Available online at www.scholarsresearchlibrary.

com

Scholars Research Library

Archives of Applied Science Research, 2011, 3 (1): 214-221

(http://scholarsresearchlibrary.com/archive.html)
ISSN 0975-508X CODEN (USA) AASRC9

On the nature of vibrational bands in the FTIR spectra of medicinal plant leaves
1

Mitali Konwar and 2G D Baruah

Department of Physics1, Moran College, Sibsagar-785670, Assam, India Department of Physics, Dibrugarh University, Dibrugarh-786004 Assam, India ______________________________________________________________________________
2

ABSTRACT Fifteen samples of dried leaves of medicinal values from different plants are selected for study of their FTIR spectra. The main purpose of their study is to observe the salient features exhibited by the IR spectra.All the fourteen bands originating from different groups (N-H, O-H, C-H, C=O, C=C and C=N) may be termed as key bands and one can follow their absorption characteristics and differentiate individual plant leaves or tissues. These observations also make it evident that the manifestation of bands in the infrared region results primarily from the chlorophyll molecules masking or suppressing the appearance of all other bands due to other molecular origin, which of at all appear, it does with extremely low intensities. ______________________________________________________________________________ INTRODUCTION In the present work we dealt with the nature of the visible absorption and fluorescence spectra of acetone extract of some medicinal plant leaves. It emerged that in the absorption spectra belonging to different medicinal plant leaves there is a continuous absorption below 5000. This is due to heavy absorption maxima of chlorophyll a and b at 4050, 4325 and 4625. A comparative studies of the absorption spectra of the plant leaves exhibited show that most of the absorption bands exhibit strong absorption in the region 6100-6700.The absorption bands are presumably due to chlorophyll-a and chlorophyll-b. There are also few additional absorption bands that appear in the spectrograms. These bands are due to the presence of unknown molecules. In general the intensities of the chlorophyll bands vary from species to species. In the present workr we shall consider the FTIR spectra of the eushed and dried samples of the plant leaves in the form of thin pellek. The primary aim to study the intensity variations of the prominent absorption bands and also look for additional bands which are characteristics of a particular leaf. From the survey of available literature it is reasonable to believe that the type of study being presented here has not been made in any of the earlier works. It is worthwhile to note
214 Scholar Research Library

Mitali Konwar et al Arch. Appl. Sci. Res., 2011, 3 (1):214-221 _____________________________________________________________________________________ that the chlorophyll molecules ( a and b) exhibits many absorption bands in the infrared region(4000-400cm-1 ).These absorption bands are chararacteristics of the molecular vibrational groups associated with the molecular structure of chlorophyll a and b .As in the case of absorption spectra of the acetone of the medicinal plant leaves, the infrared spectra of these plant leaves are expected to exhibit the characteristic bands of the chlorophyll molecules and their variations with the environments. An extensive literature now exists on the visible, infrared (IR), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPQ) spectroscopy of chlorophyll solutions [1-6]. The infrared spectra of dry chlorophyll a in non polar solvents generally are characterized by an absorption peak at about 1653cm-1 in aliphatic hydrocarbons, which has been generally assigned to C-9 ketone oxygen co coordinated to magnesium : C=O Mg. As this is the interaction that leads to dimmer formation,the absorption peak at ~ 1650cm-1 has been termed an aggregation peak; the short wavelength shoulder in aliphatic hydrocarbon solvents can be taken to indicate the presence of higher aggregates. Free ketone carbonyl absorption is found at 1695cm-1,and easter carbonyl absorption at 1735 cm-1. Hydrated chlorophyll a in CCl4 or benzene solution shows a typical dimmer IR spectrum, but with increased absorption at 1695cm1 it indicates some disaggregation to monomer. In aliphatic or alicyclic hydrocarbon solution of hydrated chlorophyll, however, a strong absorption is observed at 1638cm-1,which is no doubt similar to the absorption peak of hydrated chlorophyll in Nujol observed by Sherman and Wang [5], but which was not differentiated by them from the 1650-1660cm-1 aggregation peak. In solutions of hydrated chlorophyll in aliphatic hydrocarbon solvents, the 1695 cm-1 absorption peak, which is considered as due to a small amount of free carbonyl in the absorbing species. Therefore the absorption peak at 1638cm-1 is assigned to Ring vibration ketone oxygen, which is hydrogen, bonded to water coordinated to the central magnesium atom of another chlorophyll molecule:

O H

Mg

This assignment follows from the near absence of free keto carbonyl in the infrared spectrum, from the evidence in the chlorophyll, and from the water spectrum in the OH stretch region. Three Absorption peaks corresponding to water coordinated to magnesium, to hydrogen-bonded ester carbonyl, and to hydrogen bonded keto carbonyl can be seen in the OH stretch region of the IR (3600cm-1, 3460cm-1, 3250cm-1), and these correspond very well to the assignments made in the 1600-1800 cm-1 of the IR spectrum. Studies with models show that a water molecule coordinated to the magnesium atom of one chlorophyll molecule can be oriented to form hydrogen bonds simultaneously to the ketone oxygen ( at C-10) carnonyl functions of another chlorophyll molecule. Particular of this easter carbonyl is established by the splitting of the ester carbonyl absorptions into two peaks now appearing at 1743 and 1727cm-1.The interactions described here is compatible with the absolute configuration of chlorophyll established by Fleming[6]. Thus, the 1660cm-1 peak (chlorophyll n-mer), the 1653cm-1 peak (chlorophyll dimmer),and the 1638cm-1 peak (chlorophyll-water aggregates) are aggregation peaks, but their origin and significance are quite different. It is worthwhile to tabulate the prominent wave numbers and vibrational modes of the major spectral regions (I-V) between 1760 and 1500cm-1 in the FTIR spectrum of chlorophyll photosystem II (segui et.al)[14] .
215 Scholar Research Library

Mitali Konwar et al

Arch. Appl. Sci. Res., 2011, 3 (1):214-221 _____________________________________________________________________________________

The dimerization of photo chlorophyll pigments in non-polar solvents was earlier investigated by Rasquin et. al. They showed that the infrared spectra in the region 1600-1800 cm-1 clearly show the existence of a coordination interaction between the C-9 ketone oxygen function of one molecule and central magnesium atom of another molecule. Similarly the infrared spectra in the OH stretching region (3200-3800cm-1) providea valuable teat of water content in the samples. Thus in polar solvents chlorophyll-a displays a nonbonded C 9=O stretching vibration (16951700cm-1) which shifts when aggregates form, as already known from IR investigations. The intensified absorption between 1600 to1620cm-1 is due to conjugated C=C bonds in the phenyl group. Fifteen samples of dried leafs of medicinal values from different plants are selected for study of their FTIR spectra. The main purpose of this study is to observe the salient features exhibited by the IR spectra of the plant leaves and the differences if any among the IR bands and make a possible correlation between the individual samples. The investigation on the absorption characteristics of the leaves described earlier suggested to the author that an examination of their FTIR spectra might yield results of interest. This has indeed proved to be the case. MATERIALS AND METHODS Experimental: The sample of individual leaves are adequately dried in an oven to avoid their moisture content and subsequently crushed into fine powders. The FTIR spectra are recorded in KBR by a sophisticated computer-controlled FTIR Perkin Elmer spectrometer with He-Ne laser as reference. Fig 1 ( a,b,.) shows the spectra for a direct comparison of the characteristic features. RESULTS AND DISCUSSION:

The complexity of the spectra below 1600cm-1 is so great that we have not attempted to assign the bands. Absorption bands are presented from 1000 to 3860 cm-1 in order to make a reasonable comparison. The bands lying in these region is sufficiently intense and therefore it is always
216 Scholar Research Library

