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Vibrational biospectroscopy: what can we say about the surface wax layer of Norway spruce needles?
Journal of Molecular Structure 565±566 (2001) 305±310
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Vibrational biospectroscopy: what can we say about the surface wax layer of Norway spruce needles? P. MateÏjka a, L. PlesÏerova a, G. BudõÂnova a, K. HavõÂrÏova a, X. Mulet b,1, F. SkaÂcel c, K. Volka a,* a
Department of Analytical Chemistry, Institute of Chemical Technology, Technicka 5, Prague 6, CZ 166 28, Czech Republic b Imperial College of Technology and Medicine, London SW7 2AZ, UK c Department of Gas, Coal and Air Protection, Institute of Chemical Technology, Technicka 5, Prague 6, CZ 166 28, Czech Republic Received 31 August 2000; revised 22 November 2000; accepted 22 November 2000
Abstract NIR-excited FT-Raman spectra of the Norway spruce (Picea abies (L.) Karst.) needles were measured by microscopic and macroscopic techniques. From the NIR micro-Raman spectra of the stomatal and interstomatal area, the spectrum of the epistomatal wax was extracted. The effect of the cellular substrate of the needles on this spectrum is shown and its variability demonstrated by NIR macro-Raman spectra. Strong variability in the spectra of the spruce needles is observed even for needles of the same age from the same branch. One of the dominant features is variation in intensity of the carotenoid bands. q 2001 Elsevier Science B.V. All rights reserved. Keywords: NIR FT-Raman spectroscopy; Microspectroscopy; Spruce needles; Stomatal wax; Chemometrics
1. Introduction The surface of the spruce needle is covered by a cuticle (1±5 mm thick) consisting of two elements, the cutin (a polyester built up by di- and trihydroxy fatty acids) and cuticular waxes (a complex mixture of lipids, i.e. long-chain secondary alcohols, diols and free fatty acids as major classes, n-alkanes, n-alkenes, primary alcohols, a,v-diols, ketones and v-hydroxyacids as minor classes) [1,2]. Waxes deposited onto the outer surface of the cuticle are denoted as * Corresponding author. Tel.: 1420-2-2435-4056; fax: 1420-23112828. E-mail address: [email protected] (K. Volka). 1 IAESTE student at the Department of Analytical Chemistry in Summer 2000.
epicuticular wax, waxes within the inner cutin layer as intracuticular wax. Epicuticular wax occurring in the stomatal entrances is denoted as epistomatal wax. The morphology of the epistomatal wax is very characteristic and changes dramatically under natural and anthropogenic factors. The tubular forms are with increasing environmental stress transformed into the melted, tabular ones. The morphology of the wax is evaluated for a de®ned number of stomas by numerical values between 1.0 (unaffected) and 5.0 (heavily affected) called wax quality. The micro-morphology of the stomatal wax now serves as a biomarker of air pollution [1]. These morphological changes are strongly connected with chemical changes and this has been the reason why we were interested in the potential of NIR-excited Raman (NIR Raman hereafter) spectroscopy as an alternative to scanning
0022-2860/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0022-286 0(00)00929-7
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P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
electron microscopy, which is rather dif®cult to use and also non-speci®c. Our preliminary experiments showed some signi®cant differences in the NIR Raman spectra of the Norway spruce (Picea abies (L.) Karst.) needles sampled in two different localities of the Czech Republic [3]. This work highlights some dif®cult points in NIR Raman spectroscopic evaluation of the epicuticular wax and also in the quanti®cation of the method. 2. Experimental A branch of Norway spruce was taken in the Velke Losenice area (Czech Republic) in December 1998. Individual twigs and separated needles were packed in aluminum foil and then put into polyethylene bags. The bags were placed in desiccators and stored in a refrigerator (at ca. 48C). The extracts of the spruce needles were prepared by two methods. In method I, spruce needles in a mixture of chloroform±acetone (1:1 vol.) were mechanically homogenized for 5 min (Turax) and then extracted under soni®cation for 5 min. 2,6-Di-t-butyl-4-methylphenol (0.1 wt.