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Fourier-transform Raman spectroscopic study of surface of Norway spruce needles
Journal of Molecular Structure 480–481 (1999) 547–550
Fourier-transform Raman spectroscopic study of surface of Norway spruce needles J. Krˇ´ızˇova´, P. Mateˇjka*, G. Budı´nova´, K. Volka Dept. of Analytical Chemistry, Institute of Chemical Technology, Technicka´ 5, CZ-166 28 Prague 6, Czech Republic Received 24 August 1998; received in revised form 30 September 1998
Abstract The potential of Raman spectroscopy and chemometrics for environmental monitoring was investigated. Needles of Norway spruce [Picea abies (L.) Karst.] were studied without any chemical treatment by Fourier-transform Raman spectroscopy. Two different forest areas of Czech Republic were chosen. Samples of twigs were taken in June (1997) and in September (1997). FTRaman spectra obtained were analysed by cluster analysis and further chemometric evaluation was performed. The influences of two factors (“sample area”, “needle age”) were primarily described. 䉷 1999 Elsevier Science B.V. All rights reserved. Keywords: FT-Raman spectroscopy; Picea abies; Cuticular wax; PCA; SIMCA
1. Introduction Needles play an important role in the physiology of conifers. The surface of needles is covered by a cuticle (a waxy layer) which forms an interface between the epidermal surface and the atmosphere protecting the underlying cells [1,2]. The micromorphology of cuticle is well described for conifers from both clean and polluted areas by the scanning electron microscopy (SEM) [2,3]. The observed alterations of structure should be reflected by modification of the wax composition. Chemical analyses of cuticle are generally based on chromatographic separation processes of various organic extracts of needles [1,2]. We focus our effort on Fourier-transform (FT) Raman spectroscopic analysis of needles, because Raman spectroscopy should provide information on chemical composition of the surface of needles * Corresponding author. Tel.: ⫹ 420-2-2435-4281; fax: ⫹ 4202-311-2828. E-mail address: [email protected] (P. Mateˇjka)
without any chemical treatment of the material. The near-infrared excitation enables to obtain spectra of biomaterials [4] (e.g. wood [5,6], animal and human tissues [7]) overcoming the problem of fluorescence and/or photodecomposition. The interpretation of vibrational spectra of complex biological samples is very complicated and the chemometric evaluation of the data seems to be very powerful tool to obtain information on such materials. 2. Experimental 2.1. Materials Norway spruce [Picea abies (L.) Karst.] was the tree species investigated, because it has a widespread distribution and it has been reported as an air pollution bioindicator [1]. The samples were taken at the end of June (1997) and in the middle of September (1997). Two sites were chosen in different parts of Czech Republic, the first one named Rˇepice is located in
0022-2860/99/$ - see front matter 䉷 1999 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(98)00735-2
548
J. Krˇ´ızˇova´ et al. / Journal of Molecular Structure 480–481 (1999) 547–550
2.2. Instrumentation FT-Raman spectra were collected using a Fourier transform near-infrared (FT-NIR) spectrometer Equinox 55/S (Bruker) equipped with FT-Raman module FRA 106/S (Bruker). The type of focus of laser beam and the magnitude of laser power were tested in the first period. Then the samples were irradiated by the focused laser beam with a laser power 50 mW of Nd-YAG laser (1064 nm, Coherent). The scattered light was collected in backscattering geometry. Quartz beamsplitter and Ge detector (liquid N2 cooled) were used to obtain interferograms. 1024 interferograms were co-added and then processed by the Fourier transformation with Blackman–Harris 4-term apodization a zerofilling factor 8 to obtain final FT-Raman spectra in the range 4000 – (⫺1000) cm ⫺1 with 4 cm ⫺1 resolution. The Bruker software OPUS 2.3 was used to control the spectrometer and to process the spectra obtained. The software module OPUS-IDENT 2.0 was used for cluster analysis of the spectra. Both the original and the vector-normalized spectra were classified. The Raman spectra were also exported to J-CAMP DX format for further chemometric evaluation. Fig. 1. FT-Raman spectra of current-year needles of Norway spruce. The samples of twigs were taken in September 1997 from A-Repice, B-Prosˇtı´pena´.
the south Bohemia in the district Strakonice (ST), the second one named Prosˇtı´pena´ is in the south Moravia in the district Uherske´ Hradisˇteˇ (UH). The twigs with current-year needles, the one-year old needles and the two-year old needles were sampled separately and were stored in air-permeable bags in desiccator filled with silica gel as a desiccant. The desiccator was stored in a refrigerator (ca. 5⬚C). The basic sets of data were collected within one month from taking the samples. The individual needle was torn off the twig immediately before measurement and it was placed in special sample holder. The FT-Raman spectra were measured on both abaxial and adaxial side of the needle. 24 needles of each age were used to obtain basic data set for the particular site, i.e. 72 needles (144 spectra) per site for one date of sampling were analysed.
