The Characterization and Differentiation of Higher Plants byFourier Transform Infrared Spectroscopy

SEVGI TURKER GORGULU, MUSA DOGAN, and FERIDE SEVERCAN*Department of Biology, Middle East Technical University, 06531 Ankara, Turkey

Several techniques have been used to identify and classify plants. We

proposed Fourier transform infrared (FT-IR) spectroscopy, together with

hierarchical cluster analysis, as a rapid and noninvasive technique to

differentiate plants based on their leaf fragments. We applied this

technique to three different genera, namely, Ranunculus (Ranunculaceae),

Acantholimon (Plumbaginaceae), and Astragalus (Leguminoseae). All of

these genera are angiosperms and include a large number of species in

Turkey. Ranunculus and Acantholimon have ornamental importance,

while Astragalus is an important pharmaceutical genus. The FT-IR

spectra revealed dramatic differences, which indicated the variations in

lipid metabolism, carbohydrate composition, and protein conformation of

the genera. Moreover, cell wall polysaccharides including diverse groups

could be identified for each genus. Acantholimon was found to have the

highest hydrogen capacity in its polysaccharide and proteins. A higher

lignin content and a lower occurrence of decarboxylation and pectin

esterification reactions were appointed for Ranunculus and Astragalus

compared to Acantholimon. All these results suggested that FT-IR

spectroscopy can be successfully applied to differentiate genera, as

demonstrated here with Ranunculus, Astragalus, and Acantholimon. In

addition, we used this technique to identify the same species from different

geographical regions. In conclusion, the current FT-IR study presents a

novel method for rapid and accurate molecular characterization and

identification of plants based on the compositional and structural

differences in their macromolecules.

Index Headings: Ranunculus; Acantholimon; Astragalus; Fourier trans-

form infrared spectroscopy; FT-IR spectroscopy; Plant characterization;

Leaf; Lipid; Protein; Carbohydrates; Cluster analysis.

INTRODUCTION

Plant taxonomy is the science of describing and naming anygroup of plant to represent a distinct taxonomic unit.1

Taxonomists classify plants while trying to understandevolutionary aspects of plants according to not only the degreeof similarity between their morphological and anatomicalcharacteristics, but also any other molecular or even chemicalproperties.2,3 By using these properties, plant taxonomists havebeen establishing taxonomic markers and named taxa.However, there are still disagreements among taxonomistswith regard to the naturalness of plant groups.1 Since plantscomprise a wide range of different molecular componentsvarying from one group to another, the data obtained from thesestructures may bring fundamental evidence for characterizationof plants. Therefore, the techniques giving information at themolecular level may also be used to improve plant systematics.

Fourier transform infrared (FT-IR) spectroscopy is moresensitive, rapid, and more environmentally friendly comparedto other chemical methods.4 It is also a noninvasive techniquethat gives not only qualitative and quantitative informationabout the chemical structure, but also information about

molecular conformation and interaction between neighboringmolecules. Thus, this technique detects macromolecularcompounds such as proteins, lipids, carbohydrates, and nucleicacids, simultaneously.4–9 FT-IR spectroscopy has been widelyused for microbial and bacterial characterization.10–13 On theother hand, the application of this method to plant sciences islimited. In one of those studies, FT-IR spectroscopy was usedto distinguish fossil gymnosperm leaf remains of Cordaitesprincipalis and Cordaites borassifolus.14 The other studydiscriminated cell walls of apple from other fruits andvegetables by only monitoring signals arising from polysac-charides.15 Furthermore, Kacurakova et al.16 studied charac-terization of plant carbohydrates by FT-IR spectroscopytogether with microscopy. Recently, this technique was alsoapplied to monitor salt stress effects on the changes in chemicalcomposition in the leaves of some plants.17

In the present work, we aimed to provide molecularinformation for the identification of plants at the genus level.For this purpose, FT-IR spectroscopy was used to characterizedifferent genera by using its ability to simultaneously probedifferent functional groups belonging to different biomole-cules.