%) and calcium carbonate were added. In method II, an extract in a hexane±acetone mixture (3:2 vol.) was prepared from the whole needles by 5min soni®cation. Solvents were evaporated at 508C before measurement of the spectra. NIR Raman spectra were collected using a Fourier transform near-infrared spectrometer Equinox 55/S (Bruker) equipped with FT Raman module FRA 106/S (Bruker). In the case of macro-measurement, a needle placed vertically in an adapted holder was irradiated ca. 2± 3 mm from its tip by the focused laser beam with a power 50 mW of Nd±YAG laser (1064 nm, Coherent). Both top and bottom sides of a needle were examined at the same distance from the tip by turning the sample holder, i.e. four spectra were obtained per needle. The scattered light was collected in back-scattering geometry. 1024 interferograms were co-added and processed to obtain Raman spectra with 4 cm 21 resolution. In the case of the micro-experiment, a needle was placed on a movable table of microscope Ð Ramanscopee (Bruker) equipped with Nikon objectives (10 £ , 20 £ , 40 £ , 100 £ ). The image of a needle
was monitored by color CCD camera (Sony) connected to a computer that made it possible to save the visual micro-images. Three pairs of stomatal and interstomatal points were selected on a needle (ca. 2 mm from a tip, in the middle, and ca. 2 mm from the base of the needle). The excitation radiation (laser power less than 30 mW) was supplied by optical ®ber (diameter 5 mm). The 40 £ objective was used for Raman measurement. Two times 1024 scans were collected at each position of the needle to verify the repeatability of data collection. In sum, 12 Raman spectra with 4 cm 21 resolution were obtained per needle. The software opus 2.3 (Bruker) was used to control the spectrometer and to process the spectra obtained using ªrubber bandº baseline (100 points), Min/Max and vector normalization. Chemometric evaluation of the data using principal component analysis (PCA) and the soft-independent modeling of class analogy (SIMCA) were done by unscrambler 7.5 (CAMO, Norway). 3. Results and discussion The dif®culty of a measurement of the NIR Raman spectrum of the epicuticular wax is given by its very non-uniform distribution on the needle and low content. The main portion of the wax is located in the stoma of a diameter of several tens of micrometers. Outside of the stomatal areas, this type of coverage disappears; only in rare cases did we observe a visible structure beyond the boundary of about 1.5 m diameter of the stoma. The needles are covered with stomata in about three parallel lines on both sides, but there are many irregularities in their distribution. The content of the wax can be estimated by its extraction and amounts from 0.5 to over 2% dry weight of the needles [1]. In NIR macro-Raman measurement, the diameter of the focused laser beam is about 0.8 mm. So NIR micro-Raman measurement is needed to gain information about the epistomatal wax only. The diameter of the focused laser beam on a ¯at surface is about 5 mm. The combination of visual microscopy with NIR Raman microscopy is substantial: there are many microscopic objects of a comparable size on the surface, which can make the microscopic
P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
measurement quite erratic. They are inorganic powder, pollen, fungi, etc. NIR micro-Raman spectra of the stomatal (spectrum A) and interstomatal area (spectrum B) are given in Fig. 1 along with their difference (spectrum C). Two ªnegativeª bands at about 1530 and 1160 cm 21 correspond to carotenoids [4]. The NIR excitation penetrates through the epicuticular wax to the cellular substrate, so the cellular substrate is also excited along with the wax. Lower intensity of the carotenoid bands in the
307
stomatal areas in comparison with interstomatal ones can be easily elucidated by a shielding of the cellular substrate by the wax in the areas of stoma. The well-preserved epistomatal wax structures strongly scatter the light and so attenuate the excitation and emitted radiation very effectively. The NIR Raman spectra of stomatal wax alone is characterized ®rst of all by the bands corresponding to aliphatic moiety (bands at 2882, 2845 and about 1456 cm 21, Fig. 1C). The difference spectrum (Fig. 1C) can be compared with the NIR Raman
Fig. 1. NIR micro-Raman spectra of a Norway spruce needle: A ± stomatal area; B ± interstomatal area; C ± difference of spectrum A minus spectrum B.
