Non-destructive NIR FT Raman analysis of plants

Non-destructive NIR FT Raman analysis of plantsq

B. Schradera,* , H.H. Klumpb, K. Schenzelc, H. Schulzd

aInstitut fur Physikalische und Theoretische Chemie, Universita¨t Essen, D-45117 Essen, GermanybDepartment of Biochemistry, University of Cape Town, Private Bag Rondebosch 7700, Cape Town, South Africa

cInstitut fur Acker- und Pflanzenbau, Martin-Luther-Universita¨t, D-06099 Halle, GermanydBundesanstalt fu¨r Zuchtungsforschung an Kulturpflanzen, Institut fu¨r Qualitatsanalytik, D-06484 Quedlinburg, Germany

Received 15 March 1999; accepted 14 April 1999

Abstract

Non-destructive analyses of animal and plant cells and tissues by ‘classical’ Raman spectroscopy with excitation in thevisible range have not been possible since the samples are destroyed photochemically or their fluorescence conceals the Ramanspectra completely. When excited with the Nd:YAG laser line at 1064 nm fluorescence-free Raman spectra of animal or plantcells and tissues can be recorded without special preparation. In this paper we concentrate on plants and its constituents:essential oils, natural dyes, flavors, spices, alkaloids and fibers can be characterized. The spectra allow the observation ofbiochemical processes, to observe the distribution of natural products, application to taxonomy, optimizing plant breeding, theharvesting time and control of food—everything non-destructively in living plants!q 1999 Elsevier Science B.V. All rightsreserved.

Keywords:Non-destructive analyses of plants; Biochemical processes; Fluorescence-free Raman spectroscopy; Natural products of plants;Food quality control

1. Introduction

Cells can only live with the help of a complexmachinery. Most of the necessary enzymes and coen-zymes absorb in the visible range of the spectrum —consequently they show fluorescence and are photo-chemically sensitive. Since these are essentialcompounds, they are not permitted to be removedby tricks like ‘burning out’ or chemical or physicalprocessing. Therefore, Raman spectroscopy withexcitation in the visible range is not applicable. The

cells would be destroyed and the Raman spectraare overlaid by the fluorescence of manycompounds, even when they are present only astraces. This is due to the fact that the quantumyield of fluorescence is of the order of 1, thequantum yield of Raman spectroscopy is, however,about 6–10 orders of magnitude smaller [1,2].Since fluorescence is a consequence of the absorp-tion spectrum, it is reduced when exciting radia-tion is used, the wavelength of which is long,beyond the range of electron absorption spectra.By excitation with the radiation of the Nd:YAGlaser at 1064 nm fluorescence and photochemicaldegradation can be effectively eliminated. Theseconditions mark a global optimum for the non-destructive recording of the Raman spectra of livingcells [2,3]. ‘Non-destructive analysis’ means analysis

Journal of Molecular Structure 509 (1999) 201–212

0022-2860/99/$ - see front matterq 1999 Elsevier Science B.V. All rights reserved.PII: S0022-2860(99)00221-5

www.elsevier.nl/locate/molstruc

q Dedicated to Professor Peter Klæboe, University of Oslo, on his70th birthday. One of the authors (B.S.) is looking back to manyyears of personal friendship and fruitful scientific cooperation.

* Corresponding author. Soniusweg 20, D-45259 Essen,Germany. Tel.:1 49-201-460638; fax:1 49-201-466650.

E-mail address:[email protected] (B. Schrader)

with no mechanical, chemical, photochemical orthermal decomposition.

We have reported in several publications someresults of our exploration of NIR FT Raman spectro-scopy in medical diagnostics [4–6] and have alreadyshown some application to the analysis of plants [3],e.g. fruit, herbs, and demonstrated taxonomy ofneedle trees by Raman spectroscopy of their needles[7]. Analyses of natural products in green plants is noproblem anymore. Urlaub et al. [8] studied the distri-bution of alkaloids inliana plants using micro NIR FTRaman spectroscopy. By combining excitation in thevisible range with surface enhancement with silversols Rosch et al. [9] were able to study the distributionof origanum and thyme oil inLamiaceaeplants.