The genus Acantholimon (Plumbaginaceae) contains about196 species and is centered especially in the Irano-TuranianPhytogeographic Region.18 The genus has important ecologicalroles in preventing erosion. Some of the species are also widelyused as ornamentals because of their nicely colored and long-lasting flowers.18,19

Ranunculus (Ranunculaceae) covers about 400 species andcomprises the crowfoots characterized by glossy yellowflowers.20 They are especially common in the woods andfields of the northern temperate region.20,21

Astragalus (Leguminosae or Fabaceae), which is one of thelargest groups of vascular plants on Earth, is represented by2500 documented species. This genus includes herbaceousplants, some of which are merely ornamental and others ofwhich are medicinal or poisonous.22–24 They are widelydistributed throughout the temperate region of the world andlocated principally in Europe, Asia, and North America.22–25

Although a plant can easily be identified by its phenotypiccharacteristics, it is not always possible to characterize the plantfrom a small portion of leaves. We have chosen here threedifferent genera to test the discrimination potential of FT-IRspectroscopy based on biochemical profiles of their leaves. Ifwe succeed in differentiating the plants using their infraredspectra, this technique can be applied in the future as a rapidand sensitive method to distinguish different taxa where onlyplant parts are available rather than the whole plant.

MATERIALS AND METHODS

In order to obtain molecular information, the leaves of plantsspecimens were used. The specimens of the genera Astragalus

Received 4 October 2006; accepted 14 January 2007.* Author to whom correspondence should be sent. E-mail: feride@metu.edu.tr.

300 Volume 61, Number 3, 2007 APPLIED SPECTROSCOPY0003-7028/07/6103-0300$2.00/0

� 2007 Society for Applied Spectroscopy

and Ranunculus were collected from Eastern Anatolia andCyprus by the late professor P. H. Davis from EdinburghUniversity in the years of 1966 and 1957, respectively. For thecomparison of the genera, Astragalus species (n¼ 5) collectedfrom Eastern Anatolia were used. Acantholimon specimenswere collected from Central Anatolia in the year 2000 byProfessor Musa Dogan of the Middle East Technical University(Turkey) and Dr. Galip Akaydin from Hacettepe University(Turkey). The species names are as follows: Ranunculusbrutius, R. brachylobus, R. cuneatus, R. fenzli, R. oreophilus,Acantholimon caserium, A. confertiflorum, A. halofilum, A.

puberulum, A. armenum, Astragalus bicolour, A. asciocalyx, A.angistiflorus, A. camplosema, A. cinereus, A. caprinus, and A.macrocarpus.

The samples were prepared using the potassium bromide(KBr) pellet technique. The leaves were grounded in an agatemortar containing liquid nitrogen for 5 minutes to obtain apowder form. Small quantities of the powder form were thenmixed with KBr at a 1/75 ratio to produce a homogenouspowder. This mixture was subsequently subjected to a pressureof 1100 kg/cm2 for 8 minutes to produce a thin KBr disk orpellet. We prepared three different pellets from the same

FIG. 1. The average FT-IR spectra of (A) Ranunculus, (B) Astragalus, and (C) Acantholimon in the 4000–410 cm�1 region.

TABLE I. General band assignments of the average FT-IR spectrum of plants based on the literature.

Peak # Frequency (cm�1) Definition of the Spectral Assignments

1 3380 O–H and N–H group stretching vibration: carbohydrates. protein2 2957 CH3 asymmetric stretching: mainly lipids with a little contribution from proteins, carbohydrates, and nucleic acids3 2922 CH2 asymmetric stretching: mainly lipids with a little contribution from proteins, carbohydrates, and nucleic acids4 2873 CH3 symmetric stretching: mainly protein with a little contribution from lipids, carbohydrates, and nucleic acids5 2852 CH2 symmetric stretching: mainly lipids with a little contribution from proteins, carbohydrates, and nucleic acids6 1733 Saturated ester C¼O stretch: phospholipids, cholesterol esters, hemicellulose, and pectin7 1651 Amide I (protein C¼O stretch): protein and pectin8 1601 C¼O aromatic stretching: lignin9 1555 Amide II (C¼N and N–H stretching): mainly proteins

10 1446 C–H: cell wall polysaccarides1415 O–H bending: cell wall polysaccarides, alcohols, and carboxylic acids1250 Pectic substances