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P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
spectra (macro-measurement) of spruce needles (Fig. 2A) and of two extracts into chloroform±acetone (method I) (Fig. 2B) and/or hexane±acetone (method II) (Fig. 2C) showing the highest similarity of spectra 1C and 2C. The carotenoid bands (1530 and 1160 cm 21) are missing in spectrum 2C suggesting that the carotenoids are not extracted using method II. But they are observed in spectrum 2B even with a comparable intensity to the spectrum of the whole needle (Fig. 2A). We conclude that method II extracts stomatal wax selectively while the extract of method I also includes subcuticular components.
The crucial question connected with direct measurement of the spruce needles (Fig. 2A) is the variability of the spectrum of the cellular substrate. Our estimation of the surface coverage of a needle by epistomatal wax is only about 2±3% and so a basic piece of information about the variability of the spectrum of the cellular substrate can be obtained by NIR macro-Raman measurement of the needle. The 404 spectra of the needles of nine 1-year, two 2-year and one 3-year-old twigs were measured. The evaluation of the similarity of the spectra in the range 3050±2650 and 1800±720 cm 21 is based on the
Fig. 2. NIR Raman spectra (macro) of a Norway spruce needle (A) and extracts of needles in chloroform±acetone mixture (method I, spectrum B) and hexane±acetone mixture (method II, spectrum C).
P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
No manipulation of initial data
1 2 3 4 5 6 8 9 10 12 13 14
1 1 2 2 3 3 2 2 2 2 5 9 5
2 3 4 2 2 3 1 2 2 2 1 3 2 3 1 2 2 2 2 2 2 5 4 6 3 3 3 3 3 3 7 11 5 7 12 4 4 4 3
5 3 2 2 2 1 2 4 3 3 9 9 5
6 8 9 2 2 2 2 5 3 2 4 3 2 6 3 2 4 3 1 5 2 5 1 3 2 3 1 2 3 2 5 18 6 7 18 8 4 9 5
Baseline corrected initial data
10 12 13 14 2 5 9 5 3 7 7 4 3 11 12 4 3 5 4 3 3 9 9 5 2 5 7 4 3 18 18 9 2 6 8 5 1 6 7 3 6 1 4 4 7 4 1 4 3 4 4 1
1 1 2 2 5 3 3 20 2 4 3 7 5
MinMax normalization of initial data
1 2 3 4 5 6 8 9 10 12 13 14
1 1 2 3 4 5 3 3 1 2 4 7 5
2 2 1 2 3 3 2 6 3 2 6 6 4
3 3 2 1 3 4 2 5 3 3 8 9 4
4 4 3 3 1 2 2 9 5 3 5 4 3
5 5 3 4 2 1 2 8 5 3 7 6 3
6 8 9 3 3 1 2 6 3 2 5 3 2 9 5 2 8 5 1 5 3 5 1 3 3 3 1 2 3 2 4 7 5 5 10 6 3 10 6
1 1 2 3 3 4 2 2 2 3 6 8 4
10 12 13 14 2 4 7 5 2 6 6 4 3 8 9 4 3 5 4 3 3 7 6 3 2 4 5 3 3 7 10 10 2 5 6 6 1 5 5 4 5 1 3 5 5 3 1 4 4 5 4 1
2 3 4 5 6 8 9 10 12 13 14 2 3 3 4 2 2 2 3 6 8 4 1 2 2 2 2 3 2 3 8 9 3 2 1 4 4 3 4 4 5 12 13 4 2 4 1 2 2 4 3 3 9 8 3 2 4 2 1 2 4 4 4 12 9 4 2 3 2 2 1 3 2 3 6 7 4 3 4 4 4 3 1 2 2 8 11 5 2 4 3 4 2 2 1 3 7 10 4 3 5 3 4 3 2 3 1 8 8 5 8 12 9 12 6 8 7 8 1 4 9 9 13 8 9 7 11 10 8 4 1 6 3 4 3 4 4 5 4 5 9 6 1
2 2 1 3 2 2 2 22 3 4 4 5 3
3 2 3 1 4 2 2 22 2 5 4 8 5
4 5 2 4 1 2 3 31 4 5 5 5 3
5 3 2 2 2 1 2 24 3 4 5 6 3
6 3 2 2 3 2 1 25 2 4 4 6 3
8 20 22 22 31 24 25 1 26 3 15 36 11
9 2 3 2 4 3 2 26 1 4 3 6 5
10 12 13 14 4 3 7 5 4 4 5 3 5 4 8 5 5 5 5 3 4 5 6 3 4 4 6 3 3 15 36 11 4 3 6 5 1 5 7 4 5 1 4 4 7 4 1 3 4 4 3 1
