Fourier Transform Raman Spectroscopy of Honey

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thanks PROPP-UFJF for fi nancial support. The authors also thank Mr. D. W. Farwell for helpful discussions. 1. H. M. Ransome, The Sacred Bee in Ancient Times ...
Fourier Transform Raman Spectroscopy of Honey LUIZ FERNANDO C. DE OLIVEIRA,* ROSANA COLOM BARA, and HOW ELL G. M . EDW ARDS Department of Chemical and Forensic Sciences, University of Bradford, Bradford, W est Yorkshire, BD7 1DP, UK (L.F.C.d.O., H.G.M .E.); Departamento de Quõ´mica, Instituto de Cieˆ ncias Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, 36036-330, Brazil (L.F.C.d.O., R.C.); and School of Pharmacy, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK (R.C.)

In this work we present the Fourier transform Raman spectra of several commercial samples of honey in different states, comprising nine crystallized and three  uid samples. The measured water content of the specimens bears no relationship to their  uid behavior. The relative intensities of the vibrational bands in the C–H stretching region of the FT-Raman spectra are found to be sensitive to the observed physical states of the specim ens. Several observed vibrational bands in the region 500–1800 cm 2 1 could be identiŽ ed as Ž ngerprints of the two major com ponents in honey, fructose, and glucose, and at least one vibrational band was characteristic of sucrose. A relationship between the bandshape of the C–H stretching bands of honey specimens and their  uid properties was noted; crystallized samples show a well-resolved distinctive spectrum in this region, whereas the  uid samples do not exhibit this pattern. Some minor differences in the FT-Raman spectra of the honey specimens are discussed in terms of composition (saccharides and water) and the  uid state of the samples. Index Headings: FT-Raman spectroscopy; Honey; Saccharides.

INT RODUCTIO N Honey has been used as food and as medicine since ancient times in several folklore cultures. 1 The analytical literature relevant to honey can be divided in to two main themes: the Ž rst is the determination of the geographical and botanical origin of the specimens by chemical composition, the identiŽ cation of adulteration, and a certiŽ cation of quality of the product. 2 The second theme concerns the identiŽ cation of active components that could be responsible for medical properties of honey. Some of the therapeutic properties of honey includes its antibacterial, anti-in ammatory, and antioxidant effects and its role in the stimulation of cell growth activities.3 Honey is a very complex m atrix comprising a supersaturated sugar solution containing approximately 80% carbohydrates (glucose, fructose, and sucrose) in composition, and also minor quantities of vitamins, m inerals, proteins, and amino acids.4 Due to its complex composition, there is a large variability in honey types globally, and it has been shown that it is impossible to provide answers to the chemical or m edical questions using a single analytical approach.5 Generally, analytical methods such as liquid and gas chromatography, together with mass spectrometry, 6 are cited in the literature, and infrared spectroscopy provides the m ajor spectroscopic approach.7,8 Copious literature relates to the vibrational spectroscopic analysis of carbohydrates.9 –19 Mono and oligosaccharides are compounds that play an important role in Received 26 July 2001; accepted 5 November 2001. * Author to whom correspondence should be sent.

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several biological activities, and these are also the major components in honey. In a recent paper, Arboleda and Loppnow 20 applied Raman spectroscopy to the identiŽ cation of some carbohydrate m ixtures present in an unknown sugar sample. In other work, So¨ derholm and coworkers studied the role of water content on several Raman bands from amorphous and crystalline fructose and glucose. 21 There are also some recent studies in the literature concerning the use of infrared techniques for the analysis of the physical properties of honey; these are related mainly to the characterization of the major components of honey for the detection of adulteration using multidimensional analysis.8,22–24 In one of these studies, 22 the authors used FT-IR spectroscopy to analyze fructose and glucose in honey; a good statistical analysis was reported for the major components using near-infrared trans ectance spectroscopy. In studies of beet inverted sugar,8,23 and also sugar cane, 24 Sivakesava and Irudayaraj applied attenuated total re ectance spectroscopy combined with multivariate statistics to the analysis of honey, giving rise to a means for the nondestructive spectroscopic detection of adulteration of honey. However, honey has not been studied hitherto by Raman spectroscopy using visible excitation probably due to the high  uorescence background that is intrinsic in many natural products. Therefore, Fourier transform Raman spectroscopy provides an excellent tool for investigating this type of material; here, the near-infrared excitation wavelength can be used to obtain the Raman spectrum without  uorescence. In recent years, this technique has demonstrated its ability in the study of a range of different materials from natural Ž bers, 25 ancient resins, 26 natural dyes, 27 and artwork pigments 28 to the quality control of bricks; 29 all of these materials exhibit strong  uorescence emission to varying degrees in the visible range. In this work we report for the Ž rst time the results obtained from the nondestructive Fourier transform Raman spectroscopic analysis applied to the identiŽ cation of the main constituents in several honey specimens; the results will be compared with previously reported FT-IR spectroscopic investigations. EXPERIMENTAL Samples. Twelve different samples of commercial honey were analyzed without further treatment. Some of these were crystallized and others were  uid. The main  oral type descriptions present on the labels of comm ercial honey are depicted in Table I, together with their geographical source. It is worth noting that some commercial honey samples do not present a description of the

