosen that utilized pyrolysis containers that enabled exact counting of the pollen/spore sample inside (Figure 1). It was observed that pollen counting inside the pyrolysis container itself afforded the most accurate analysis, since the loss of individual sample entities was noticed to occur upon manipulation. Since low numbers of pollen or spores are used, loss of for example a few pollen will greatly influence the analytical sensitivity and reproducibility. For the analysis of pollen, spores, and other organic remnants such as seeds, the samples were placed inside quartz pyrolysis tubes with an internal diameter of 0.9 mm. For optimization of the pyrolysisGC/MS system fresh Alnus glutinosa pollen were used. Alnus pollen are useful for this purpose as they are easily recognizable under the microscope and have an intermediate size (Figure 1) (8) Wampler, T. P. J. Anal. Appl. Pyrolysis 1989, 16, 291-322. (9) Challinor, J. M. J. Anal. Appl. Pyrolysis 2001, 61, 3-34. (10) Lehtonen, T.; Peuravuori, J.; Pihlaja, K. J. Anal. Appl. Pyrolysis 2003, 6869, 315-329.
relative to the grains of other taxa. Inserting the Alnus pollen into the pyrolysis tubes was done using a micromanipulator (Leica microsystems), carefully avoiding spreading of the pollen grains throughout the tube. A small as possible patch of grains avoids the unequal or incomplete wetting of the grains with reagent. Since in practice it is impossible to homogeneously cover all pollen in the pyrolysis tubes with an exact amount of TMAH, an excess is used to ensure complete wetting and complete conversion of the analytes to their methylated analogues. An incubation time of 20 min at room temperature followed by 2 h at 70 °C was observed to suffice for complete penetration of the methanolic reagent solution into the samples. Split or Splitless Mode. Considering the internal volume of the pyrolysis chamber and length of the transfer lines (∼45 cm), ∼500 µL of He gas is needed to transport the pyrolysis products to the GC injector. At instantaneous pyrolysis, it will take ∼30 s at 1 mL/min gas flow to transfer the products to the GC injector. Running in split mode will decrease the amount of analyte entering the GC, but will increase the transfer speed and efficiency, due to the lower residence time of the analytes in the pyrolysis unit. The optimum split ratio for the pyrolysis system was tested with fresh Alnus pollen. Figure 2 illustrates that the most efficient sample transfer takes place at a split ratio of >10. Although sample loss occurs in splitless mode, the absolute amount of material transferred onto the GC column is still higher than in split mode, which is preferred for the lower detection limit and thus will have a smaller sample requirement. Running in splitless mode will result in increased memory effects, since the sample transfer speed toward the GC column will allow more contact time of the
Figure 2. Split ratio corrected pCA and FA signals vs split ratio, illustrating the incomplete transfer of analytes at lower spit ratios.
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Figure 3. pCA signal vs pyrolysis temperature.