5 6 8 9 3 2 14 2 2 2 23 3 3 2 16 3 2 2 16 4 1 2 7 5 2 1 17 3 7 17 1 19 5 3 19 1 3 3 4 4 4 3 11 4 5 5 30 5 4 3 14 6
10 12 13 14 5 3 5 4 6 4 4 5 5 4 5 4 4 5 5 3 3 4 5 4 3 3 5 3 4 11 30 14 4 4 5 6 1 5 7 5 5 1 3 4 7 3 1 4 5 4 4 1
MinMax normalization of baseline corrected data 1 1 2 2 3 3 2 14 2 5 3 5 4
Vector normalization of baseline initial data
1 2 3 4 5 6 8 9 10 12 13 14
309
2 2 1 2 2 2 2 23 3 6 4 4 5
3 2 2 1 3 3 2 16 3 5 4 5 4
4 3 2 3 1 2 2 16 4 4 5 5 3
Vector normalization of baseline corrected data
1 2 3 4 5 6 8 9 10 12 13 14
1 1 2 2 3 3 2 6 2 3 3 5 4
2 2 1 2 2 2 2 8 3 4 4 4 4
3 2 2 1 4 3 3 7 4 5 5 6 4
4 3 2 4 1 2 2 9 4 4 5 5 3
5 3 2 3 2 1 2 8 5 4 5 5 4
6 8 9 10 12 13 14 2 6 2 3 3 5 4 2 8 3 4 4 4 4 3 7 4 5 5 6 4 2 9 4 4 5 5 3 2 8 5 4 5 5 4 1 7 3 3 4 5 3 7 1 7 4 10 12 8 3 7 1 3 4 6 5 3 4 3 1 5 6 4 4 10 4 5 1 3 4 5 12 6 6 3 1 3 3 8 5 4 4 3 1
Fig. 3. Rounded values of model distances for NIR macro-Raman spectra Norway spruce needles of twigs of different age from the same branch. Age: (1±10) 1-year, (12) 2-year, (13,14) 3-year (7 and 11 not measured; for detailed description of the branch see Ref. [3]). Values greater than 3 are marked by a shadow.
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P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
models created by PCA and classi®ed by SIMCA. Model distance is one of the parameters calculated. If the model distance is higher than 3, the models are suitable for classi®cation and the models are said to be different [6]. The rounded values of these model distances are given in Fig. 3 for different methods of the preliminary treatment of the experimental data: baseline correction or normalization. Evaluating the obtained data only from the point of view of similarity or dissimilarity of the spectra, it is clear that 2- and 3year-old needles differ from 1-year-old needles in all cases without exception. On the other hand, there is a signi®cant difference in 1-year-old needles even for very closely placed twigs. Twig no. 8 shows an exceptional spectrum. A deeper analysis is needed to account for the differences observed for different preliminary treatment of the spectra. 4. Conclusions Direct measurement of the stomatal wax on a needle is possible, but strong in¯uence of the substrate is observed, variability in carotenoid bands being the most pronounced. A selective information on stomatal wax can be obtained by comparison of micro-Raman
spectra of stomatal and interstomatal areas. Spectra of extracts can be complicated by co-extracted intracuticular components; an optimization of the extraction conditions is needed. A deeper insight into the origin of the different information gained for different treatments of the initial spectral data is needed.
Acknowledgements Financial support of the Ministry of Environment of the Czech Republic (grant ªEvaluation of the State of the Environment: Monitoring of Contaminants in Food Chainsº, No. MR/14/95) is gratefully acknowledged.
