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  • FTIR de Polisacaridos.

    Transféré par

    Henry Lambis Miranda
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    Descripción de Espectros FTIR de Polisacaridos.

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    Descripción de Espectros FTIR de Polisacaridos.

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    © All Rights Reserved
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    Signaler comme contenu inapproprié
    21 vues8 pages

    FTIR de Polisacaridos.

    Transféré par

    Henry Lambis Miranda
    Descripción de Espectros FTIR de Polisacaridos.

    Droits d'auteur :

    © All Rights Reserved
    Téléchargez comme PDF ou lisez en ligne sur Scribd
    Signaler comme contenu inapproprié
    sur 8
    ACTA BIOCHIMICA POLONICA vel. 27 1900 No. 2 ANDRZEJ GALAT SFUDY OF THE RAMAN SCATTERING AND INFRARED ABSORPTION SPECTRA OF BRANCHED POLYSACCHARIDES* Institute of Biology, Teachers’ Training College, Zolnierska 14; 10-561 Olsziyn, Poland Raman scattering and infrared absorption spectra of glycogen and starch were recorded over the range of 4000-50 cm-*, and the observed bands were correlated with the vibrational modes. It was shown that the spectral regions within 1050-970 em" and 900-780 cm~', where the specific Raman scattering and infrared bands of solvated hydroxy! groups and glycosidic linkages are found, can be applied success fully in the studies on the conformation and structure of branched polysaccharides. Raman scattering and infrared absorption spectroscopy have been recently applied for the elucidation of the structure and conformation of carbohydrates (Cael et al., 1975; Tu et al., 1977; Gatat & Popowicz, 1978a,b). However, a number of the polysaccharides commonly occurring in nature, e. g. dextran, glycogen and starch contain cross-linked chains. While dextran has been thoroughly investigated by Vasko et al, (1971) and Heyn (1974), only fragmentary information is available concerning the structure and conformation of the storage polysaccharides, such as starch and glycogen. These compounds usually occur as compact structures, which exhibit considerable resistance against temperature, various chemicals and radiation. ‘Though starch and glycogen may vary from species to species, the essential feature of their structure and conformation presumably remains the same irrespective of thieir diverse biological origin. In this paper the infrared and Raman scattering spectra of starch from potatoes and glycogen from rabbit liver are analysed and discussed, EXPERIMENTAL, The Raman scattering spectra were measured over the range of 3800 - 200 cm~* with a CARY 82 spectrometer, in solid state, using an argon ion laser (5145 A), at the power level of 100 mW. The samples were irradiated in a laser beam for 0.5 - 1h, * This paper is’ based on work performed under the contract R.TIT.13.3.4, 1135) 1136 A. GaLar 1980 then the Raman spectra were recorded. The spectral bandwidth was kept within 6-45 cm-". ‘The infrared spectra were recorded over the range of 4000-50 cm=?, with a Perkin-Elmer 180 spectrophotometer in KBr pellets (4000 - 400 m="), poly(chloro- trifluoroethylene) oil on CsI plates (4000 - 1300 cm~!), Nujol om Csl plates (1300 - 400. cm~#) and in Nujol in polyethylene cells in the far infrared region (450 - 50.cm~"). In this study highly purified samples of starch from potatoes (B.D.H., England), and glycogen from rabbit liver (Biomed, Krakéw, Poland) were used. RESULTS AND DISCUSSION The infrared absorption aad Raman scattering spectra of glycogen and starch are shown in Figs. 1-4. The band frequencies and intensities, with the suggested assignments to bond modes, are summarized in Table 1. Wad 0 Transmittance (%) F a ae 205 ‘000 0 Weave number (om) Fig. 1. Infrared spectrum of glycogen recorded within the range 4000 - 50-cm~', 000 Unsubstituted carbohydrates exhibit only a few bands in the spectral region within 4000 - 1500 cm}, Essentially in this region only hydrogen-bonded OH groups and skeletal CH and CH, may be expected. The hydtoxyl groups self-associated by hydrogen bonds give a strong and broad band, with the center of gravity within 3500 - 3200 cm~1. Howevel, it is still extremely difficult to find a reasonable corte- lation between these intense OH stretching modes and vibrational states, due to the complicated network of inter- and intra-moleculat hydrogen bonds occurring in polysaccharide structure. ‘A second group of bands, strong in Raman spectra and of moderate intensity in IR speetra, is found within 3000 - 1800 cm-1; they are due to the CH and CH; symmetrical and antisymmetrical stretching vibrations, and extensive studies have Vol. 27 RAMAN AND IR SPECTRA OF BRANCHED POLYSACCHARIDES 337 Transmittance (%) a z ait 00 00 0 205 Wave number em) Fig. 2. Infrared spectrum of starch recorded within the range 4000-50 m=‘. been performed to correlate the infrared and Raman bands of this region to bond. modes (Liang & Marchessault, 1959; Galat & Popowicz, 1978a,b). Another distinct band, which is usually recorded at about 1635 cm™, is related to the vibrations of bound water molecules (Heyn, 1974; Tu e7 al., 1978). In the solid state the observed intensity of this “water band” is dependent on the number of water molecules asso- ciated with polysaccharide particles, and inherently bound to the molecules “crystal water” (Heyn, 1974). 