Mitali Konwar et al Arch. Appl. Sci. Res., 2011, 3 (1):214-221 _____________________________________________________________________________________ possible to make a comparative study. Due to this reason we have also not indicated the relative intensities. The most familiar amongst all absorption bands of molecular group vibrational origin all therefore the first to claim our attention is that exhibited by the OH group. Its spectral nature can easily be studied. In all the samples the wave numbers characteristics of this group lies within the range 3400 to 3421 cm-1. In some cases like Hibiscus rosasinensis L two close bands are observed at 3421 cm-1 and 3362cm-1.The next band of interest is the strong band at 2916cm-1, which is presumably due to C-H stretching mode of vibration. This band is invariably present in the FTIR spectra of all the samples. But the other band, which we also assign to C-H stretching mode, is absent in many samples. It is an indication of structural difference. We should note that in the higher wave number side and to the left hand side of the hydroxyl group vibration,there are present a strong band at 3650cm-1 in the sample Paederia foclida, a medium strong band at 3600cm-1 in the sample clerodendron colebrokianum, a pair of band at 3750 cm-1 in the spectrum of Occimum canum sims.syn. From the available literature it is identified as due to the stretching mode of N-H. This band is conspicuous by its absence in the spectra of other samples. We shall next consider the case in which the band of wave number 2131cm-1 appear with extremely feeble intensity in the spectra of most of the samples, does not appear at all in the spectra of few samples, but it appears with a strong intensity in the spectra of cinnamonum tamala nees and Monochoria vaginalis presel. This band appears in a region where other bands do not appear and therefore this band is easy to be identified. The assignment for this band is not made. It may be noted that the band at 2131cm-1 falls in the region of N=C=N- stretching, isocyanides and N3 stretching. It may be inferred from table 6-2 that there are four C=O stretching mode of vibrations originating from four different sources. The wave numbers in the region 1718cm-1 to 1740cm-1 are due to C=O stretching mode but there are originating from ester group. This band is strong and appears as shoulder in some spectra but in most of them this ban is well resolved. But exception can be seen in the spectra of Curcuma Longa L, Terminalia chebula Retz and Zingiber officinalis Rox. The first and most obvious difference is the complete absence of the general shape of the spectra. These spectra are completely different as compared to the spectra of other plant leaves. It is not surprising and also not unexpected as the spectra are recorded for the dried seeds of the samples. In case of Curcuma Longa L the most intense band is one at 1024cm-1. The spectrum of Terminalia Chebula Retz presents another interesting characteristic feature. As may be seen from the spectrum the bands at 1718cm-1, 1618cm-1, 1340cm-1, 1213cm-1 and 1027cm-1 are of almost equal intensities. This feature is not to be seen in any of the spectra we have recorded thus far. Similarly we note that the FTIR spectra of Curcuma Longa L and Zingiber officinalis Rex possess striking similarly. It may be noted that the FTIR spectra of Curcuma Longa L in KBR were earlier investigated by several workers [23] and the shape and nature of the spectra were exactly identical to what we have worked out and reproduced here. This is naturally expected because the FTIR spectrum of a particular plant leaf is generally considered to be a fingerprint spectrum, a but a careful examination the spectra in different samples indicate differences which need attention. In fact during last three years near infrared spectroscopy (NIRS) and FT-Raman spectroscopy have exploited this minor change in the spectra as an useful tool for rapid and reliable analyses of numerous valuable substances in spice and medicinal plants [24]. Recently also various application of IR spectroscopy in combination with sophisticated chemometric algorithms were successfully introduced for qualitative and quantitative evaluation of essential medicinal plants [25,26]. Mostly the obtained spectra present significant and well-resolved key bands of individual components. As an
217 Scholar Research Library