11 1235 Amide III (C¼N and N–H stretching): mainly proteins12 1145 Cellulose (b-1.4 glucan)13 1101 Antisymmetric in-phase: pectic substances

1073 Rhamnogalactorunan. b-galactan14 1064 C–O stretching: cell wall polysaccarides (glucomannan)15 1035 OH and C–OH stretching: cell wall polysaccarides (arabinan)16 895 Arabinan

893 Galactan17 873 b-D-Fructose

APPLIED SPECTROSCOPY 301

species whose FT-IR spectra were similar, and thereby anaverage spectrum was obtained. We performed the sameprocedure for each different species (n ¼ 5) belonging to thesame genus whose spectra were also identical and averaged.Finally, these average spectra belonging to each differentgenera were then used for visual comparison.

Infrared spectra were obtained with a BOMEM 157 FT-IRSpectrometer (Michelson Series, Bomem, Quebec, Canada)equipped with a deuterated triglycine sulfate (DTGS) detector.The spectrometer was continuously purged with dry air toeliminate atmospheric water vapor. The FT-IR spectra wererecorded in the region between 4000 cm�1 and 410 cm�1 atroom temperature using a Grasebay/Specac temperaturecontroller unit. A hundred scans were taken for eachinterferogram at a resolution of 4 cm�1.

For digital data processing, Win Bomem Easy software wasused. All spectra were baseline corrected in the region between4000 and 410 cm�1. The band positions were measuredaccording to the center of weight. The spectra were normalizedwith respect to specific bands for visual presentation of the

changes. However, each original baseline-corrected spectrumwas considered for accurate determination of the variations.Fourier self-deconvolution (FSD), which increases the resolu-tion of intrinsically overlapped bands by narrowing thebandwidth of individual components, was applied to the C–Hstretching region (3050–2800 cm�1) with an 0.7 gamma factor.After FSD application, the frequency values were measured.The lipid-to-protein ratio was calculated by taking the ratio ofthe intensity values of the CH2 symmetric to the CH3

symmetric bands.26,27

Statistical Test. For the statistical comparison of genera, weselected the peaks that were present in the spectra of all genera.The peaks, which are specific to a genus, were not evaluatedstatistically. These specific peaks, which were not in commonin all genera, were located especially in the region between1200 and 410 cm�1. These bands were further identified andassigned for each genus according to the literature. The resultswere expressed as mean 6 standard deviation. The differencesin the means of the Astragalus, Ranunculus, and Acantholimonwere compared using the analysis of variance (ANOVA)statistical test. A p value of less than 0.05 was consideredsignificant (p , 0.05*, p , 0.01**, and p , 0.001***).

Cluster Analysis. For cluster analysis, the spectra werevector normalized over the investigated frequency range.Hierarchical cluster analysis was performed using the clusteranalysis module of OPUS 5.5 (Bruker Optic, GmbH). It wasapplied to distinguish the spectrum of each genus using afrequency range between 4000 and 410 cm�1. For hierarchicalcluster analysis, Ward’s algorithm was used to constructdendrograms.28 As a distance measure, Pearson’s product–moment correlation coefficient was used.28,29

RESULTS AND DISCUSSION

This study was carried out with FT-IR spectroscopy todetermine whether this technique can identify plants fromdifferent genera, namely, Astragalus, Acantholimon, andRanunculus.

Figure 1 shows average FT-IR spectra of each genus(Astragalus, Acantholimon, and Ranunculus) in the 4000–410

FIG. 2. The dendrogram of a hierarchical cluster analysis of Astragalus,Ranunculus, and Acantholimon.

FIG. 3. The average FT-IR spectra of Acantholimon, Ranunculus, andAstragalus in the 3950–2800 cm�1 region. The spectra were normalized withrespect to the CH2 asymmetric stretching band, which is around 2925 cm�1.

302 Volume 61, Number 3, 2007

cm�1 region. As can be seen from the figure, they are complexspectra representing many different functional groups of lipids,carbohydrates, and proteins. The position and assignments ofthe bands are listed in Table I. Hierarchical clusteringcontained in the 4000–410 cm�1 region was used as aclassification method, resulting in the dendrogram displayedin Fig. 2. As seen from this figure, these three genera weresuccessfully differentiated by their infrared spectra, and threedistinct clusters representing each genus were produced.