109 — 8 Intensity (%) a 2000 Wave nuaiber (oni?) Fig, 3. Raman scattering spectrum of glycogen in the solid state, Within the range 2000 - 50 cm=" ‘the spectrum was recorded using a higher intensity of an argon laser (see upper panel of this spectrum), 138 A. GALAT 1980 00 Wave number (on) Fig. 4, Raman scattering spectrum of starch in the solid state. Within the range 2000 - 50 em=* the spectrum was recorded using a higher intensity of an argon Taser (see upper panel of this spectrum). Several bands observed in the range 1506 - 1000 cm™* correspond to the CH and CH, in-plane bending and wagging vibrations, and to the COH deformation modes. ‘The C-OH deformation modes mostly occur between 1250 - 970 cm=?. The infrared bands of this spectral region related with the vibration modes of CH and CH, fairly correspond with the Raman scattering bands, while a part of the C-OH group vibrations are absent from the Raman spectra. These facts are fully related with the symmetry of each sugar unit, which is low in the inherently asymmetrical poly- saccharide structure. Especially interesting is the spectral region from 1050 to 970 cm~', where infrared spectroscopy offers an advantage over the Raman spectro- scopy. In this region glycogen exhibits two distinct and well resolved infrared bands, while starch shows only one broad band. Neither of these bands coriesponds to any Raman band. in the case of glycogen the bands at about 1025 and 1000 cm~* cotrelate with the C-OH stretching vibrations of solvated alcohol groups (one water molecule), and with the stretching vibrations of highly solvated alcohol groups, respectively (Heyn, 1974), whereas starch exhibits only one band of considerable intensity at about 1015 cm7*, These facts led to the conclusion that in starch all the hydroxyl groups are essentially random solvated and therefore exhibit a wide spectrum of solvated states (broad IR band), while in glycogen the anisotropically solvated alcohol groups are present, leading to two different vibrational modes (two distinct LR bands). Furthermore, if these bands are related to the vibrational modes of solvated hydroxyl groups of polysaccharide, then their absence in the Raman scattering spectra is fully explained. Thus, the spectral region within 1050 and 970 cm? may serve as an approximate criterion of polysaccharide heterogeneity. The band detected at about 930 cm~! in the infrared spectra, and at about 940 cm-? in the Raman spectra of polysaccharides and oligosaccharides was assigned Vol. 27 - RAMAN AND IR SPECTRA OF BRANCHED POLYSACCHARIDES 139 Table 4 The band frequencies (em-), intensities and band assignments of infrared absorption and Raman scattering spectra of glycogen and starch The bands are designated as:s, strong: m, moderate; w, weak; YW, very Weak; br, broad: sh, shoulder. Starch ; ig) Raman Assignments 3300 s¢br) 3300 s(br) | 3300 ni(br) | Hydrogen bonded OH stretching 2920s | 2930 mésh) | 2920s | 2930 sh)._| (CH and CHa 2870 mh) | 2910s | 2880 m | 2910s stretching 2860 mish) vibrations 1635 m 163m | water band 1460s | 140m | 455s 140s CH bending in plane 1375s | 1388 m_—| 1375;s 13805 CH bending in plane 1365 mish) | | 1340 m— | 1340. m_—_ | 1335 mGh) | 1335 8 COH bending 1300 w 100w | CH bending I 1265 m(br) 1260 m , ‘ 1240 w | 1240 mish) | 1240 w | 1240 méohy | |CH2OH related modes | 1205 w | 1220 wish) | 1200 w | 1210 m CORE deforamtion, 1450 811125 s(be) | 1180 8 | 1150 mt | JCCORE ansisrmmetsic ridge sree i |e i , ! | 1c.0-€ vibrations 1100s | 1085 m_ | 1100 w(sh) | 1125 mh) 1075s | 1065 mish) | 1075s | 1110 m__| {COM stretching, . i | Hoes | [antisymmetric in plane ring | | | 1065 s | [stretching 1025 stbr) | 1015 s(or) | COH solvated {1000 amish) COH highly solvated 960 wish) | | | 930m | 90m | 930m | 9408 ring modes \ 895 mh) 890 «895m 900 w | CCI+H(G) bending modes 45m | 80s | Sm | ROSS CAHI@) bending, modes s00w | Ss | water band | 790 vw | 780 m(br) | 790 ww | COC stretching j 760 m 755m | 165m | CHa rod 13s | mom | T188 715 m eee am | Se Jout-of-phase bending of hydrogen | Sw m | ef bonded hydroxyl 60m | iw cosw — | Giow —_| | bonded hydrons! groups | 515s | 585 m 570m 380m | | 525m | 515 mash) $20 mobo) | 525 w 440 m(br) | 445 m 410 w 408 | 410 m 410m 350 w 345w || 360m —_—_ different skeletal modes. | 228m | 34m | 320 m i | 270m | 280 wien | 243 m 23w 228 wibs) | | | bw ont w 140 A. GARAT. 