0003-7028 / 02 / 5603-0306$2.00 / 0 q 2002 Society for Applied Spectroscop y

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TABLE I. Water contents (analyzed by refractive index at 20 8C), main  oral description, and geographical source of the several investigated honeys. Honey samp le Honey Honey Honey Honey Honey Honey Honey Honey Honey Honey Honey Honey a

1 2 3 4 5 6 7 8 9 10 11 12

Floral type

Geographical source

Eucalyptus sp. Alternanthera sp. Schinus sp. Baccharis sp. Astronium sp. Schinus sp. Trifolium sp. Silvester Native Acacia sp. No description No description

Minas Gerais Brazil Minas Gerais Brazil Minas Gerais Brazil Santa Catarina Brazil Minas Gerais Brazil Minas Gerais Brazil New Zealand Santa Catarina Brazil Santa Catarina Brazil More than one country a Santa Catarina Brazil Santa Catarina Brazil

Water content (%) 18.6 19.8 15.8 16.6 15.0 19.0 16.6 19.4 20.2 17.2 23.8 .25

There is no description of geographic source; it may probably be imported by the dealer from different countries and blended in the UK.

main  oral type, as, for example, honey specimens 8, 9, 11, and 12 studied here. Honey specimens 11 and 12 differ in the collector bee species, and honey specimens 8 and 9 are described as probably a m ixture of several  oral species present in the geographical region where the honey was collected. Some crystallized honey specimens were also analyzed in a  uid form by warming in a water bath to a temperature of about 50 8C for about 20 min. Crystalline standard saccharides (Sigma-Aldrich) such as glucose, fructose, and sucrose were also used as models to assist in the assignment of the Raman vibrational bands. Instrumental. FT-R aman spectra were obtained using a Bruker IFS66 optical bench with an FRA 106 Raman accessory, a liquid nitrogen cooled germ anium detector and excitation at 1064 nm from a Nd:YAG laser. The laser power was set at 80 mW or less to minimize possible specimen degradation, and 2000 scans were accumulated with a spectral resolution of 4.0 cm 2 1 . The honey and standard specimens were examined in NM R glass tubes with a 1808 scattering geometry. The Raman spectra were obtained from at least two specimen aliquots to conŽ rm the relative band intensities and wavenumber positions. The moisture content was m easured following the AOAC OfŽ cial Method 969.38, where the refractive index of honey is related to the water content at 20 8C.30

F IG . 1. FT-Raman spectra of honey specimens 2 (19.8% water, A) and 3 (15.8% water, B), showing the changes in resolution in the CH region (see text).

tween this parameter and the degree of crystallization; for example, honey specimen 10 is  uid and contains 17.2% water while honey specimen 9, a crystallized sample, contains 20.8% water. There is some discussion on this subject in the literature,31–33 which addresses the in uence of the fructose/glucose ratio on the crystallization of honey, in addition to the speciŽ ed water content. Rheological measurem ents on several different types of Australian,34 Saudi,35 and Chinese 36 honey specimens show that they follow a Newtonian  uid behavior, and the viscosity can be predicted using an Arrhenius-type relationship if the respective constants are known for a particular species. Another fact that affects the viscosity of honey is the amount and type of colloidal material present because these particles can act as crystallization seeds.36 The FT-R aman spectra of some of the honey specimens investigated are shown in Figs. 1 and 2. The main vibrational bands of these representative specimens are displayed in Table II, with their respective assignments, based on the literature data, along with the standard spec-

RESULTS AND DISCUSSIONS Twelve different types of honey were investigated, with differences in geographical origin and physical state; honey specimens 1 to 9 were crystallized and 10 to 12 were  uids. The geographical source and  oral type of all honey specimens can be seen in Table I. The results of moisture measurem ents show a wide range of water content, as also shown in Table I. The water content can be related to several physical properties of honey and it also deŽ nes the requirements for storage. As can be seen in Table I, the sample with the least quantity of water is honey specimen 5, containing 15.0% water. On the other hand, the highest water content was observed in honey specimen 12, containing more than 25% water. Despite the expectation that a  uid honey should have the highest water content, there is actually no direct correlation be-

F IG . 2. FT-Raman spectra of honey specimens 10 (17.2% water, A) and 12 (up to 25% water, B), showing the change in relative intensity of the CH region (see text).