In order to investigate the structural and compositionalvariations of macromolecules in the plants, detailed spectralanalyses were additionally performed in the whole spectralrange by dividing it into three distinct frequency regions whereall the bands were located. These regions are 3950–2800 cm�1,3050–2800 cm�1, and 2000–410 cm�1.

Figure 3 shows the average infrared spectra of Astragalus,Ranunculus, and Acantholimon in the 3950–2800 cm�1 region.The spectra were normalized with respect to the CH2

asymmetric stretching mode, which is observed around 2925cm�1. In this region, the broad peak (Amide A) located around3370 cm�1 arises mainly from the N–H and the O–H stretchingmodes of proteins and polysaccharides and intermolecular Hbonding.6,9,30 In the present study, water was largely removedin the preparation of the plant specimens. Therefore, thecontribution of water molecules to this band could be neglectedand only the contribution of polysaccharides and proteins werebe considered. It is seen from the figure that the intensity/areavalues of the Amide A band of Acantholimon (0.55 6 0.25/149.56 6 0.21) (p , 0.05) is lower than Astragalus (0.72 60.14/174.25 6 0.65) (p , 0.05) and Ranunculus (0.68 6 0.10/171.21 6 1.23) (p , 0.01). This indicated possible differencesin the concentration of polysaccharides and proteins present inthe samples.31,32 In addition, it has also been reported that thebroadness of this band results from the extent of hydrogenbonding in the molecule.33 The band was more broad forAcantholimon compared to Astragalus and Ranunculus, whichimplied that Acantholimon showed more hydrogen bondingcapacity in polysaccharides and proteins than the other genera.

The infrared spectra of Astragalus, Acantholimon, andRanunculus in the region between 3050 and 2800 cm�1 areshown in Fig. 4. The spectra were normalized with respect tothe CH2 asymmetric stretching band centered at around 2925cm�1. This region contains four bands, which are the CH2 andthe CH3 symmetric and asymmetric stretching modes. The CH3

and the CH2 asymmetric stretching bands are mainly due tolipids and the CH3 symmetric stretching bands are due toproteins. As seen from Fig. 4, the frequency and intensity/areavalues of these bands are different for each genus. This may bedue to differences in lipid metabolism of plants. The CH3

asymmetric stretching mode is correlated with the order of thedeep interior of the membrane, and an increase in the frequencycorresponds to increasing freedom of the acyl chain in thecenter of the bilayer.9,34 This band was positioned at 2954.326 0.05 cm�1, 2956.53 6 0.39 cm�1, and 2956.80 6 0.14cm�1, for Acantholimon, Astragalus, and Ranunculus, respec-tively. This result indicated a higher disorder of the interior partof the lipid chains for Ranunculus and Astragalus incomparison to Acantholimon.

FIG. 4. The average FT-IR spectra of Astragalus, Acantholimon, and Ranunculus in the 3050–2800 cm�1 region. The spectra were normalized with respect to theCH2 asymmetric stretching band, which is around 2925 cm�1.

TABLE II. The frequency values of the FT-IR bands for Acantholimon,Astragalus, and Ranunculus.

Acantholimon, n ¼ 5 Astragalus, n ¼ 5 Ranunculus, n ¼ 5

Band # Frequency Frequency Frequency

1 3373.93 6 0.84 3376.60 6 1.3* 3375.12 6 0.75*2 2954.32 6 0.05 2956.53 6 0.39 2956.80 6 0.143 2925.22 6 0.22 2924.40 6 0.26 2926.62 6 0.464 2872.92 6 0.64 2870.14 6 0.16 2870.18 6 0.055 2851.17 6 0.71 2853.64 6 0.52 2855.57 6 0.89*6 1736.31 6 0.01 1732.82 6 0.71* 1732.17 6 0.18*7 1647.70 6 0.78 1651.81 6 0.60 1652.28 6 0.258 1614.70 6 0.60 1601.64 6 0.96** 1604.22 6 1.23 **9 1538.11 6 0.14 NOT OBSERVED NOT OBSERVED