We especially want to show the kind of results onecan get from the original samples without any specialpreparation—extraction or separation, since thiswould result in a destruction of the cells and someof its air or light sensitive components. Of courseRaman spectroscopy is not a method for traceanalyses. Most bands in the spectra are produced bygroups of compounds in a concentration of more than1%. Only substances, which exhibit a pre-resonanceenhancement of Raman spectra, as the carotenoidsmay be observed in a somewhat lower concentration.

2. Experimental

The usual sampling technique in NIR FT Ramanspectroscopy has been modified somewhat in order todeal with the special properties of plants.

Some samples were investigated in a ‘sample cup’,a cylinder of 10 mm diameter, 10 mm long, ofaluminum with a parabolic highly polished cavitywith a diameter of about 5 mm, covered with awindow of CaF2, 1 mm thick (Fig. 1(a)).

In order to record representative Raman spectra ofinhomogeneous samples like leaves, flowers andseeds without expensive preparation we have put thesamples loosely in sample tubes in the center of aspherical mirror (HK in Fig. 1(b)). Behind theentrance optics we have arranged a notch filter N,[1], which reflects the exciting radiation, emergingfrom the sample with a reflectivity of more than 99%.

This arrangement provides a multiple reflectioncell, which integrates the Raman spectra fromdifferent parts of the sample and, at the same time,enhances its intensity considerably, it works as thespherical cuvette [1]. If the overall reflectivity of thesample arrangement isr , then the enhancement factorF is

F � 1=�1 2 r�:This means, forr � 80%; F � 5.

Since a Raman spectrum excited at 1064 nm iscovering a spectral range, where NIR absorptionspectra of the overtones and combinations ofinfrared bands are recorded, it is necessary todiscuss if and how NIR Raman spectra aredisturbed by the NIR absorption spectra. Themain component of living tissues is water, thereforeit seems to be sufficient to discuss the properties of theabsorption spectrum of water in this range [3]. We seethat the exciting radiation of 1064 nm is situated at a

B. Schrader et al. / Journal of Molecular Structure 509 (1999) 201–212202

Fig. 1. Sample arrangement for Raman spectroscopy of plant tissues. (a) Sample cup, a parabolic highly polished insertion in an aluminumcylinder, closed with a window of CaF2, illuminated with the beam of a Nd:YAG laser, L, with a wavelength of 1064 nm. The diffusely reflectedlaser radiation, emerging from the sample is reflected by the notch filter, N, which transmits the Raman radiation, RA. (b) The sample in a testtube is adjusted in the center of a half spherical mirror, HK, which reflects the exciting and Raman radiation, emerging from the sample back tothe diffusely reflecting sample.

minimum of the water absorption spectrum. This hastwo consequences:

• The penetration depth of the exciting radiation inpure water is several cm (37% transmission for athickness of 4 cm), therefore large volumina maybe illuminated uniformly, also, the danger of over-heating the sample by the exciting radiation isminimal.

• The depth, from which Raman signals areobserved, is for the range 700–1800 cm21 onlysome millimeters—this means that mainlysignals from near the illuminated surface areobserved. However, signals with Raman shiftssmaller than 700 cm21 may give information ofdeeper layers.

The Raman spectra in this paper were recordedusing an NIR FT Raman Spectrometer BRUKERRFS 100 and another especially designed smallBruker NIR FT Raman spectrometer with a diode-pumped Nd:YAG laser, emitting a light flux of up to350 mW at 1064 nm and observation by a Germaniumdetector, cooled with liquid nitrogen. Most spectrahave been recorded with a resolution of 4 or 8 cm21

with a laser power of up to 350 mW using an unfo-cused laser beam with recording times between 8 and45 min.