2.3. Chemometrics The principal component analysis (PCA) represents the fundamental method of the evaluation of both the measured and vector-normalized spectra. Afterwards the classification method SIMCA (soft independent modeling of class analogy) was used especially to compare the data obtained for the samples taken in June and in September. Chemometric calculations were performed using the Unscrambler ver. 6.1 (Camo AS, Norway).
3. Results and discussion 3.1. Spectroscopic data The FT-Raman spectra of the needles of Norway spruce of both forest areas (Fig. 1) are dominated by the similar spectral features. Nevertheless, the relative intensities of some bands are very different, e.g. the relation of intensities of ca. 1525 ⫺ cm ⫺1 and ca.
J. Krˇ´ızˇova´ et al. / Journal of Molecular Structure 480–481 (1999) 547–550
549
Fig. 2. Simplified dendrogram of clustering of Raman spectra of Norway spruce needles. The presented analysis includes 288 vector-normalized spectra of 144 needles of three various ageing from two sites. The samples were taken in September 1997.
1605 ⫺ cm ⫺1 bands. This effect does not depend on the age of needles. The factor of “needle age” exhibits similar effects by comparing the spectra of various ageing of needles for the individual sampling sites, e.g. the relative slight decrease of 2934 ⫺ cm ⫺1 and 1440 ⫺ cm ⫺1 bands attributable to saturated aliphatic chains. This observation can be ascribed to the loss of some saturated aliphatic components within ageing of the needles. The spectra of both abaxial and adaxial side of the needle are very similar. The differences of spectra obtained on the top and on the base of the needle will be further studied.
spectra show generally better clustering than the raw data. Two main clusters (Fig. 2) based on forest area are formed for all type of experiments, (i.e. for data taken in June, in September, for all the data together), independently on the number of spectra per needle and on the age of needles. The formation of clusters on relatively lower level of heterogeneity is dependent on the age of needles. Although the separation of the individual ages is not complete (Fig. 2), the mix of all three ages was never observed. The clustering based on the effect of the adaxial and abaxial side of needle cannot be distinguished. The effect of various parts along the needle (from the base to the top) will be presented in forthcoming study.
3.2. Cluster analysis
3.3. Principal component analysis
The cluster analysis was made on both measured and vector-normalized data. The vector-normalized
The results of PCA analysis are generally analogous to the results of cluster analysis. The separation
550
J. Krˇ´ızˇova´ et al. / Journal of Molecular Structure 480–481 (1999) 547–550
of data of various age of needles is even better than the before described clustering. The adaxial and abaxial sides of needles cannot be resolved even by PCA. 3.4. Evaluation by soft independent modeling of class analogy The classification method SIMCA was used to compare data of the needles taken in June and in September. Two types of models based on forest areas were created from spectra of the needles taken in June. The spectra of needles taken in September were used to test the two models. The 100% spectra of ˇ epice and from Prosˇtı´pena´ were needles both from R attributed to the correct models The SIMCA models based on ageing were created for the individual forest areas. While 87.5% of spectra from Rˇepice was assigned to the correct model, only assignment of 79% of spectra from Prosˇtı´pena´ was correct. This result corresponds to result of cluster analysis (Fig. 2). 4. Conclusions All the statistical methods used show the primary difference in the spectra based on forest sites. The age of needles leads to rather lower level clustering of spectra for each site separately. The statistical results reflect the variation of relative intensities of many
bands in the spectra. The interpretation based on assignment of these bands will provide further information on changes of chemical composition of needle surface with respect both to the site and age.
Acknowledgements Financial support of the Ministry of Environment of the Czech Republic (grant “Evaluation of the State of the Environment: Monitoring of Contaminants in Food Chains”, No. MR/14/95) is gratefully acknowledged.
References B. Pru¨gel, P. Loosveldt, J.P. Garrec, Trees 9 (1994) 80. C. Lu¨tz, U. Heinzmann, Environ. Pollut. 64 (1990) 313. C. Trimbacher, O. Eckmu¨llner, Eur. J. For. Path. 27 (1997) 83. B. Schrader, G. Baranovic, A. Epding, G.G. Hoffmann, P.J.M. van Kan, S. Keller, P. Hildebrandt, C. Lehner, J. Sawatzki, Appl. Spectrosc. 47 (1993) 1452. [5] D. Stewart, H.M. Wilson, P. J. Hendra, I.M. Morrison, J. Agric. Food Chem. 43 (1995) 2219. [6] U.P. Agarwal, S.A. Ralph, Appl. Spectrosc. 51 (1997) 1648. [7] S. Keller, B. Schrader, A. Hoffmann, W. Schrader, K. Metz, A. Rehlaender, J. Pahnke, M. Ruwe, W. Budach, J. Raman Spectrosc. 25 (1994) 663.