References [1] M. GrodzinÂska-Jurczak, Acta Soc. Bot. Pol. 67 (1998) 291. [2] B. Prugel, G. Lognay, Phytochem. Anal. 7 (1996) 29. [3] L. PlesÏerovaÂ, G. BudõÂnovaÂ, K. HavrÏrovaÂ, P. MateÏjka, F. SkaÂcel, K. Volka, J. Mol. Struct., in press. [4] B. Schrader, B. Dippel, I. Erb, S. Keller, T. LoÈchte, H. Schulz, E. Tatsch, S. Wessel, J. Mol. Struct. 480±481 (1999) 21. [6] The unscrambler (User Manual), CAMO (Norway), 1998.
www.elsevier.nl/locate/molstruc
Vibrational biospectroscopy: what can we say about the surface wax layer of Norway spruce needles? P. MateÏjka a, L. PlesÏerova a, G. BudõÂnova a, K. HavõÂrÏova a, X. Mulet b,1, F. SkaÂcel c, K. Volka a,* a
Department of Analytical Chemistry, Institute of Chemical Technology, Technicka 5, Prague 6, CZ 166 28, Czech Republic b Imperial College of Technology and Medicine, London SW7 2AZ, UK c Department of Gas, Coal and Air Protection, Institute of Chemical Technology, Technicka 5, Prague 6, CZ 166 28, Czech Republic Received 31 August 2000; revised 22 November 2000; accepted 22 November 2000
Abstract NIR-excited FT-Raman spectra of the Norway spruce (Picea abies (L.) Karst.) needles were measured by microscopic and macroscopic techniques. From the NIR micro-Raman spectra of the stomatal and interstomatal area, the spectrum of the epistomatal wax was extracted. The effect of the cellular substrate of the needles on this spectrum is shown and its variability demonstrated by NIR macro-Raman spectra. Strong variability in the spectra of the spruce needles is observed even for needles of the same age from the same branch. One of the dominant features is variation in intensity of the carotenoid bands. q 2001 Elsevier Science B.V. All rights reserved. Keywords: NIR FT-Raman spectroscopy; Microspectroscopy; Spruce needles; Stomatal wax; Chemometrics
1. Introduction The surface of the spruce needle is covered by a cuticle (1±5 mm thick) consisting of two elements, the cutin (a polyester built up by di- and trihydroxy fatty acids) and cuticular waxes (a complex mixture of lipids, i.e. long-chain secondary alcohols, diols and free fatty acids as major classes, n-alkanes, n-alkenes, primary alcohols, a,v-diols, ketones and v-hydroxyacids as minor classes) [1,2]. Waxes deposited onto the outer surface of the cuticle are denoted as * Corresponding author. Tel.: 1420-2-2435-4056; fax: 1420-23112828. E-mail address: [email protected] (K. Volka). 1 IAESTE student at the Department of Analytical Chemistry in Summer 2000.
epicuticular wax, waxes within the inner cutin layer as intracuticular wax. Epicuticular wax occurring in the stomatal entrances is denoted as epistomatal wax. The morphology of the epistomatal wax is very characteristic and changes dramatically under natural and anthropogenic factors. The tubular forms are with increasing environmental stress transformed into the melted, tabular ones. The morphology of the wax is evaluated for a de®ned number of stomas by numerical values between 1.0 (unaffected) and 5.0 (heavily affected) called wax quality. The micro-morphology of the stomatal wax now serves as a biomarker of air pollution [1]. These morphological changes are strongly connected with chemical changes and this has been the reason why we were interested in the potential of NIR-excited Raman (NIR Raman hereafter) spectroscopy as an alternative to scanning
0022-2860/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0022-286 0(00)00929-7
306
P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
electron microscopy, which is rather dif®cult to use and also non-speci®c. Our preliminary experiments showed some signi®cant differences in the NIR Raman spectra of the Norway spruce (Picea abies (L.) Karst.) needles sampled in two different localities of the Czech Republic [3]. This work highlights some dif®cult points in NIR Raman spectroscopic evaluation of the epicuticular wax and also in the quanti®cation of the method. 2. Experimental A branch of Norway spruce was taken in the Velke Losenice area (Czech Republic) in December 1998. Individual twigs and separated needles were packed in aluminum foil and then put into polyethylene bags. The bags were placed in desiccators and stored in a refrigerator (at ca. 48C). The extracts of the spruce needles were prepared by two methods. In method I, spruce needles in a mixture of chloroform±acetone (1:1 vol.) were mechanically homogenized for 5 min (Turax) and then extracted under soni®cation for 5 min. 2,6-Di-t-butyl-4-methylphenol (0.1 wt.