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TABLE II. Raman wavenumbers (in cm 2 1) of several different honey samples, crystallized (honey samples 1 to 4) and  uid (honey samples 10 and 11), excited at 1064 nm, and their respective tentative assignments. Honey 1 336 vw 421 m 450 sh 521 m 565 596 630 708 774 820 862 917

w w m w w wm wm wm

980 1026 1060 1075 1125 1150 1265 1332 1361 1457 2890 2902 2937

w sh sh m m sh wm wm wm m sh s vs

2998 sh

Honey 2

Honey 3

Honey 4

338 369 420 450 519

vw vw m sh m

339 369 421 456 520

vw vw m sh m

338 369 421 451 519

vw vw m sh m

563 594 629 707 774 821 859 917 924 980 1022 1060 1074 1125 1153 1265 1332 1361 1457 2885 2903 2938 2961 2974 2987

w w m w w wm m wm sh w sh sh m m sh wm wm wm m sh s vs sh sh sh

565 594 629 708 774 821 858 918 924 981 1027 1061 1074 1127 1157 1266 1333 1361 1457 2886 2903 2937 2962 2973 2990

w w m w w wm wm wm sh w sh sh m m sh wm wm wm m sh s vs sh sh sh

563 592 629 707 774 819 860 917

w w m w w wm wm wm

980 1021 1062 1074 1126 1155 1265 1333 1361 1457 2888 2902 2937 2960 2972 2989

w sh sh m m sh wm wm wm m sh s vs sh sh sh

Honey 10 239 331 353 423 446 521 540 560 595 630 707 778 821 868 917

vw w w s sh s sh sh w s w w m m wm

Honey 11 331 352 423 448 521 540 560 595 630 707 778 821 869 918

vw w s sh s sh sh w s w w m m wm

Assignment

d(C–C–O) 1 d(C–C–C) d(C–C–O) 1 d(C–C–C)

n(C–O–C)

979 w

979 w

1064 sh 1080 s 1126 s

1064 s 1077sh 1124 s

n(C–C) 1 n(C–O) 1 d(C–O–H) n(C–C) 1 n(C–O) 1 d(C–O–H) n(C–C) 1 n(C–O) 1 d(C–O–H)

1264 1340 1368 1459

1264 s

d(C–C–H) 1 d(O–C–H) 1 d(C–O–H)

1368 m s 1459 s

d(C–C–H) d(CH) 1 d(CH 2 ) 1 d(C–O–H) n(CH) n(CH) n(CH) n(CH) n(CH) n(CH)

m sh m s

2906 sh 2942 m

2900 sh 2942 m

tra from saturated solutions and those of solid individual sugars (Figs. 3 and 4, respectively). It was found that all twelve honey samples studied here could be represented by the FT-Raman spectra depicted in Figs. 1 to 5, as well as the assignments shown in Table I. At Ž rst sight, there is a reasonably good similarity between the Raman spectra of crystallized versus  uid honey and also between honey specimens with different water contents. The Raman spectra reveal some substantial spectroscopic differences between crystallized and  uid

honey specimens, m ainly in the C–H stretching region above 2700 cm 2 1. The relative intensities of some vibrational bands in this region change slightly but signiŽ cantly according to physical state and water content of the species. A comparison between the Raman and infrared spectra of honey shows that the former presents a better resolved and clearer picture compared with the infrared spectra. Garcia-Alvarez and co-workers 22 and Sivakesava and Irudayaraj, 8,23,24 who used FT-IR techniques, analyzed only a sm all region of the spectra of honey, and mathematical procedures were used to sub-

F IG . 3. FT-Raman spectra in the range 1700 –100 cm 2 1 of saturated solutions of fructose (A), glucose (B), sucrose (C ), and  uid honey specim en 12 (greater than 25% water, D ).