10 1447.38 6 1.66 NOT OBSERVED NOT OBSERVED11 1238.48 6 0.19 1264.32 6 0.38** 1248.73 6 1.44**12 1145.13 6 0.18 NOT OBSERVED NOT OBSERVED13 1104.64 6 0.70 1104.15 6 0.54 1098.15 6 0.71*14 1064.27 6 0.58 NOT OBSERVED NOT OBSERVED15 1036.19 6 0.25 NOT OBSERVED NOT OBSERVED16 895.34 6 0.14 NOT OBSERVED NOT OBSERVED17 873.88 6 1.2 NOT OBSERVED NOT OBSERVED

APPLIED SPECTROSCOPY 303

The CH2 asymmetric stretching band also monitors theorder/disorder state of membrane lipids. It is known that theband frequency increases with the introduction of gaucheconformers into the fatty acyl chains.8,27,35 Our results revealedthat the frequency of this band was at a higher value forRanunculus (2926.62 6 0.46 cm�1) when compared toAcantholimon (2925.22 6 0.22 cm�1) and Astragalus(2924.48 6 0.26 cm�1). This result might imply thatRanunculus had a more disordered membrane structurebecause it was composed of more gauche conformers in thefatty acyl chains of membrane phospholipids.

As reported in Table I, the CH2 symmetric stretching modeis positioned at 2851.77 6 0.53 cm�1 for Acantholimon,2853.64 6 0.52 cm�1 for Astragalus, and 2855.57 6 0.89cm�1 (p , 0.05) for Ranunculus. The peak position of thisband also gives information about the chain flexibility, which isrelated to the order of the membrane structure.9,27 Since thefrequency of this band for Ranunculus was a higher value, thisobservation supports the previously mentioned conclusion thatRanunculus has a more disordered membrane structure than theother genera.

According to the empirical rule, the differences in thebandwidth values of the C–H stretching bands reflect thevariation in the dynamics of cell membrane lipids.8,9,35 As seenfrom Fig. 4, the bandwidth value of the CH2 asymmetricstretching mode was higher for Ranunculus when compared tothe others. This result revealed that membrane lipids ofRanunculus were more mobile in comparison to Astragalusand Acantholimon. Similar behavior could also be concludedfrom the bandwidth value of the CH2 symmetric stretchinglocated at 2855 cm�1 (Fig. 4).

The intensity/area values of the CH2 symmetric stretchingband were lower for Astragalus and Ranunculus when

compared to Acantholimon, as can be seen from Table III.This finding suggested a low proportion of the CH2 groups inRanunculus and Astragalus, and this implied a low content ofphospholipids and fatty acyl chains in these genera, assuggested by Jackson et al.31 Although there are severaltechniques such as gas chromatography and Lowry proteinassays for macromolecule determination, the signal intensi-ty8,36–38 and band area9,36,37 of FT-IR bands give rapid andsensitive information about the relative quantity of macromol-ecules.

Table IV shows the lipid-to-protein ratio that was calculatedfrom ratio of intensity of the CH2 symmetric stretching band tothe CH3 symmetric stretching band. As seen from the table, thelipid-to-protein ratio is low for Astragalus and Ranunculuscompared to Acantholimon. This difference implied a highamount of protein or a low amount of lipid, supporting thefindings derived from the CH2 stretching band intensities orboth27,31,38 in Astragalus and Ranunculus.

The fingerprint region between 2000–410 cm�1 contains themodes of several different groups that are present in thesamples (Fig. 5). It is clearly observed from all spectra thatthere are several distinctions reflecting the differences in themolecular structure of Ranunculus, Astragalus, and Acantho-limon. These differences are summarized in Tables II and III interms of wavenumber and peak intensity/area values.

The absorption band around 1735 cm�1 is assigned to theC¼O group, which represents ester-containing compounds inmembrane lipids (cholestrol esters), cell wall pectin, andhemicellulose.6,27,39 As reported in Table III, the intensity/areavalues of the C¼O ester stretching are lower for Astragalus andRanunculus in comparison to Acantholimon. This implied thatthe concentration of molecules affecting this band was mostabundant in Acantholimon, which further supported high lipidcontent. The frequency values of this band are 1736.31 6 0.01cm�1 for Acantholimon, 1732.17 6 1.39 (p , 0.05) cm�1 forRanunculus, and 1732.17 6 0.71 cm�1 (p , 0.05) forAstragalus. The lower frequency value for Ranunculus andAstragalus indicated a high number of hydrogen bonds, whichcould be formed between the C¼O group of lipids andpolysaccharides and other chemicals containing O–Hgroup.40–42 Therefore, it could be concluded that Astragalus

TABLE III. Intensity/area values of FT-IR bands for Acantholimon, Astragalus, and Ranunculus.