%) and calcium carbonate were added. In method II, an extract in a hexane±acetone mixture (3:2 vol.) was prepared from the whole needles by 5min soni®cation. Solvents were evaporated at 508C before measurement of the spectra. NIR Raman spectra were collected using a Fourier transform near-infrared spectrometer Equinox 55/S (Bruker) equipped with FT Raman module FRA 106/S (Bruker). In the case of macro-measurement, a needle placed vertically in an adapted holder was irradiated ca. 2± 3 mm from its tip by the focused laser beam with a power 50 mW of Nd±YAG laser (1064 nm, Coherent). Both top and bottom sides of a needle were examined at the same distance from the tip by turning the sample holder, i.e. four spectra were obtained per needle. The scattered light was collected in back-scattering geometry. 1024 interferograms were co-added and processed to obtain Raman spectra with 4 cm 21 resolution. In the case of the micro-experiment, a needle was placed on a movable table of microscope Ð Ramanscopee (Bruker) equipped with Nikon objectives (10 £ , 20 £ , 40 £ , 100 £ ). The image of a needle
was monitored by color CCD camera (Sony) connected to a computer that made it possible to save the visual micro-images. Three pairs of stomatal and interstomatal points were selected on a needle (ca. 2 mm from a tip, in the middle, and ca. 2 mm from the base of the needle). The excitation radiation (laser power less than 30 mW) was supplied by optical ®ber (diameter 5 mm). The 40 £ objective was used for Raman measurement. Two times 1024 scans were collected at each position of the needle to verify the repeatability of data collection. In sum, 12 Raman spectra with 4 cm 21 resolution were obtained per needle. The software opus 2.3 (Bruker) was used to control the spectrometer and to process the spectra obtained using ªrubber bandº baseline (100 points), Min/Max and vector normalization. Chemometric evaluation of the data using principal component analysis (PCA) and the soft-independent modeling of class analogy (SIMCA) were done by unscrambler 7.5 (CAMO, Norway). 3. Results and discussion The dif®culty of a measurement of the NIR Raman spectrum of the epicuticular wax is given by its very non-uniform distribution on the needle and low content. The main portion of the wax is located in the stoma of a diameter of several tens of micrometers. Outside of the stomatal areas, this type of coverage disappears; only in rare cases did we observe a visible structure beyond the boundary of about 1.5 m diameter of the stoma. The needles are covered with stomata in about three parallel lines on both sides, but there are many irregularities in their distribution. The content of the wax can be estimated by its extraction and amounts from 0.5 to over 2% dry weight of the needles [1]. In NIR macro-Raman measurement, the diameter of the focused laser beam is about 0.8 mm. So NIR micro-Raman measurement is needed to gain information about the epistomatal wax only. The diameter of the focused laser beam on a ¯at surface is about 5 mm. The combination of visual microscopy with NIR Raman microscopy is substantial: there are many microscopic objects of a comparable size on the surface, which can make the microscopic
P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
measurement quite erratic. They are inorganic powder, pollen, fungi, etc. NIR micro-Raman spectra of the stomatal (spectrum A) and interstomatal area (spectrum B) are given in Fig. 1 along with their difference (spectrum C). Two ªnegativeª bands at about 1530 and 1160 cm 21 correspond to carotenoids [4]. The NIR excitation penetrates through the epicuticular wax to the cellular substrate, so the cellular substrate is also excited along with the wax. Lower intensity of the carotenoid bands in the
307
stomatal areas in comparison with interstomatal ones can be easily elucidated by a shielding of the cellular substrate by the wax in the areas of stoma. The well-preserved epistomatal wax structures strongly scatter the light and so attenuate the excitation and emitted radiation very effectively. The NIR Raman spectra of stomatal wax alone is characterized ®rst of all by the bands corresponding to aliphatic moiety (bands at 2882, 2845 and about 1456 cm 21, Fig. 1C). The difference spectrum (Fig. 1C) can be compared with the NIR Raman
Fig. 1. NIR micro-Raman spectra of a Norway spruce needle: A ± stomatal area; B ± interstomatal area; C ± difference of spectrum A minus spectrum B.