3. Pigments of plants

For many years the study of the vibrational spectraof chlorophyll in its various functions in the photo-synthetic process has found great interest, especiallyin situ in its natural environment. Applying resonanceRaman spectroscopy (RRS) to the photosyntheticmembrane and its subunits results in several difficul-ties, two of which (thermal degradation and lumines-cence) are typical in studies of any biological materialby this technique [10]. Surface-enhanced Ramanspectroscopy (SERS) may be useful for reducingfluorescence problems [11], however this techniquecannot be applied non-destructively to living tissues.The most important compound of plants, chlorophyll,could not be investigated by ‘classical’ Raman spec-troscopy due to thermal degradation, luminescenceand photooxidation [10]. However, spectra, excitedwith radiation of 1064 nm show an enhancement ofthe intensity of the chlorophyll bands by a pre-reso-nance Raman effect [12].

Fig. 2 shows plant material with a large concentra-tion of chlorophyll: broccoli, grass and spinach. Somebands of low intensity at 1602, 1326, 1287 and744 cm21 can be assigned to chlorophyll [7,10]. Thestrongest bands however are due to carotenoids: at1157 and 1526 cm21. Carotenoids can be detected

B. Schrader et al. / Journal of Molecular Structure 509 (1999) 201–212 203

Fig. 2. Raman spectra of plant material with a large concentration of chlorophyll: (a) broccoli; (b) grass; (c) spinach.

quite easily employing these bands, for instance intagetesflower, Fig. 6(a), red paprika andsafran, thestamen of crocus [3].

Every autumn one can observe the beautiful color

changes preceding the loss of leaves of deciduoustrees. Chlorophyll breakdown is a typical feature ofsenescence and programmed cell death—essentialaspects of plant development [13]. Matile and


Fig. 3. Raman spectra of maple leaves (Acer platanoidesL.): (a) red leaves in autumn; (b) green summer leaves; difference spectrum(c� a2 b), positive bands mean increase of concentration.

Fig. 4. Raman spectra of the South-African xerophyte (Myrothamnus flabellifolia): (a) green leaf; (b) brown bud; difference spectrum(c� a2 b), positive bands mean increase of concentration.

Krautler [14,15] have pointed out that—after theperiod of photosynthetic activity—chlorophyllwould irreversibly damage the cells because of itsphotodynamic properties. This would hinder thenormal decay of the cell material, necessary for therecycling of proteins and carbohydrates. Thereforechlorophyll is deactivated by a ring cleavage into aphotochemically inactive catabolite. Directlyconnected with the activity of chlorophyll is that ofthe carotenoids: They act as antennae pigments andfavor energy transfer in the cell. They also protect thephotosynthetic apparatus by quenching the excitedtriplet states of the chlorophyll as well as singletoxygen. They are antioxidants, anticarcinogens andantimutagens [16,17]. After the decay of chlorophyllthe colors of carotenoids and flavonoids become visible.

Fig. 3(b) shows Raman spectra of green summerleaves of maple, (a) the red autumn leaves, and (c)the difference spectrum, (after2 before); positivebands mean increase of the intensity). Surprisinglybands, due to chlorophyll do not seem to decrease,however some bands at 1610 and 1690 cm21 increase.A comparison with the spectra of plant pigments inFig. 6 suggests that these are bands of the ring vibra-tions of flavonoids.

We have also studied two examples of the inverseprocess, the development of chlorophyll in drybrown plants. A South-African xerophytic plantMyrothamnus flabellifolia develops green leaves(Raman spectra in Fig. 4(a)) from brown buds (Fig.4(b)) within 5 h after being watered. NIR Ramanspectroscopy suggests that the chlorophyll of thegreen leaves was already present in the dry plant.Thus, the development of the green leaves seems tobe mainly a physical effect. This is confirmed by thefact that the plants develop green leaves, even whenwatered and kept in a dark room. However, a negativedifference band at 1607 suggests a decrease of theflavonoids when the brown bud develops to greenleaves.