[1] [2] [3] [4]
Fourier-transform Raman spectroscopic study of surface of Norway spruce needles J. Krˇ´ızˇova´, P. Mateˇjka*, G. Budı´nova´, K. Volka Dept. of Analytical Chemistry, Institute of Chemical Technology, Technicka´ 5, CZ-166 28 Prague 6, Czech Republic Received 24 August 1998; received in revised form 30 September 1998
Abstract The potential of Raman spectroscopy and chemometrics for environmental monitoring was investigated. Needles of Norway spruce [Picea abies (L.) Karst.] were studied without any chemical treatment by Fourier-transform Raman spectroscopy. Two different forest areas of Czech Republic were chosen. Samples of twigs were taken in June (1997) and in September (1997). FTRaman spectra obtained were analysed by cluster analysis and further chemometric evaluation was performed. The influences of two factors (“sample area”, “needle age”) were primarily described. 䉷 1999 Elsevier Science B.V. All rights reserved. Keywords: FT-Raman spectroscopy; Picea abies; Cuticular wax; PCA; SIMCA
1. Introduction Needles play an important role in the physiology of conifers. The surface of needles is covered by a cuticle (a waxy layer) which forms an interface between the epidermal surface and the atmosphere protecting the underlying cells [1,2]. The micromorphology of cuticle is well described for conifers from both clean and polluted areas by the scanning electron microscopy (SEM) [2,3]. The observed alterations of structure should be reflected by modification of the wax composition. Chemical analyses of cuticle are generally based on chromatographic separation processes of various organic extracts of needles [1,2]. We focus our effort on Fourier-transform (FT) Raman spectroscopic analysis of needles, because Raman spectroscopy should provide information on chemical composition of the surface of needles * Corresponding author. Tel.: ⫹ 420-2-2435-4281; fax: ⫹ 4202-311-2828. E-mail address: [email protected] (P. Mateˇjka)
without any chemical treatment of the material. The near-infrared excitation enables to obtain spectra of biomaterials [4] (e.g. wood [5,6], animal and human tissues [7]) overcoming the problem of fluorescence and/or photodecomposition. The interpretation of vibrational spectra of complex biological samples is very complicated and the chemometric evaluation of the data seems to be very powerful tool to obtain information on such materials. 2. Experimental 2.1. Materials Norway spruce [Picea abies (L.) Karst.] was the tree species investigated, because it has a widespread distribution and it has been reported as an air pollution bioindicator [1]. The samples were taken at the end of June (1997) and in the middle of September (1997). Two sites were chosen in different parts of Czech Republic, the first one named Rˇepice is located in
0022-2860/99/$ - see front matter 䉷 1999 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(98)00735-2
548
J. Krˇ´ızˇova´ et al. / Journal of Molecular Structure 480–481 (1999) 547–550
2.2. Instrumentation FT-Raman spectra were collected using a Fourier transform near-infrared (FT-NIR) spectrometer Equinox 55/S (Bruker) equipped with FT-Raman module FRA 106/S (Bruker). The type of focus of laser beam and the magnitude of laser power were tested in the first period. Then the samples were irradiated by the focused laser beam with a laser power 50 mW of Nd-YAG laser (1064 nm, Coherent). The scattered light was collected in backscattering geometry. Quartz beamsplitter and Ge detector (liquid N2 cooled) were used to obtain interferograms. 1024 interferograms were co-added and then processed by the Fourier transformation with Blackman–Harris 4-term apodization a zerofilling factor 8 to obtain final FT-Raman spectra in the range 4000 – (⫺1000) cm ⫺1 with 4 cm ⫺1 resolution. The Bruker software OPUS 2.3 was used to control the spectrometer and to process the spectra obtained. The software module OPUS-IDENT 2.0 was used for cluster analysis of the spectra. Both the original and the vector-normalized spectra were classified. The Raman spectra were also exported to J-CAMP DX format for further chemometric evaluation. Fig. 1. FT-Raman spectra of current-year needles of Norway spruce. The samples of twigs were taken in September 1997 from A-Repice, B-Prosˇtı´pena´.
the south Bohemia in the district Strakonice (ST), the second one named Prosˇtı´pena´ is in the south Moravia in the district Uherske´ Hradisˇteˇ (UH). The twigs with current-year needles, the one-year old needles and the two-year old needles were sampled separately and were stored in air-permeable bags in desiccator filled with silica gel as a desiccant. The desiccator was stored in a refrigerator (ca. 5⬚C). The basic sets of data were collected within one month from taking the samples. The individual needle was torn off the twig immediately before measurement and it was placed in special sample holder. The FT-Raman spectra were measured on both abaxial and adaxial side of the needle. 24 needles of each age were used to obtain basic data set for the particular site, i.e. 72 needles (144 spectra) per site for one date of sampling were analysed.