308
P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
spectra (macro-measurement) of spruce needles (Fig. 2A) and of two extracts into chloroform±acetone (method I) (Fig. 2B) and/or hexane±acetone (method II) (Fig. 2C) showing the highest similarity of spectra 1C and 2C. The carotenoid bands (1530 and 1160 cm 21) are missing in spectrum 2C suggesting that the carotenoids are not extracted using method II. But they are observed in spectrum 2B even with a comparable intensity to the spectrum of the whole needle (Fig. 2A). We conclude that method II extracts stomatal wax selectively while the extract of method I also includes subcuticular components.
The crucial question connected with direct measurement of the spruce needles (Fig. 2A) is the variability of the spectrum of the cellular substrate. Our estimation of the surface coverage of a needle by epistomatal wax is only about 2±3% and so a basic piece of information about the variability of the spectrum of the cellular substrate can be obtained by NIR macro-Raman measurement of the needle. The 404 spectra of the needles of nine 1-year, two 2-year and one 3-year-old twigs were measured. The evaluation of the similarity of the spectra in the range 3050±2650 and 1800±720 cm 21 is based on the
Fig. 2. NIR Raman spectra (macro) of a Norway spruce needle (A) and extracts of needles in chloroform±acetone mixture (method I, spectrum B) and hexane±acetone mixture (method II, spectrum C).
P. MateÏjka et al. / Journal of Molecular Structure 565±566 (2001) 305±310
No manipulation of initial data
1 2 3 4 5 6 8 9 10 12 13 14
1 1 2 2 3 3 2 2 2 2 5 9 5
2 3 4 2 2 3 1 2 2 2 1 3 2 3 1 2 2 2 2 2 2 5 4 6 3 3 3 3 3 3 7 11 5 7 12 4 4 4 3
5 3 2 2 2 1 2 4 3 3 9 9 5
6 8 9 2 2 2 2 5 3 2 4 3 2 6 3 2 4 3 1 5 2 5 1 3 2 3 1 2 3 2 5 18 6 7 18 8 4 9 5
Baseline corrected initial data
10 12 13 14 2 5 9 5 3 7 7 4 3 11 12 4 3 5 4 3 3 9 9 5 2 5 7 4 3 18 18 9 2 6 8 5 1 6 7 3 6 1 4 4 7 4 1 4 3 4 4 1
1 1 2 2 5 3 3 20 2 4 3 7 5
MinMax normalization of initial data
1 2 3 4 5 6 8 9 10 12 13 14
1 1 2 3 4 5 3 3 1 2 4 7 5
2 2 1 2 3 3 2 6 3 2 6 6 4
3 3 2 1 3 4 2 5 3 3 8 9 4
4 4 3 3 1 2 2 9 5 3 5 4 3
5 5 3 4 2 1 2 8 5 3 7 6 3
6 8 9 3 3 1 2 6 3 2 5 3 2 9 5 2 8 5 1 5 3 5 1 3 3 3 1 2 3 2 4 7 5 5 10 6 3 10 6
1 1 2 3 3 4 2 2 2 3 6 8 4
10 12 13 14 2 4 7 5 2 6 6 4 3 8 9 4 3 5 4 3 3 7 6 3 2 4 5 3 3 7 10 10 2 5 6 6 1 5 5 4 5 1 3 5 5 3 1 4 4 5 4 1
2 3 4 5 6 8 9 10 12 13 14 2 3 3 4 2 2 2 3 6 8 4 1 2 2 2 2 3 2 3 8 9 3 2 1 4 4 3 4 4 5 12 13 4 2 4 1 2 2 4 3 3 9 8 3 2 4 2 1 2 4 4 4 12 9 4 2 3 2 2 1 3 2 3 6 7 4 3 4 4 4 3 1 2 2 8 11 5 2 4 3 4 2 2 1 3 7 10 4 3 5 3 4 3 2 3 1 8 8 5 8 12 9 12 6 8 7 8 1 4 9 9 13 8 9 7 11 10 8 4 1 6 3 4 3 4 4 5 4 5 9 6 1
2 2 1 3 2 2 2 22 3 4 4 5 3
3 2 3 1 4 2 2 22 2 5 4 8 5
4 5 2 4 1 2 3 31 4 5 5 5 3
5 3 2 2 2 1 2 24 3 4 5 6 3
6 3 2 2 3 2 1 25 2 4 4 6 3
8 20 22 22 31 24 25 1 26 3 15 36 11
9 2 3 2 4 3 2 26 1 4 3 6 5
10 12 13 14 4 3 7 5 4 4 5 3 5 4 8 5 5 5 5 3 4 5 6 3 4 4 6 3 3 15 36 11 4 3 6 5 1 5 7 4 5 1 4 4 7 4 1 3 4 4 3 1