F IG . 4. FT-Raman spectra in the CH stretching region of cr ystalline sucrose (A), fructose (B), glucose (C ), and crystallized honey specimen 3 (D ).

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tract the water spectrum and the individual saccharide contributions to the overall band contour. Generally the infrared spectra of honey is dominated by the OH vibrational modes and hydrogen bond features,8,22 and this is clearly not the case for the Raman spectra reported here. In Table II, only the most intense Raman bands are tentatively assigned. There are several vibrational bands that could be proposed as Ž ngerprints for the main saccharide components (fructose and glucose) identiŽ ed in honey. Skeletal vibrational m otions dominate the region between 200 and 600 cm 2 1 , with m ajor contributions from the deformation m odes of the C–C–C, C–C–O, C– C, and C–O portions of the sugar m olecules. From the detailed studies of a-D-glucose and b-D-glucose, carried out by M athlouthi and Luu 12 and So¨ derholm and coworkers, 21 the Raman band at 540 cm 2 1 in the spectrum of  uid honey can be assigned to a C2–C1–O1 bending vibration. However, this band is absent in the Raman spectrum of crystallized honey, suggesting that this band could possibly have a dependence on the water content, as proposed by So¨ derholm and co-workers. 21 The vibrational band at 630 cm 2 1 can also be discussed in terms of specimen composition, as it is not present in the Raman spectrum of glucose. This Raman band is present as an intense Raman mode in the spectrum of crystalline fructose centered at 627 cm 2 1 and at 638 cm 2 1 in amorphous fructose. The vibrational Raman band at 917 cm 2 1 is characteristic of glucose and is present in all the spectra obtained here. However, honey specimens 3 and 4 exhibit a shoulder at 924 cm 2 1 , a band present only in the spectrum of crystalline fructose. This vibrational band can be assigned as a coupled mode, consisting of contributions from C-O stretching, C–C–H deformation, C–C stretching, and C–C–O deform ation. 21 However, in terms of relative band intensities, this band is not so intense in the spectrum of crystalline fructose, and the spectrum of amorphous fructose does not exhibit this feature at all. Since this m edium-intensity Raman band is present in all our Raman spectra of honey, we can assign its presence unequivocally to glucose. The Raman bands at 1060 –1064 cm 2 1 and 1074 –1080 cm 2 1 can be assigned as coupled vibrations involving contributions of the C–C and C–O stretching m odes. In the spectrum of cr ystallized honey, the most intense band is that at 1074 cm 2 1, and the band at 1060 –1062 cm 2 1 shows up as a shoulder. In the  uid honey specimens (samples 10 to 12, see Table II), these bands are shifted to 1080 –1077 and 1064 cm 2 1, respectively. In the case of honey specimen 11 there is also an inversion of band intensity ratios in this wavenumber region, with the feature at 1080 cm 2 1 being the m ore intense. So¨ derholm and co-workers, 21 in their study of the Raman spectra of amorphous fructose and glucose, pointed out that considerable changes in these two bands occurred when water was added to their samples. Their conclusions were that these bands are very sensitive to water content, and that an increase in water content leads to a decrease in the intensity of the two Raman bands. They also proposed that the most important contributions to these bands must be from the n(C–OH), n(C–C), and d(C–O–H) vibrations. Fructose also features these bands at 1061 and 1082 cm 2 1 , which are shifted by 4 cm 2 1 with increasing water