2.3. Chemometrics The principal component analysis (PCA) represents the fundamental method of the evaluation of both the measured and vector-normalized spectra. Afterwards the classification method SIMCA (soft independent modeling of class analogy) was used especially to compare the data obtained for the samples taken in June and in September. Chemometric calculations were performed using the Unscrambler ver. 6.1 (Camo AS, Norway).
3. Results and discussion 3.1. Spectroscopic data The FT-Raman spectra of the needles of Norway spruce of both forest areas (Fig. 1) are dominated by the similar spectral features. Nevertheless, the relative intensities of some bands are very different, e.g. the relation of intensities of ca. 1525 ⫺ cm ⫺1 and ca.
J. Krˇ´ızˇova´ et al. / Journal of Molecular Structure 480–481 (1999) 547–550
549
Fig. 2. Simplified dendrogram of clustering of Raman spectra of Norway spruce needles. The presented analysis includes 288 vector-normalized spectra of 144 needles of three various ageing from two sites. The samples were taken in September 1997.
1605 ⫺ cm ⫺1 bands. This effect does not depend on the age of needles. The factor of “needle age” exhibits similar effects by comparing the spectra of various ageing of needles for the individual sampling sites, e.g. the relative slight decrease of 2934 ⫺ cm ⫺1 and 1440 ⫺ cm ⫺1 bands attributable to saturated aliphatic chains. This observation can be ascribed to the loss of some saturated aliphatic components within ageing of the needles. The spectra of both abaxial and adaxial side of the needle are very similar. The differences of spectra obtained on the top and on the base of the needle will be further studied.
spectra show generally better clustering than the raw data. Two main clusters (Fig. 2) based on forest area are formed for all type of experiments, (i.e. for data taken in June, in September, for all the data together), independently on the number of spectra per needle and on the age of needles. The formation of clusters on relatively lower level of heterogeneity is dependent on the age of needles. Although the separation of the individual ages is not complete (Fig. 2), the mix of all three ages was never observed. The clustering based on the effect of the adaxial and abaxial side of needle cannot be distinguished. The effect of various parts along the needle (from the base to the top) will be presented in forthcoming study.
3.2. Cluster analysis
3.3. Principal component analysis
The cluster analysis was made on both measured and vector-normalized data. The vector-normalized
The results of PCA analysis are generally analogous to the results of cluster analysis. The separation
550
J. Krˇ´ızˇova´ et al. / Journal of Molecular Structure 480–481 (1999) 547–550
of data of various age of needles is even better than the before described clustering. The adaxial and abaxial sides of needles cannot be resolved even by PCA. 3.4. Evaluation by soft independent modeling of class analogy The classification method SIMCA was used to compare data of the needles taken in June and in September. Two types of models based on forest areas were created from spectra of the needles taken in June. The spectra of needles taken in September were used to test the two models. The 100% spectra of ˇ epice and from Prosˇtı´pena´ were needles both from R attributed to the correct models The SIMCA models based on ageing were created for the individual forest areas. While 87.5% of spectra from Rˇepice was assigned to the correct model, only assignment of 79% of spectra from Prosˇtı´pena´ was correct. This result corresponds to result of cluster analysis (Fig. 2). 4. Conclusions All the statistical methods used show the primary difference in the spectra based on forest sites. The age of needles leads to rather lower level clustering of spectra for each site separately. The statistical results reflect the variation of relative intensities of many
bands in the spectra. The interpretation based on assignment of these bands will provide further information on changes of chemical composition of needle surface with respect both to the site and age.
Acknowledgements Financial support of the Ministry of Environment of the Czech Republic (grant “Evaluation of the State of the Environment: Monitoring of Contaminants in Food Chains”, No. MR/14/95) is gratefully acknowledged.
References B. Pru¨gel, P. Loosveldt, J.P. Garrec, Trees 9 (1994) 80. C. Lu¨tz, U. Heinzmann, Environ. Pollut. 64 (1990) 313. C. Trimbacher, O. Eckmu¨llner, Eur. J. For. Path. 27 (1997) 83. B. Schrader, G. Baranovic, A. Epding, G.G. Hoffmann, P.J.M. van Kan, S. Keller, P. Hildebrandt, C. Lehner, J. Sawatzki, Appl. Spectrosc. 47 (1993) 1452. [5] D. Stewart, H.M. Wilson, P. J. Hendra, I.M. Morrison, J. Agric. Food Chem. 43 (1995) 2219. [6] U.P. Agarwal, S.A. Ralph, Appl. Spectrosc. 51 (1997) 1648. [7] S. Keller, B. Schrader, A. Hoffmann, W. Schrader, K. Metz, A. Rehlaender, J. Pahnke, M. Ruwe, W. Budach, J. Raman Spectrosc. 25 (1994) 663.
[1] [2] [3] [4]