A similar behavior of the ‘False Rose of Jericho’,Selaginella lepidophyllacan also be followed directly.This brown ball unfolds into an open structure withgreen leaves, during a few hours (Raman spectra inFig. 5(b) and (a)). Again, there is a decrease of theband at 1605 cm21 and an increase of the carotenoidbands, at 1157 and 1526 cm21.

Fig. 6 shows typical examples of plant pigments,assumed to be responsible of the yellow and red colorsof the autumn leaves, carotenoids (Fig. 6(a), tagetes),


Fig. 5. Raman spectra of a pteridophyte (Selaginella lepidophylla) the ‘false rose of Jericho’): (a) green leaf after unfolding of plant; (b) brownplant.

and different flavonoids, universally present [16] in allspecies of land plants (Fig. 6(b) apple skin, (c) rasp-berry and (d) rose). All flavonoids are derived from astructural unit C6H5–CH2–CHyCH–C6H5, in whichCH groups are exchanged against OH groups. Allshow typical benzene ring vibrations at 1606–1610 cm21. They are the main pigments of leaves,fruits and flowers.

4. Essential oils and drugs

We have already discussed the NIR Raman spectraof green dill leaves and seeds, of the herbs showingseveral bands of lower intensity, which seem to

characterize the essential oils, especially in therange above 1600 cm21 [3].

Fig. 7 shows the Raman spectra of powders ofrosemary leaves with—from bottom to top—increasing concentration of carnosic acid (0.4–7.8%), a valuable diterpene substance with antiox-idative properties. A clear dependence of any bandintensity from the determined concentration is notyet visible. This may be due to different reactionproducts appearing at the same time in differentconcentrations [18]. Also, the possibility ofsystematic and statistical errors and artifactsduring the determination of the carnosic acid contentas well as during the measurement of the Ramanspectra have to be investigated. A more detailed inter-pretation should be possible in the near future, when


Fig. 6. Raman spectra of plant colors: (a) petal of tagetes (T. patulusL.); (b) red skin of a Boskop apple; (c) raspberry fruit; (d) petal of red rose.

Raman spectra of pure carnosic acid, isolated fromrosemary extracts, are available.

NIR Raman spectra of camomile flowers with high,small and medium concentration of matricine areshown in Fig. 8(a)–(c). For comparison in Fig. 8(d)a Raman spectrum of a matricine crystal is shown.Matricine is regarded to be one of the most importantantiphlogistic components of the camomile drug [19].Unfortunately, no clearly isolated bands of the matri-cine spectrum appear in the NIR Raman spectrum ofthe powdered flowers. Only the weak bands at1444 cm21 could be assigned to this molecule; otherstronger bands are more or less superimposed by thespectral influences of the plant matrix. Furthermore,there may occur artifacts during the measurement ofthe Raman spectra. It has been found that driedchamomile flowers lost about 70% of their matricinecontent within 2 years, even when stored undercontrolled conditions [20].

In Fig. 9(a) the spectra of the yellow tubular flowersand in Fig. 9(b) the spectrum of the bottom of a flowerhead are shown. The difference between both spectrais significant. Especially, Fig. 9(b) shows Ramanlines of the (CxC)2 residue resulting fromcis- andtrans-spiroethers, which, beside matricine are known

to occur in the extracts of flower heads in relativelyhigh amounts [21].

Some discrepancies must obviously be removed,however, we believe that NIR Raman spectroscopyis a powerful tool, able to study the concentration ofcomponents at different parts of the plant.

5. Optimization of plant fiber

The recent development of environmentallyfriendly alternatives to petrochemical-derivedproducts has led to an enhanced awareness of thevalue of renewable plant sources. The annual cropplant Linum usitatissimumL. provides an alternativesource of both, oil and fiber in the form of linseed oiland flax fiber. The quality of the flax fiber is deter-mined by graders and buyers using subjective eye andhand evaluations [22].