content, accompanied by a change in the intensity pattern, and the band at low wavenumber position increases in intensity with increasing water content. The presence of these two Raman bands in the honey spectra can be interpreted as a m ixture of the two carbohydrates glucose and fructose; however, the wavenumber positions of these bands are actually m ore similar to those of glucose. The intensity pattern observed in the honey spectra also shows a dependence on water content. Increasing the water content leads to a subtly different behavior of these two Raman bands. The Raman band at 1025–1027 cm 2 1, which is present in all honey spectra, can be assigned to a coupled n(C–C) and n(C–O) vibration of glucose, as fructose does not have any bands in this region. The Raman band at 1265 cm 2 1 in honey can be assigned as a coupled d(CH), d(CH2), and d(COH) vibration. Cr ystalline fructose shows this Raman band at 1266 cm 2 1 and the glucose bands at 1273 cm 2 1, so this band can be ascribed here to the presence of fructose in the honey specimens. The Raman band at 1332 cm 2 1 is present in crystalline fructose as a weak and broad feature; this occurs in the glucose spectrum as a shoulder on the more intense band at 1346 cm 2 1 , so it may be reasonably assigned here to the presence of fructose. The band at 1361 cm 2 1 (in honey specimens 1 to 4 and 1368 cm 2 1 in honey specimens 10 and 11, Table II) is not present in the spectra of fructose or glucose, but is present in the spectra of some disaccharides such as 1-O-methyl-a-Dglucoside.12 This observation suggests that the Raman band at 1361 cm 2 1 could be assigned to the presence of sucrose, the most common disaccharide present in honey.4 The Raman band at 1457–1459 cm 2 1 (calculated as a pure CH 2 group vibration 21) can be correlated with fructose because the cr ystalline species shows this band at 1461 cm 2 1, whereas glucose shows this Raman feature at 1471 cm 2 1 with a shoulder at 1455 cm 2 1 . There are other vibrational bands that can be proposed as the basis of ‘‘Ž ngerprints’’ of individual sugars present in honey. The stack plot of the Raman spectra of concentrated solutions of fructose, glucose, and sucrose can be seen in Fig. 3, together with the spectrum of a typical  uid honey (honey specimen 10). Vibrational bands of glucose can be observed at 1366, 896, and 420 cm 2 1 ; the vibrational bands that can be directly correlated with the presence of fructose are the ones at 816, 863, 703, 625, and 520 cm 2 1. Some of these Raman features can be common to both species, as, for example, the band at ;1460 cm 2 1 , as discussed above. However, due to the low natural species concentration it is m ore difŽ cult to assign speciŽ c vibrational bands of sucrose in the Raman spectra of the honey investigated other than the band at 1361 cm 2 1 discussed above. The Raman band at ;1335 cm 2 1 is present as a shoulder in the spectrum of honey specimens, as can be seen in Fig. 3; this band is also present in the Raman spectrum of glucose, although weak in intensity. In the honey spectrum, a Raman band of medium intensity at 918 cm 2 1 is related to sucrose content. In this region only fructose and sucrose show Raman bands; both are of weak intensity, but because in fructose this Raman band is very weak, it can reasonably be proposed as a contribution of sucrose to the Raman spectra of honey. The m ost signiŽ cant differences in the honey spectra APPLIED SPECTROSCOPY

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can be seen in the n(C–H) vibrational region between 2850 and 3100 cm 2 1. In Fig. 4, the Raman spectrum of a typical cr ystallized honey, together with the Raman spectra of crystalline fructose, glucose, and sucrose are shown. The crystallized honey shows the strongest bands at 2937 and 2902 cm 2 1 , respectively the symmetric and anti-symmetric stretching CH modes, with several prominent shoulders being observed. In the more viscous ( uid) samples, these bands can be seen at 2942 and ;2900 cm 2 1 , the latter as a shoulder, but both features show a decrease in intensity compared with the other crystallized samples. As can be seen in Figs. 1 and 2, the bands in the CH stretching region are ver y sensitive to the crystalline state of the honey; these bands are the most intense in the Raman spectrum of crystallized samples, and several resolved features can also be observed as shoulders. However, the m ore  uid samples show only two bands in the same region and without the presence of shoulders. Another interesting observation relates to the water content; on increasing the water content of crystallized honey, a reduction in the number of resolved vibrational bands in the CH region occurs, whereas in the  uid samples, the increase of water content leads to a decrease in intensity of these bands. The relationship between band shape and intensity of the CH vibrational bands and water content in the investigated honey is important. Carbon– hydrogen stretching band shapes show a dependence on the crystallized behavior of the honey studied, and it can be seen clearly that the most  uid samples show a poor spectrum in this region. Figure 4 also shows that the two most intense bands in the honey spectrum can be assigned to glucose and fructose, respectively; however, the most intense CH stretching band observed in the honey spectrum is at 2936 cm 2 1 and probably has contributions from both fructose and glucose. It must be realized that we are comparing here the Raman spectra of cr ystalline sugars with crystallized honey, and the crystallized solids in honey may not exhibit the same behavior as the standards selected. According to So¨ derholm and co-workers, 21 sugar solutions show a pattern that could be understood as a loss of crystallinity, when compared with the crystalline state, and several vibrational bands show some modiŽ cations in intensity due to the increase of water content. As the honey specimens studied here have almost the same average composition based on the saccharides (with a predominance of fructose and glucose) and water content, it is difŽ cult to explain the differences between the spectra in the region above 2800 cm 2 1 when we compare the crystallized honey with the more  uid honey. In an attempt to understand the spectral changes in this wavenumber region, some temperature tests were carried out with honey specimen 1 and the results obser ved can be seen in Fig. 5. First, the honey was warmed at 50 8C, obtaining a  uid sample whose spectrum is shown in Fig. 5B. Water was then added to the sample, and the Raman spectrum shown in Fig. 5C was obtained. These results demonstrate that when the crystallized honey becomes  uid, the Raman spectrum does not change signiŽ cantly, except in the C–H stretching region. In this spectral region, both of the samples that were warmed and diluted show a very poor resolution of the vibrational bands compared with the crystallized species, and also, a 310