Mitali Konwar et al Arch. Appl. Sci. Res., 2011, 3 (1):214-221 _____________________________________________________________________________________ alternative analytic option, also several Raman spectroscopy methods have been developed during last few years to predict simultaneously the amount of different valuable substances also directly in plant leaves and tissues [27]. Using Nd : YAG laser excitation at 1064 it is possible to excite non-destructively characteristic key Raman bands of valuable substances also directly in the plant leaves. Therefore, in principle a qualitive determination of different chemotypes among the same species can be performed without applying any statistical calculations. It is worthwhile to add here that there exists an increasing interest in producing plant-derived dyestuffs. Recently a new FT-IR and FT-Raman spectroscopy methods were developed to determine the individual dyes ( e.g.indigo,bixin,b-carotine, lycopene, crocetin, curcumin)directly in the raw materials or in the obtained solvent extracts. Furthermore, the distribution of natural pigments in the plant tissue (leaves,roots) are also analyzed applying 2-dimensional Raman mapping technique[28,29]. Various considerations indicate that the C=O stretching mode originating from aldehyde group lies in the range, 1618cm-1 to 1653cm-1 as shown in table 6-2 while the C=O stretching vibrations due to ketone group lies within 1685cm-1 to 1690cm-1.Similarly the chelated C=O stretching vibrations lie towards lower wave number side, that is, within the range 1621cm-1 to 1635cm-1.In the C=C stretching region we have assigned three bands while in general the C=C stretching region falls within the range 1511cm-1 to 1561cm-1 as indicated in Table 6-2. Another key I.R. band is the one around 1060cm-1, which is also very strong and has multiple components. The particular vibrational frequency undergoes substantial intensity alternation in the FTIR spectra of various samples. It is noteworthy that the relative intensity of this band does not depend on the concentration of the sample of medicinal plant leaves under study. The next key FTIR band is the one at 1349cm-1 for the sample Occimum cucum syms,syn and reasonably strong band around this wave number for other samples, as may be seen in Table 6-2(column 12). The band is identified as due to C N stretching mode. One of the salient features of this band is that the band appears invariably in the IR spectra of all the sample but the intensity undergoes rapid changes. As for example for the sample Hibiscus rosa-sinensis L the intensity of this band is unusually strong. We may appropriately sum up the results, which emerge from the foregoing studies. Studies in the manner described, the nature and origin of the key FTIR bands become clear. All the fourteen bands originating from different groups (N-H, O-H, C-H, C=O, C=C and C=N) may be termed as key bands and one can follow their absorption characteristics and differentiate individual plant leaves or tissues. These observations also make it evident that the manifestation of bands in the infrared region results primarily from the chlorophyll molecules masking or suppressing the appearance of all other bands due to other molecular origin, which of at all appear, it does with extremely low intensities. CONCLUSION We may appropriately sum up the results, which emerge from the foregoing studies. Studies in the manner described, the nature and origin of the key FTIR bands become clear. All the fourteen bands originating from different groups (N-H, O-H, C-H, C=O, C=C and C=N) may be termed as key bands and one can follow their absorption characteristics and differentiate individual plant leaves or tissues. These observations also make it evident that the manifestation of bands in the infrared region results primarily from the chlorophyll molecules masking or suppressing the

218 Scholar Research Library

Mitali Konwar et al

Arch. Appl. Sci. Res., 2011, 3 (1):214-221 _____________________________________________________________________________________

Table 2 Infrared absorption bands of medicinal plant leaves frequencies (cm-1 ) in KBr Compound Paederia foelida Vinca major Linn Cinnamonum tamala Nees Centella asiatica Clerodendron colebrookianum Mesua ferrea Linn Occimum canum sims.syn Hibiscus rosasinensis Linn Monochoria vaginalis presl Clitoria ternatea Linn Terminalia arjuna(Roxb) Spilathes acmella murr. syn Curcuma longa Linn Terminalia chebula Retz Zingiber officinalis Rox NH 3650 OH 3402 3411 3421 3402 3600 3750 3860 3750 3860 3424 3421 3421 C=O Ester 1739 1735 1740 1720 1740 1715 Ketone 1690 1685 Aldehyde 1650 1653 1653 1655 1653 1725 1735 1685 1651 1652 Chelate