Prior investigations by chemical, microscopic andinstrumental analyses are showing significant differ-ences between high- and low-quality flax fibers [23–25]. Above all NIR FT Raman spectroscopy seems tobe a suitable objective method to detect the major


Fig. 7. Raman spectra of rosemary (Rosmarinus officinalisL.) powder with different concentration of carnosic acid: (a) 0.4, (b) 0.6, (c) 1.3, (d)2.0, (e) 2.4, (f) 3.06, (g) 3.96, (h) 4.73, (i) 5.5, (j) 6.0, (k) 6.44, and (l) 7.8. All spectra were normalized for thed(CH) range (1420–1480 cm21).


Fig. 9. Raman spectra of (a) the camomile tubular flowers, (b) the bottom of the flower heads, variety ‘Manzana’; insert: structural formula ofen-in-spiroether.

Fig. 8. Raman spectra of powdered camomile flower heads (Matricaria chamomillaL.): (a) variety ‘Manzana’; (b) Egyptian type; (c) Germantype; (d) crystals of matricine; insert: structural formula of matricine.

chemical components and their relative amounts offlax stems and their anatomically parts in situ [26,27].

We observed and discussed the changes of thevibrational behavior of non-retted flax fibers oflinseed (Linum usitatissimumL., cv. Gold Merchant),with relatively short fibers, in relation to the numberof the mechanical treatments releasing bast fibersfrom the woody part of the stem. The plant materialwas not retted after pulling, it was loosely shelvedunder dry conditions. The shives (broken woodyparts of the flax stem) were removed by mechanicalprocessing. Fibers obtained by this procedure differ intheir properties depending on the number of passes ofthe mechanical treatment. The natural fibers havebeen studied without any chemical pre-treatments.

The flax fibers consist mainly of cellulose (65–70%, w/w) but also of hemicellulose (16%, w/w),lignin (3%, w/w) and pectic material (3%, w/w).These are the major components besides smallamounts of fats and waxes, protein and residual ash[28,29] which can also be detected by FT Ramanspectroscopic investigations of drew retted flax [26].

All Raman spectra of this paragraph are normalizedfor then (CH) range, 3040–2780 cm21. Fig. 10 illus-trates the Raman spectra of green linseed flax during

mechanical processing. Hereby the woody parts of theflax stems were broken. Bast fibers treated this wayare a composition of phloem fibers, parenchyma, cuti-cular and epidermal tissue [26]. FT Raman spectra ofthe bast fibers clearly indicate the ongoing disappear-ance of the lignin parts. The Raman lines at3069 cm21 (n (CHarom)), 2937 cm21 (n(CHaliph)) andthe doublet at 1657 cm21/1600 cm21, arising fromthe aromatic ring stretch (1600 cm21) with an accom-panying band (1657 cm21) assigned to the coniferal-dehyde carbonyl and ethylenic double bond inconiferyl alcohol [26,30], lose their intensitydepending on the number of the mechanical treat-ments. Also the weak band at 1737 cm21 assigned tothe acetylated hemicellulosic polysaccharides locatedin the xylem tissue are disappearing by the mechan-ical isolation of the bast fibers.

Already after three times passing the mechanicalprocedure the FT Raman spectra of the isolated bastfibers appear to be primarily that of cellulose, see Fig.11. However, the signal intensities at 1604 cm21,1530 cm21 and a broad shoulder of the very strongband at 2894 cm21 indicate that there are smallamounts of lignin, pectins and hemicellulosic poly-saccharides associated with cellulose. The vibrational


Fig. 10. FT Raman spectra of mechanically isolated flax fiber after: (a) one, (b) three, (c) seven, and (d) 10 mechanical treatments. The spectraare normalized for the range 2800–3000 cm21.

modes of theb (1! 4) glycosidic linkage of theb -d-glucopyranosyl units of cellulose,nas (C–O–C) at1096 cm21 and n s (C–O–C) at1121 cm21 coupledwith the nas (CC) of the ring breathing at 1153 cm21

[31–33], show a sensitive response to the mechanicaltreatments, see Fig. 12.