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F IG . 5. FT-Raman spectra of honey specim en 1 at room temperature (A) and warmed at 50 8C (B), and after addition of water (C ).

change in the relative intensities is observed. It is interesting to note that the warm ed sample shows a spectrum that is very similar to the spectrum of the  uid sample at room temperature (see Fig. 2). In the case of the warm ed sample, we observe a loss of the glassy state that is common in honey, with a consequent decrease in viscosity, and the same explanation could apply for water addition. This conclusion is rather different from the observations of So¨ derholm and co-workers, 21 as they discuss the amorphous behavior of sugars only in the sense of water content, and we see here that the same behavior is present in the different glassy states of honey. Comparison of the results reported in this work with similar investigation using infrared techniques 7,8 shows that the same qualitative results can be achieved. However, future work on the development of quantitative determinations must be undertaken to provide a more suitable analysis of honey using Raman spectroscopy. The comparison of our results with the work of Cadet and Offmann 7 must be done with some care because they were working with several characteristic mono and disaccharides as standards instead of the three standards we are dealing with in this investigation. However, after the works of Cadet and Offmann 7 and also Sivakesava and Irudayaraj,8,23,24 it is possible to apply vibrational techniques in the qualitative and quantitative analyses of honey. The general difŽ culty that infrared techniques are subject to, i.e., water content, is not applicable to the FTRaman technique, and this could be an important consideration in future Raman investigations using FT-R aman spectroscopy. However, the presence of relatively m assive quantities of saccharides with their rich Raman spectra swam ps the obser vation of other minor chemical species, which may also be present in honey. In this sense, this investigation shows that it is possible to see some spectral differences between crystallized and  uid honey, but it does not address the differences between the analyzed honey specimens, as all them show similar Raman spectra dominated by the vibrational bands of fructose, glucose, and sucrose. Finally, it is important to note that if we compare in a general way the Raman spectra of honey in the same physical state (crystallized or  uid), it can be argued that

the variation in water content leads to a decrease in the relative intensity of the CH stretching bands. This leads also to a decrease in spectral resolution in the same bands. This latter fact can be understood in the sense that solutions do not present a well-resolved vibrational spectrum when compared with spectra obtained from the crystalline species; a possible explanation for this effect could be attributed to the in uence of hydrogen bonding between the CH groups and the hydroxyl oxygen atoms of the saccharide molecules. In the infrared spectra of honey specimens, the CH stretching bands are heavily swamped by the OH stretching modes and the effects noted here have not been reported in the infrared. Clearly, from the breadth and intensity of the infrared spectra in the OH stretching region, hydrogen bonding is very important in honey specimens and m ust also affect the CH Raman band intensities in this region. CONCLUSION We can conclude that FT-R aman spectroscopy has the potential for accom plishing the analytical characterization of the major components in real honey samples. Several vibrational bands have been identiŽ ed as speciŽ c Ž ngerprints of fructose and glucose in the analyzed samples. Also, at least one Raman feature can be assigned to the presence of sucrose. The water content in the samples plays an important role in the obser ved Raman spectra, as some vibrational bands of the saccharides exhibit subtle relative intensity changes with water content. The band shape of the C–H stretching bands also clearly shows a dependence on the  uid characteristics of the honey. The identiŽ cation of other components, such as other saccharides,  avonoids, and carboxylic acids is masked by the high concentration of the major saccharides. Clearly, future work on the rheological behavior of viscous honey correlated with changes in molecular structure and different water content will need the input of vibrational spectroscopy and rheo-optical techniques on m odel and real systems. ACK NOW LEDGM ENTS L.F.C.O. and R.C. gratefully acknowledge CAPES (Brazil) for the grant of post-doctoral fellowships during the time of this work. R.C. thanks PROPP-UFJF for Ž nancial support. The authors also thank Mr. D. W. Farwell for helpful discussions.

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