analytes with the pyrolysis chamber and the transfer lines. The memory effect due to the long residence time of the analytes in the pyrolysis system was shown by running a blank containing TMAH only, which shows that about 5-10% p-coumaric acid was retained when 1000 or more Alnus pollen were analyzed. A blank run containing only the THM reagent TMAH following a blank TMAH run at high split (200:1) did not shown any remaining pCA or FA and thus suffices to clean the system. Other precautions avoiding memory effects involved a high interface temperature (280 °C) above the elution temperature of methylated pCA (178 °C) and FA (200 °C) on the HP-5 MS column and a 20-s postrun cleaning step at 1200 °C to the vent of the pyrolysis system. Pyrolysis Temperature and Ramping Rate. Pyrolysis temperatures and ramping rates were tested. It was observed that there was almost doubling of the analyte signal with a temperature increase from 300 to 500 °C, while further increase of the pyrolysis temperature to 700 °C resulted in a leveling off of the signal, with a consequent improvement of ∼10% compared to 500 °C (Figure 3). Using higher temperatures will decrease the lifetime of the pyrolysis filament and furthermore result in higher fragmentation rates upon pyrolysis. Slow or fast ramping of the pyrolysis temperature did not significantly increase the pyrolysis yield. However, qualitatively, the analysis with lower ramping rates showed a slight increase in chemical background noise due to the thermal generation of various side products. Slow ramping rates allow the compounds in the sample to react prior to thermal cracking and vaporization; therefore, the fastest heating regimes have often been chosen in previous studies.11 Pyrolysis systems in which the sample is placed on a Curie point or resistively heated wire have major advantages over pyrolysis system involving glass sample holder, since the heat transfer to samples is more direct and thus faster. However, in practice, no significant difference is observed between filament and Curie point systems when maximum heating rates are used for the former.12 Nevertheless, Curie point systems often do not allow an accurate quantification and placement of microscopic samples and are therefore not practically usable for the purpose of quantifying UV-B-absorbing compounds in very small amounts of pollen and spores. MS Conditions. The three most intense mass fragments were tested for optimal sensitivity and selectivity (see Figure 4 for the (11) Irwin, W. J. In Treatise on Analytical Chemistry, Part 1; Winefordner, J. D., Kolthoff, I. M., Elving, P. J., Eds.; Wiley: New York, 1993; Vol. 13, p 309. (12) Stankiewicz, B. A.; van Bergen, P. F.; Smith, M. B.; Carter, J. F.; Briggs, D. E. G.; Evershed, R. P. J. Anal. Appl. Pyrolysis 1998, 45, 133-151.
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Figure 4. Mass spectra of pCA and FA.
Figure 5. Peak-to-peak signal-to-noise ratio vs pollen number.
mass spectra of methylated pCA and FA). The molecular ions m/z 192 and 222 were found to give the lowest background and highest signal for pCA and FA, respectively. To further improve the sensitivity of the method, pCA and FA were analyzed in selective ion monitoring mode (SIM) at a dwell times of 800 ms, which was shown to increase the sensitivity without increasing the background noise. The dwell time of 800 ms (1.22 cycles/s) still gives more that 5 scans/peak, allowing accurate peak integration. Detection Limit. Applying the conditions and settings described above, the detection limit of the method was tested with fresh Alnus pollen. A series with a range from 50 to 1100 pollen grains was counted under the microsope and analyzed for the presence of pCA and FA. The largest source of error in the analysis was found empirically to be the pollen number, especially at higher counts. Alnus pollen tended to aggregate together at numbers higher than ∼400, which complicates counting under the microscope. Even in SIM mode, the background noise was high. Figure 5 shows the peak-to-peak signal-to-noise ratio of FA. Increasing the sample size results in a flattening out of the S/N ratio above ∼500 pollen, after which the chemical noise becomes more significant. Applying the described conditions, a S/N ratio better than 100 for FA is not feasible for fresh Alnus pollen. Based on the S/N ratios, the detection limit of pCA and FA in Alnus pollen with the methodology described above is 57 and 12 pollen, respectively (Table 1). However, owing to relatively large
Table 1. Detection Limits (Alnus Pollen Numbers) LOD S/N ) 3 RMSE
pCA 57 101
Table 2 pollen no.
pCA (counts)
FA (counts)
ratio
229 114 303 606 588
122 469 53 810 138 946 260 585 224 738
109 810 52 419 109 475 273 517 255 266
FA 12 253
average RSDa (%) a
Figure 6. Method linearity for the analysis of pCA and FA within the range of ∼50-∼1000 Alnus pollen.