The ratio of the intensities,R� I �1121 cm21�=I �1096 cm21�, of the fiber modes decreases withincreasing number of mechanical treatments. Table1 represents the trend of the intensity ratiosR of thedeconvoluted triplets depending on the number of themechanical procedures.

This confirms the results of IR investigations ofnative cellulose that the bands in the skeletal region,including the above-observed ones, are very sensitiveagainst changes of cellulose crystallinity. Increasingband intensities indicate an increase of cellulose crys-tallinity and consequently a decrease of amorphousregions in the fibrillar structure of cellulose [34].Relative intensity measurements carried out on theNIR FT Raman spectra of microcrystalline cellulose,a cellulose with a relatively high cellulose crystalli-nity, yield to an intensitiy ratioR� 0:45^ 0:05.Cellulose samples of decreasing crystallinity obtainedby milling the microcrystalline cellulose showedincreasing intensity ratiosR.

It can be concluded from our NIR FT Ramanspectroscopic investigations that the mechanicalprocessing brings about an alteration of the cellu-lose crystallinity of the bast fibers. The increasingband sharpness and increasing band intensities ofthe skeletal modesn s/as (C–O–C) during the step-wise mechanical treatment indicate an increase ofcellulose crystallinity by mechanical processing.The change in intensity ratio R�I �1121 cm21�=I �1096 cm21� illustrate that the cellu-lose crystallinity of the mechanically treated fibersapproaches the crystallinity of a microcrystallinecellulose with increasing number of mechanicalfiber processing passes.


Table 1Relative band intensities versus the number of the mechanical fibertreatments

R� I(1121 cm21)/I(1096 cm21)

Number of mechanicalfiber treatments

1.3 11.0 30.8 70.6 10

Fig. 11. FT-Raman spectrum of microcrystalline cellulose.

6. Conclusions

• By excitation with the radiation of the Nd:YAGlaser at 1064 nm fluorescence and photochemicaldegradation of living cells can be effectively elimi-nated. These conditions mark a global optimum forthe non-destructive analysis of the Raman spectra.This means analysis with no mechanical, chemical,photochemical or thermal decomposition.

• The metamorphosis of pigments in plant cells canbe observed directly: the decay of chlorophyll andthe development of the beautiful color changes inautumn that precede the loss of leaves from decid-uous trees; inversely, the development of chloro-phyll can also be followed.

• NIR FT Raman spectra of green plants are domi-nated by the bands of the carotenes and chloro-phyll. Spectra of herbs show in addition thebands of the essential oils. Since their concentra-tion depends on the individual genotype, growingconditions and harvest time, Raman analysis maybe a helpful tool for optimizing breeding and culti-vation of plants. Also, an application to taxonomyof plants is possible.

• Also the fruits of these plants show the spectra ofthe essential oils, however, they are often super-imposed by broad bands produced by the plantmatrix and the seed coats.

• The study of the distribution of natural productsover the different parts of the plant by NIR FTRaman spectroscopy can be of great help in plantprocessing.

• Raman spectra of biomaterials can further beemployed to control of food [35]. to study productsand artifacts of biomaterials which can be of helpin characterization, dating, for restoration andconservation, even in archaeology [36].

• The qualitative and quantitative evaluation of theRaman spectra needs spectra of standardsubstances for calibration. Since there are onlyfew of them published, we ask the interested scien-tific community to publish spectra of puresubstances of interest in plant physiology and tocooperate in setting up an international collectionof Raman spectra of molecules of biologicalinterest in plant physiology, however, as alreadypointed out in prior publications, also for applica-tion in medical diagnostics.

The present paper intends to demonstrate the powerof non-destructive analysis of living cells. However,there is, of course, a much larger field of applying NIRFT Raman spectroscopy to any biochemical analysisof cells and tissues in combination with the other well-established methods of analysis.

Acknowledgements

Financial help of the Deutsche Forschungsge-meinschaft and the Fonds der Chemischen Industrieand instrumental support by Bruker Optik, Karlsruheis gratefully acknowledged.

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Non-destructive NIR FT Raman analysis of plants