C-H 2916 2916 2916 2926 2916 2916 2916 2860 2857 2847

C=C 1627 1631 1561 1561

C=N

C=N 1066 1060 1056 1061 1059 1051

2857 2837

2133 2130 2131

1635 1631 1632 1626 1621 1349 1313 1318

3421 3362 3401

2960 2926 2916

2857

2130

1735 1740 1734 1719 1734

1650

1631

2153

1655 1645 1656 1655 1651 1635 1618 1646

1630

1317

3372 3421 3421 3334 3401 3404

2926 2916 2916 2911 2926 2931

2857 2857 2846 2143

1737 1719 1735 1718 -

1621

1318

1105 1066 1026 1076 1059 1035 1105 1055 1031 1058 1105 1105

1335 1340 1340

219 Scholar Research Library

Mitali Konwar et al Arch. Appl. Sci. Res., 2011, 3 (1):214-221 _____________________________________________________________________________________ appearance of all other bands due to other molecular origin, which if at all appear, it does with extremely low intensities. Acknowledgement The author (M.K.) is grateful to UGC for the award of a research project no. F.5-56/2008-09 (MRP/NERO)/8086. REFERENCES [1] E. Rabinowitch : Photosynthesis ( New York Interscience 1951) Vol 2 , pt 1, chap. 21 pp. 603-656. [2] J.J.Katz, L.J.Boucher and R.C.Dougherty; in The chlorophyll,ed L.P.Vernon and G.R.Seely(Academic Press,New York 1966),chap 7, pp 186-251 [3] H.H.Strain and W A Svee, in The Chlorophylls ed. L P Vernon and G. R. Seely (Academic Press, New York 1966) chap 2, pp 21-66. [4] B Ke, in The chlorophylls ed. L p Vernon and G R Seely (New york : Academic Press 1966) chap.8,pp 276-278. [5] G Sherman and S F Wang , Nature 212 ,588(1996) ; Photochem. Photobid, 6 , 239 (1997) [6] I Fleming. Nature 216 , 151 (1997). [7] L J Bellamy: Infrared Spectra of complex molecules, Methuen, London pp 1-323 (1954). [8] A S Holt and E E Jacobs: Spectroscopy of plant pigments. Amer. Jour. Bot. 4, 718 (1954). [9] E E Jacobs, A E Vatter and A S Holt: Biochem. Biophys. 53 , 228-238 (1954). [10] N J Leonard , H S Gutowsky, W J Middleton and E M Peterson: J.Amer.Chem.Soc : 74 ,4073 (1952). [11] A A Krasnovsky : Doklady Akad. Nauk. SSSR. 60 , 421-424(1948). [12] A. S. Holt and E. E. Jacobs: Plant. Physiology. Infrared absorption spectra of chlorophylls and derivatives, in Plant.Physiology,553-558(1955) D Van Nostrand, Inc. [13] W.J. Weigl and R. Livingston: Jour. Amer. Chem. Soc. 75 ,2173-2176 (1953). [14] J.A.Segui, V.Maire, I.S. Gabashvili and M.Frugata ,J.Photochem. Photobiol. B.Biology, 56 , 39-47(2000). [15] I. S. Gabashvili, A. Menikh, J A Segui and M. Fragata J.Mol. Struct 444 ,123-133 (1998) [16] F.S.Parker ; Application of Infrared,Raman and Resonance Raman Spectroscopy in Biochemistry, Plenum Press, New York 1983. [17] S Krimm and J Bandekar ; Adv.Protein.Chem.38 , 181-364 (1986). [18] J.L.R. Arrondo , A Muga,J. Castresana and F.M.Goni ; Prog. Biophys. Mol. Biol. 59 , 2356 (1993). [19] J.L.R.Arrondo, J.Castresana, J.M.Valpuesta and F.M.Goni ; Biochemistry 33 , 11650-11655 (1994). [20] J.De Las Rivas and J.Barber Biochemistry 36 , 8897-8903(1997). [21] E.Wilson Jr.,J.C.Decius and P.C. Cross.The Theory of Infrared and Raman vibrational spectra, Mc.Grow-Hill,New York,1955. [22] A Rasquain, C. Houssier and C. Sironval Biochem. Biophys. Acta; 462(3) ,622-41(1977). [23] Sutapa (Goswami) Bhattacharyya: Spectroscopic investigation of some pigment gallstone and Biological molecules, Ph.D thesis, Dibrugarh University, Dibrugarh (India)(2003). [24] H Schulz, Application in analysis of coffee, tea, cocoa, tobacco, spices and aromatic plants and related products ; in C. Roberts ; J Workman ; J.Reeves(Eds): Near infrared Spectroscopy in
220 Scholar Research Library

Mitali Konwar et al Arch. Appl. Sci. Res., 2011, 3 (1):214-221 _____________________________________________________________________________________ agriculture, American Society of Agronomy, Crop Science of America-Soil Science Society of America, Madison U S A , Agronomy monograph , No 44,(2004),1-32. [25] H. Schulz, R.Quilitzsch, H Kruger : J. Mol. Struct. 299,661(2003). [26] H. Schulz and M,Baranska Perfumer and Flavorist 30,28 (2005). [27] H. Schulz , B.Schrader,R.Quilitzsch and B.Steuer: Appl. Spectrosc.56,117 (2002) [28] H. Schulz and Malgorzata Baranska Proc.of International Conference on Perspectives in vibrational Spectroscopy, Feb 25-28,(2006) Meerut. [29] K. G. Gilbert, H. G.Maule, H. G. Rudolph, B. Lewis, E. Sales, S. Tozzi and D. T. Cooke Biotechnol. Prog 20 ,1289-1292 (2004).

221 Scholar Research Library