errors in pollen counts and variance in pyrolysis efficiencies due to differences of the spread and placement of sample and reagent in the pyrolysis tubes, the RMSE method described by the EPA13,14 provides more realistic numbers of 101 and 253 pollen for pCA and FA, respectively. The method provided a good linearity from detection limit to ∼1000 Alnus pollen (Figure 6). Ratio versus Pollen Number. UV-B radiation stimulates the phenyl propanoid pathway (PPP) key enzyme phenylalanine lyase in plants.4,15 Not all products of the PPP are affected in a similar fashion; thus, the ratio between these plant metabolites varied at different levels of UV-B radiation.3 In view of the fact that the largest error in the method described here originates from counting of the pollen prior to analysis, the use of compound ratios may increase the accuracy of the method. Furthermore, it is expected that some variation in the method will be due to the effect of discrepancies in pyrolysis efficiencies, which would affect both pCA and FA. The use of pCA/FA ratios was tested by conducting five repeat measurements using 114-606 fresh Alnus pollen. Table 2 shows covariance in the signal of pCA and FA, as depicted by the RSD of the ratio, which is in the same range as that of the individual analytes. Though using the ratio between pCA and FA does not improve reproducibility, it does not require accurate counting of the pollen or spores. It must be noted that the RSDs will decrease if the spread in pollen number is smaller than that used in this experiment. Quantitation of pCA and FA. Due to the chemical complexity of sporopollenin and the wide variety of intermolecular linkages,3,6,16,17 it is unlikely that all the polymerized pCA or FA will be released as intact monomers upon THM pyrolysis. The wide variety of intermolecular linkages will result in various pyrolysis fragments in addition to pCA and FA. Different degrees of polymerization or linkage types will result in varying amounts of (13) Corley, J. In Handbook of Residue Analytical Methods for Agrochemicals; Lee, P. W., Aizawa, H., Barefoot, A. C., Murphy, J. J., Eds.; John Wiley & Sons Ltd.: London, 2003; Vol. 2, p 1552. (14) US-EPA. http://www.epa.gov/pesticides/trac/science/trac3b012.pdf, 2000. (15) Rozema, J.; van de Staaij, J.; Bjo ¨rn, L. O.; Caldwell, M. Trends Ecol. Evolution 1997, 12, 22-28. (16) Boom, A., University of Amsterdam, Amsterdam, 2004. (17) Ahlers, F.; Thom, I.; Lambert, J.; Kuckuk, R.; Wiermann, R. Phytochemistry 1999, 50, 1095-1098.
pCA/ pollen
FA/ pollen
1,1 1,0 1,3 1,0 0,9
534 472 459 430 382
478 460 361 451 434
1.0 14
456 12
437 10
RSD, relative standard deviation.
pCA and FA estimated by THM-py-GC/MS analysis. As a consequence, it is hard to determine whether increased UV-B will directly affect the concentration of these monomers or the mode of polymerization. During analysis of fresh Alnus pollen, the amount of pCA and FA that is released upon THM pyrolysis is 1.0 and 2.5 pg/pollen, respectively. Although the pyrolytic release of pCA and FA will not be complete, variation in the chemical composition of sporopollenin due to UV differences may be recorded using this method. To illustrate the complexity of sporopollenin, an acetolyzed sample of ∼5000 A. glutinosa pollen was subjected to a full-scan (m/z 50-500) THM-py-GC/MS analysis (Figure 7). Acetolysis is a standard chemical purification method in palynology (the study of fossil pollen and spores as an aid to reconstruct past vegetation and climates), to remove organic debris18 and isolate pollen and spores prior to counting (see below). The total ion current of the acetolyzed Alnus pollen shows that indeed pCA and FA are important compounds in the resistant residue, next to a wide variety of aromatic compounds, long-chain fatty acids and various compounds that cannot be identified with standard MS databases. Earlier reports suggest that sporopollenin contains both a pCA/ FA-based aromatic component and an aliphatic part.3,6,7 Though the long-chain fatty acids in the pyrolysate do suggest an aliphatic origin, the linkages that would have connected this hydrolysisresistant material together remain unclear. Furthermore, it is uncertain if acetolysis would remove all soluble and hydrolyzable pollen content completely. Though the samples were handled with great care, the presence of hexadecanoic acid, octadecanoic acid, and squalene in Alnus pollen (Figure 7) could also indicate contamination of the sample by finger oil. For these reasons, it is hard to establish which compounds besides pCA and FA in the pyrogram really originate from a hydrolysis-resistant material. Sample Workup. As noted above, acetolysis is widely used to release and purify pollen and spores from soils and peat cores.18 However, the effect of such a treatment on the chemistry of such isolated material is unknown. Therefore, we subjected A. glutinosa pollen to a standard acetolysis procedure to test the effect of the treatment on the sporopollenin. Figure 8 shows that acetolysis drastically decreases the pCA and FA signal when compared to the fresh material. This decrease could be due to the removal of soluble pCA and FA or partial destruction of the sporopollenin structure. The observation that doubling the acetolysis time from 10 to 20 min will further decrease pCA and FA suggests that acetolysis should be used with caution. Control of the reaction (18) Erdtman, G. Svensk Botanisk Tidskrift 1960, 54, 561-564.