1980 ‘Glycogen ‘Starch ; ar | Raman IR | Raman ‘Avsigoments 195 w 195 w(bs) 188 s{br) 179 | 12m mw 1357 m 160 w 139m 142 w(be) 133 m 133 m 12m 2m | 108 w 100m | 95 m(br) 87m Bm 82m 69 m 1m | 4m to out-of-phase C-O-C ring vibrations (Heyn, 1974), whereas Cael ef al. (1974) assigned this band to skeletal modes related to a(1-»4) linkages. While, in these two conclusions similar groups of atoms were taken into account, it has not been unambigously established which one of these two vibrations might te related with the tand within 930-940 cm-!. ‘Two distinct IR bands at about 895 and 845 em-? correspond to the C(1)-H(A), “f-configuration”, and C(1)-H(q), “a-configuration” bending modes. From the measurements of the peak area (e.g. Koenig et al., 1977) for these bands, the molar raiio of P-glycosidic to a-glycosidic linkages in branched polysaccharide can be derived, Because of the roughly equal intensity of these bands in the infrared and Raman scattering spectra of glycogen and starch, it may be concluded that in these compounds f-and a-linkages between sugar units are almost equally distributed. At present it is difficult to resolve into component bands the spectral region discussed above, as it was recently done with the amide 1 and amide J overlapping bands of the infrared spectra of chitin (Gatat et al., 1979). However, this method could certainly facilitate the estimation of the average number of the interchain cross-linkages occurring in branched polysaccharides, where different f- and/or a(1+3), a(1+4), and o(1-+6) intersugar bonds may be found. On the shoulder of the distinct and sharp band situated at about 765 cm~' two very small bands were visible. positioned at about 800 and 780 cm-*. Accor- ding to Heyn (1974) these bands reflect the vibration of “organized” bound water (chain configuration), and (1-3) branching modes, respectively. A considerable number of intense and overlapping bands occurring below 700 cm™? were assigned to C-O-H rocking, out-of-plane bending modes of the hydrogen-bonded hydroxyl groups, out-of-plane CH bending and CH; deformation vibrations (Susi & Ard, 1974; Tulchinsky er af., 1976). In this spectral region also other bands certainly occur but are masked by those of higher intensity (see Table 1). This fact, unfor- Vol 27. RAMAN AND IR SPECTRA OF BRANCHED POLYSACCHARIDES Mt tunately, limits considerably the possibility of resolving the IR. and Raman spectra into component bands, and of reasonable assignment of these bands to particular bond modes. However, accumulation of more extensive data on the IR and Raman spectra of different sugars and polysaccharides (Galat, in prepatation) would facili- tate estimation of the stucture and conformation both of polysaccharides and simpler oligosaccharides with the use of computer-assisted analysis. In the far-infrared region a number of Raman bands were recorded but at pre- sent it is rather difficult to assign them to any bond modes. More bands can be expected to occur in this spectral region, especially in the IR spectra, which might be visible only when the measurements are carried out at the liquid He temperature (Hineno & Yoshinaga, 1972). We are indebted to Mr. M. Proniewicz of the Jagellonian University for recording the Raman spectra REFERENCES Cael, J.J., Koenig, J. L. & Blackwell, J. (1974) Infrared and Raman spectroscopy of carbohydrate. Part Il, Raman spectra of the polymorphic forms of amylose. Carbohydr. Res., 29, 123-134, Cael, J. J., Koenig, J. 1. & Blackwell, J. (1975) Infrared and Raman spectroscopy of carbohydrate. Part VI. Normal coordinate analysis of V-amylose. Biopolymers, 14, 1885 - 1903. Galat, A. & Popowicz, J. (1978a) Study of the infrared spectra of chitin, Bull. Acad. Polon. Sci., Ser. Sci. Biol., 26, 295-300. Galat, A. & Popowicz, J. (1978b) Study of the Raman scattering spectra of chitin, Bull, Acad. Polon. Sci., Ser. Sci. Biol., 26, 519 - 524. Koput, J. & Popowicz, J. (1979) Analyses of infrared bands of chitin, Acta Biochim, Polon., 26, 303 - 308. Heyn, A. N. 3. (1974) The infrared absorption spectrum of dextran and its bound water. Biopoly- ‘mers, 13, 475 - 506. Hineno, M. & Yoshinaga, H. (1972) Far-infrared spectra of glucose, sorbose, saccharose and cello- biose at liquid He temperature. Spectrochim, Acta, 28A, 2263 - 2268, Koenig, J.L., D'Esposito, L. & Antoon, M.K. (1977) The ratio method for analysing infrared Spectra of mixtures. Appl. Spectrose., 31, 292 - 295. Liang, C. ¥. & Marchestault, R.H. (1959) Infrared spectra of crystalline polysaccharides. I, Hydrogen bonds in native cellulose. J. Polym, Scl., 37, 385 - 395. Susi, H. & Ard, J. 8. (1974) Laser Raman spectra of lactose. Carbohydr. Res., 37, 351 ~ 354. ‘Tu, A. Dinh, N. D., She, C. Y. & Maxwell, J. (1977) Laser Raman scattering of a- and f-methyl-

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