Acantholimon, n ¼ 5 Astragalus, n ¼ 5 Ranunculus, n ¼ 5

Peak # Intensity Peak area Intensity Peak area Intensity Peak area

1 0.55 6 0.25* 149.56 6 0.21* 0.72 6 0.14** 174.25 6 0.65* 0.68 6 0.04** 171.21 6 0.232 0.34 6 0.72 99.38 6 0.12 0.31 6 0.13 102.24 6 1.34 0.34 6 0.28 101.45 6 0.323 0.43 6 1.53 142.22 6 1.34 0.36 6 0.67** 140.04 6 0.56 0.38 6 0.14** 142.32 6 0.344 0.34 6 0.06 125.25 6 0.89 0.29 6 0.57* 121.98 6 0.34 0.33 6 0.20 129.65 6 0.715 0.39 6 0.98 134.48 6 1.21 0.32 6 0.44 130.03 6 2.45 0.34 6 0.03 131.65 6 1.896 0.47 6 1.18* 135.39 6 0.16* 0.28 6 0.14** 121.36 6 0.23* 0.35 6 0.08* 135.89 6 0.297 0.72 6 1.12 159.69 6 2.12 0.83 6 0.20* 152.36 6 1.43 0.68 6 0.56* 159.56 6 1.658 0.41 6 0.49* 128.69 6 0.32* 0.73 6 1.64** 137 6 1.89* 0.58 6 0.67* 137.04 6 2.65*9 0.49 6 0.26 127.56 6 0.78 NOT OBSERVED NOT OBSERVED NOT OBSERVED NOT OBSERVED

10 0.62 6 0.48 125.25 6 0.43 NOT OBSERVED NOT OBSERVED NOT OBSERVED NOT OBSERVED11 0.61 6 0.51 122.56 6 0.23 0.31 6 0.78** 100.04 6 2.12* 0.42 6 0.11* 118.56 6 1.3412 0.66 6 0.23 135.62 6 1.43 NOT OBSERVED NOT OBSERVED NOT OBSERVED NOT OBSERVED13 0.82 6 1.03* 109.26 6 2.21* 0.46 6 0.38* 142.9 6 0.65 0.70 6 0.35 108.15 6 1.34*14 0.85 6 1.03 124.25 6 1.09 NOT OBSERVED NOT OBSERVED NOT OBSERVED NOT OBSERVED15 0.98 6 1.18 179.56 6 0.87 NOT OBSERVED NOT OBSERVED NOT OBSERVED NOT OBSERVED16 0.20 6 0.12 51.10 6 0.23 NOT OBSERVED NOT OBSERVED NOT OBSERVED NOT OBSERVED17 0.31 6 1.10 52.45 6 0.67 NOT OBSERVED NOT OBSERVED NOT OBSERVED NOT OBSERVED

TABLE IV. The lipid-to-protein ratio of Acantholimon, Astragalus, andRanunculus.

Ratio(intensity)

Acantholimon,n ¼ 5

Astragalus,n ¼ 5

Ranunculus,n ¼ 5

I2853/I2874 1.15 6 0.11 1.10 6 0.62 1.03 6 0.03*

304 Volume 61, Number 3, 2007

and Ranunculus contained more hydrogen bonds involving theC¼O groups associated with lipids and polysaccharidescompared to Acantholimon. In addition, Bestard et al.43

reported that the shifting to lower frequency values of thisband reflects a low degree of pectin esterification anddecarboxylation reactions. When compared to Acantholimon,the low frequency value of the C¼O mode for Ranunculus andAstragalus might mean a structural modification within the cellwall matrix that cannot be identified by carbohydrate analysis.