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Figure 7. Total ion current (m/z 50-500) of ∼5000 acetolyzed Alnus pollen.
Figure 8. Abundance/pollen of untreated, 10-min acetolyzed, 20min acetolyzed, and KOH-treated Alnus pollen. Figure 10. Partial total ion chromatogram (m/z 50-500) of a single fossil Juncus seed. (a) pCA; (b) FA. Open circles: fatty acid methyl esters (Cn ) carbon number). Closed circles: dicarboxylic acid methyl esters (Cn ) carbon number).
Figure 9. Integrated peak area of p-coumaric acid and ferulic acid divided by the number of pollen (between 44 and 68) of unacetolyzed V. faba. Error bars represent the standard deviation.
time is of paramount importance, since small variations have a significant effect on the analyte signal. Another standard method used in palynology is the simmering of samples in KOH, which is necessary to disaggregate organic samples. This method also reduces the pCA and FA signals, though to the same level as a 20-min acetolysis treatment, which suggests that this is the signal corresponding to a hydrolysis-resistant biomacromolecular network. More important than the possible error introduced by the chemical treatment involved in purification of fossil pollen and spores from cores is the fact that lowering the pCA and FA signal will obviously elevate the detection limit 6- or 7-fold. As a 6030 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
consequence, to obtain a reliable analysis, 1500-1800 of acetolyzed Alnus pollen have to be collected. Use of the ratio pCA/FA will avoid the error involved in counting the pollen. However, it is practically not feasible to isolate that amount of pollen or spores from a core sample. This suggests that an alternative purification method has to be developed for the analysis of fossil pollen and spores. However, when sampling material from herbarium collections, there is no requirement for sample workup, and analyses of such pollen will provide information about more recent changes in UV-B irradiance. Figure 9 shows the THM-py-GC/MS analysis of fresh pollen of Vicia faba, which was grown under high- and low-UV-B conditions, illustrating that FA and pCA are increased under high-UV-B conditions. Furthermore, pCA shows a stronger increase than FA due to UV-B-induced changes in the macromolecular composition or structure resulting in an increase in the pCA/FA ratio. Juncus effusus Seeds. To illustrate that other small fossil samples apart from pollen can be analyzed using the settings above, we used a single J. effusus subfossil seed coat from a core taken from a peat bog on Eysturoy in the Faroe Islands. The amount of organic mass per seed coat (∼0.5 × ∼0.25 mm) was enough to run in full-scan mode (m/z 50-500). This revealed that seeds also contain the UV-absorbing compounds pCA and FA, next to a myriad of other pyrolysis products (Figure 10).