5 6 8 9 3 2 14 2 2 2 23 3 3 2 16 3 2 2 16 4 1 2 7 5 2 1 17 3 7 17 1 19 5 3 19 1 3 3 4 4 4 3 11 4 5 5 30 5 4 3 14 6
10 12 13 14 5 3 5 4 6 4 4 5 5 4 5 4 4 5 5 3 3 4 5 4 3 3 5 3 4 11 30 14 4 4 5 6 1 5 7 5 5 1 3 4 7 3 1 4 5 4 4 1
MinMax normalization of baseline corrected data 1 1 2 2 3 3 2 14 2 5 3 5 4
Vector normalization of baseline initial data
1 2 3 4 5 6 8 9 10 12 13 14
309
2 2 1 2 2 2 2 23 3 6 4 4 5
3 2 2 1 3 3 2 16 3 5 4 5 4
4 3 2 3 1 2 2 16 4 4 5 5 3
Vector normalization of baseline corrected data
1 2 3 4 5 6 8 9 10 12 13 14
1 1 2 2 3 3 2 6 2 3 3 5 4
2 2 1 2 2 2 2 8 3 4 4 4 4
3 2 2 1 4 3 3 7 4 5 5 6 4
4 3 2 4 1 2 2 9 4 4 5 5 3
5 3 2 3 2 1 2 8 5 4 5 5 4
6 8 9 10 12 13 14 2 6 2 3 3 5 4 2 8 3 4 4 4 4 3 7 4 5 5 6 4 2 9 4 4 5 5 3 2 8 5 4 5 5 4 1 7 3 3 4 5 3 7 1 7 4 10 12 8 3 7 1 3 4 6 5 3 4 3 1 5 6 4 4 10 4 5 1 3 4 5 12 6 6 3 1 3 3 8 5 4 4 3 1
Fig. 3. Rounded values of model distances for NIR macro-Raman spectra Norway spruce needles of twigs of different age from the same branch. Age: (1±10) 1-year, (12) 2-year, (13,14) 3-year (7 and 11 not measured; for detailed description of the branch see Ref. [3]). Values greater than 3 are marked by a shadow.
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models created by PCA and classi®ed by SIMCA. Model distance is one of the parameters calculated. If the model distance is higher than 3, the models are suitable for classi®cation and the models are said to be different [6]. The rounded values of these model distances are given in Fig. 3 for different methods of the preliminary treatment of the experimental data: baseline correction or normalization. Evaluating the obtained data only from the point of view of similarity or dissimilarity of the spectra, it is clear that 2- and 3year-old needles differ from 1-year-old needles in all cases without exception. On the other hand, there is a signi®cant difference in 1-year-old needles even for very closely placed twigs. Twig no. 8 shows an exceptional spectrum. A deeper analysis is needed to account for the differences observed for different preliminary treatment of the spectra. 4. Conclusions Direct measurement of the stomatal wax on a needle is possible, but strong in¯uence of the substrate is observed, variability in carotenoid bands being the most pronounced. A selective information on stomatal wax can be obtained by comparison of micro-Raman
spectra of stomatal and interstomatal areas. Spectra of extracts can be complicated by co-extracted intracuticular components; an optimization of the extraction conditions is needed. A deeper insight into the origin of the different information gained for different treatments of the initial spectral data is needed.
Acknowledgements Financial support of the Ministry of Environment of the Czech Republic (grant ªEvaluation of the State of the Environment: Monitoring of Contaminants in Food Chainsº, No. MR/14/95) is gratefully acknowledged.
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