In the same region, the Amide I band at around 1650 cm�1

shows a higher value for Astragalus and Ranunculus comparedto Acantholimon (Table II). Since the position of the Amide Iband is sensitive to protein conformation,44–46 the changes inits frequency value points out conformational changes in theproteins of the sample. Our results revealed that the proteinstructure was similar for Astragalus and Ranunculus since thefrequency and spectral shape of this band were almost similar.

The band observed at around 1610 cm�1 is assigned to ligninaromatic ring stretching. This absorbance has been proven tobe useful in the detection of complex matrices by Mascarenhaset al.15 As seen from Fig. 5, the intensity/area of the band washigher for Astragalus and Ranunculus in comparison toAcantholimon. Therefore, it could be concluded that theconcentration of lignin could be higher for Astragalus andRanunculus when compared to Acantholimon.

The bands at around 1550 cm�1 and 1230 cm�1, which areassigned to proteins,5 could not be observed for Astragalus andRanunculus. Moreover, the pectin band, which could not beseen in the spectrum of Acantholimon, was located at 1258cm�1 for Astragalus and 1253 cm�1 for Ranunculus.

The band at 1446 cm�1, which is attributed to the aliphaticC–H group,33 was only observed in the Acantholimonspectrum. On the other hand, the band due to the O–H bendingvibration of alcohols and carboxylic acids33 is located at 1417cm�1 and 1414 cm�1 for Astragalus and Ranunculus,respectively.

The detailed analysis of the fingerprint region (2000–410cm�1) indicated that cell wall polysaccharides have distinctfrequency values due to the different combinations andglycosidic linkages of the monosaccharides (Table I). This

allows the identification of the cell wall polysaccharidestructure.39,45 The bands between 1160 and 1130 cm�1 havebeen described in the literature as the different glycosidiclinkages between monosaccharides forming polysaccharides.16

For instance, in the Acantholimon spectrum, there was a bandat 1146 cm�1 that can be assigned for (b-1.4) glucan units ofcellulose. This band was not observed in the Astragalus andRanunculus spectra.

The other band located at 1104 cm�1 both for Acantholimonand Astragalus and at 1098 cm�1 for Ranunculus is assigned topectic substances,16 which are composed of high homogalac-turonan content.16 The bands at 1072 cm�1 for Astragalus and1073 cm�1 for Ranunculus can be specific for bothrhamnogalactorunan and b-galactan.43 The next band waslocated at 1037 cm�1 for Astragalus and 1035 cm�1 forRanunculus and arises from b-linked arabinan.16

The spectrum of Acantholimon showed a maximum forglucomannan at 1064 cm�1 due to glucan and at 1034 cm�1

due to mannose units.16

The band assigned to galactan16 was observed at 893 cm�1

for Ranunculus and at 893 cm�1 for Astragalus. However, forAcantholimon two bands were observed at 895 cm�1 and 873cm�1 which are assigned to arabinan and b-D-fructose,respectively.15,16

Comparison of Some Astragalus Species Living inDifferent Environments. In order to test the identificationpotential of FT-IR spectroscopy at the level of species, weexamined Astragalus species collected from different geo-graphical environments. As indicated previously, all the spectraof Astragalus (n¼ 5) from Eastern Anatolia that were used inthe genera comparison are similar; therefore, they overlapped.For this reason, for visual comparison, the average of these fiveAstragalus spectra and the average of the species from theCyprus region whose spectra were also similar were used.However, for the accurate determination of the changes, eachoriginal baseline-corrected spectrum was considered.

It is known that different environmental conditions forceplants to adapt for living in that environment. This adaptationprocess causes molecular and structural variations even amongspecies from the same genus. Thus, FT-IR spectroscopy has

FIG. 5. The average FT-IR spectra of Astragalus, Acantholimon, and Ranunculus in the 2000–410 cm�1 region. The spectra were normalized with respect to theAmide I band, which is around 1650 cm�1.

APPLIED SPECTROSCOPY 305

been used to detect the changes in molecular structureassociated with environmental conditions.6,43

Figure 6 demonstrates the average spectra of Astragalusspecies that were collected from two different regions, namely,Eastern Anatolia and Cyprus. The spectra were normalizedwith respect to the N–H and the O–H stretching modes locatedat 3370 cm�1. As seen from the figure, there are differencesbetween the spectra in terms of positions and intensity/areavalues of bands, although the specimens belong to the samegenus.