CONCLUSIONS A method for the analysis of the UV-B-absorbing compounds pCA and FA in small numbers of pollen, spores, and other microscopic entities was developed. The analysis results may be expressed as amount of signal/pollen or as a pCA/FA ratio since UV-B radiation seems to affect the ratio between these sporopollenin building blocks in a different fashion. The method involves a THM-py-GC/MS technique using TMAH as a reagent. Use of quartz pyrolysis tubes enables accurate counting of the number of analyte entities prior to analysis. Running the injector of the GC in splitless mode, combined with the SIM mode of the MS operated at longer dwell times, improves the sensitivity of the method. The development of an analytical method for the analysis of pCA and FA in pollen, spores, and seeds will allow research toward the effect of increased UV-B radiation on plants and the development of a proxy to reconstruct variations in past irradiance of solar UV-B. METHODS AND MATERIALS Materials. A 25% methanolic solution of tetramethylammonium hydroxide (75-59-2), p-coumaric acid (95C-0371), and ferulic acid (1135-24-6) were obtained from Sigma Aldrich. Quartz pyrolysis tubes (10A1-3015) and filler rods (10A1-3016L) were obtained from CDS International. The pollen of A. glutinosa were collected in February 2003 from a local garden tree in Middenbeemster, The Netherlands. J. effusus seeds were taken from a peat core from Eysturoy in the Faroer islands (6°54′W, 62°10′N) (manuscript in preparation). V. faba was cultured under ambient and high-UV-B irradiation (biologically active radiation; 10 kJ‚m-2‚day-1, representing ∼30% ozone depletion) as earlier reported in ref 19. The other pollen and spore materials were collected in spring at various places in the north of The Netherlands. Sample Preparation. Pollen were suspended in demineralized water, collected using a Leica Microsystems micromanipulator, and transferred into quartz pyrolysis (CDS Analytical Inc.) tubes containing quartz filler rods (CDS Analytical Inc.). The pollen were concentrated in a small area of the tube, and after drying at 70 °C (19) Meijkamp, B. B.; Doodeman, G.; Rozema, J. Plant Ecol. 2001, 154, 135156.
in an oven, 2 µL of TMAH was added carefully ensuring complete wetting of the pollen patch. The sample was allowed to incubate for 20 min at room temperature followed by 2 h at 70 °C. J. effusus seeds were removed from the peat core without chemical pretreatment, placed in the pyrolysis tubes, covered with 5 µL of TMAH, and allowed to incubate for 20 min at room temperature followed by 2 h at 70 °C prior to analysis. Pyrolysis and GC/MS Conditions. After incubation, the samples were pyrolyzed at 700 °C at maximum ramp speed for 5 min in a CDS AS-2500 pyrolysis unit (CDS Analytical Inc.) (280 °C interface temperature) coupled to an Agilent 6890 GC equipped with an Agilent 5973 MSD. The GC oven program used was similar to standard full-scan pyrolysis runs used for routine analysis: the GC oven was programmed from 40 (6-min hold time) to 130 °C at 15 °C/min followed by 250 °C at 8 °C/min, and subsequently to 320 °C at 15 °C/min followed by 1.5 min isothermal at 320 °C. A HP5-MS (30m × 0.25 mm × 0.25 µm) capillary GC column was used applying He as a carrier gas at a constant flow of 1.2 mL/min. in splitless mode. The mass spectrometer was operated in full-scan mode (50-500 m/z) at 70eV ionization energy or in SIM mode scanning m/z 192 (16-19 min) and 220 (19-22 min) (800 m/s dwell time, 1.22 scans/s). Solvent delay was 6 min. The retention time of pCA was 18.0 min and FA 20.5 min. Chemical Degradation. A standard acetolysis procedure was performed18 using reaction times of 10 and 20 min. KOH treatment was conducted by simmering ∼100 mg of pollen in 5 mL of an aqueous KOH solution (10% m/v, 90 °C) for 1 h. Acetolysis and KOH samples were thoroughly washed five times with demineralized water, prior to placement in the pyrolysis liners. ACKNOWLEDGMENT The research of P.B. is funded by an ALW-NAAP grant (851.20.010). The research of P.B. and D.Y. was funded by a CLIVAR grant (854.00.004).
Received for review April 22, 2005. Accepted July 20, 2005. AC050696K
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