As reported previously, the broadness of the Amide A bandlocated at 3370 cm�1 gives information about the hydrogenbonding capacity in polysaccharides and proteins.33 As seenfrom the figure, Eastern Anatolia species reflected a greaterlevel of hydrogen bonding capacity in these moleculescompared to the Cyprus species.

Figure 7 shows the infrared spectra of Astragalus speciesgrowing in Cyprus and Eastern Anatolia in the region between3050 and 2800 cm�1. The spectra were normalized with respectto the CH2 asymmetric stretching band located at 2925 cm�1.

This region contains four bands that are contributed from

proteins and lipids.8,9,34 As can be seen from the figure, the

frequencies of the CH3 and the CH2 asymmetric stretching

modes and the CH2 symmetric stretching mode were not

different for the two spectra. This result might imply that there

was no difference between the species from Cyprus and

Eastern Anatolia in terms of lipid order and chain flexibili-

ty.8,9,33,34 However, a lower bandwidth value of the CH2

asymmetric stretching band for the Eastern Anatolia species

compared to the Cyprus species revealed higher membrane

fluidity for the Cyprus species.8,9,35

As explained previously, the intensity/area value of the

absorption band is directly related to the concentration of the

molecules.39,45 The intensity/area values of four bands present

in the C–H stretching region were lower for the Eastern

Anatolia species in comparison to the Cyprus species. These

differences suggested low concentration of phospholipids and

proteins for the Eastern Anatolia Astragalus species. In

addition, the low lipid content for the Eastern Anatolia species

FIG. 7. The average FT-IR spectra of Astragalus species from different regions between 3050 and 2800 cm�1. The spectra were normalized with respect to the CH2

asymmetric stretching band, which is around 2925 cm�1.

FIG. 6. The average FT-IR spectra of Astragalus species from different regions between 3900 and 410 cm�1. The spectra were normalized with respect to theAmide A band, which is around 3370 cm�1.

306 Volume 61, Number 3, 2007

was supported by the low intensity values of the C¼O group at1735 cm�1, which is assigned to cholesterol esters.6,27,39

Figure 8 belongs to the fingerprint region between 2000 and410 cm�1. The figure demonstrates the differences between theCyprus and the Eastern Anatolia species. The main differencein this region is the position and characteristic of the Amide Iand the Amide II bands that are specifically due to proteins.44,46

For the Cyprus species, the Amide I and the Amide II bands arelocated at 1651 cm�1 and 1541 cm�1, respectively. On theother hand, the Amide I band is located at 1626 cm�1 while theAmide II band disappeared for the Eastern Anatolia species.The results indicated that the concentration and conformationof proteins were very different for each species because of thevariations in the spectral shape and frequency values of thesebands. In addition, the band at 1610 cm�1 due to ligninaromatic ring stretching15 showed high intensity for the EasternAnatolia species but it could not be seen for the Cyprus species.Therefore, it could be concluded that the concentration oflignin was higher in the Eastern Anatolia species.

As can be observed from Fig. 8, the intensity/area value ofthe O–H bending vibration of alcohols and carboxylic acids33

located at 1417 cm�1 was higher for the Eastern Anatoliaspecies. However, the intensity values of the bands resultingfrom cell wall polysaccharides were higher for the Cyprusspecies. This revealed that the Cyprus species contains morecell wall polysaccharides compared to the Eastern Anatoliaspecies.

In conclusion, this study demonstrates that moleculardifferences among Acantholimon, Astragalus, and Ranunculuscan be detected using FT-IR spectroscopy by sampling a smallportion of their leaves. Furthermore, we were able todifferentiate the species of the same genus living in differentgeographical regions. This method gives not only structuralinformation such as order/disorder of membrane lipids,conformational changes of the proteins, and variations in cellwall polysaccharides, but also quantitative information regard-ing macromolecules present in the system. Therefore, FT-IRspectroscopy can provide new insight based on the plant cellstructure and composition and can be introduced as a novelmethod for discrimination of plants from their chemical

structural differences at the level of genus and even at thelevel of species living in different environmental conditions.

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