Spectral characteristics of tramadol in different solvents and β-cyclodextrin

Spectrochimica Acta Part A 74 (2009) 469–477

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Spectral characteristics of tramadol in different solvents and ␤-cyclodextrin A. Anton Smith a , R. Manavalan a , K. Kannan a , N. Rajendiran b,∗ a b

Department of Pharmacy, Annamalai University, Annamalai Nagar 608002, Tamilnadu, India Department of Chemistry, Annamalai University, Annamalai Nagar 608002, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 25 August 2008 Received in revised form 18 June 2009 Accepted 23 June 2009 Keywords: Tramadol Solvent effects ␤-Cyclodextrin Inclusion complex

a b s t r a c t Effect of solvents and ␤-cyclodextrin on the absorption and fluorescence spectra of tramadol drug has been investigated and compared with anisole. The solid inclusion complex of tramadol with ␤-CD is investigated by FT-IR, 1 H NMR, scanning electron microscope (SEM), DSC and semiempirical methods. The thermodynamic parameter (G) of inclusion process is determined. A solvent study shows (i) the spectral behaviour of both tramadol and anisole molecules is similar to each other and (ii) the cyclohexanol group in tramadol is not effectively conjugated with anisole group. However, in ␤-CD, due to space restriction of the CD cavity, a weak interaction is present between the above groups in tramadol. ␤-Cyclodextrin studies show that tramadol forms 1:2 inclusion complex with ␤-CD. A mechanism is proposed for the inclusion process. © 2009 Elsevier B.V. All rights reserved.

1. Introduction In recent years, the host–guest inclusion complexes are of very much interest in wide fields of science and technology. Cyclodextrin (CD), a cyclic oligosaccharide containing six or more d(+) glucopyranose units, is one of the most important host compounds. A variety of guest compounds comprising ionic species, organic molecules and pharmaceutical drugs can be included in the cavity of CD in aqueous solutions. The CD inclusion compound is highly stereo specific, greatly stable and less toxic or nontoxic. Hence, CDs are widely investigated as enzyme models [1] resolving agents for chiral compounds [2] and molecular capsules of pharmaceutical drugs. CDs are also utilized in photochemical reactions in an attempt to give product selectivity [3]. It is of great interest, that CDs are able to form inclusion complexes more than one type. It is well known, CDs forming inclusion complex with various guests molecule with suitable polarity and dimension because of their special molecular structured internal hydrophobic cavity and external hydrophilic surface [4]. Thus, this stability has been widely used in pharmaceutical industries [5]. Furthermore, CDs have been used as models for proteins and enzymes because CDs are interacted with many substances in a manner similar to that of proteins and enzymes. Especially, in pharmaceutical industries the inclusion process of drug molecules with CD leads to important modifications of drug properties of drug molecules [6]. In pharmaceutical more interest in CDs extends to enhance the solubility, chemical stability and bioavailability of poorly soluble drugs, to

∗ Corresponding author. Tel.: +91 94866 28800; fax: +91 4144 238080. E-mail address: [email protected] (N. Rajendiran). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.06.047

reduce the toxicity and to control the rate of release so on and forth [7]. Therefore, it is essential to comprehensively understand the effects of inclusion of drug molecules. To support the inclusion process, we also studied the solvent effect of the tramadol drug (C16 H25 NO2 , chemical name is rac-(IR, 2R)-2-(dimethylamino methyl)-I-(3-methoxyphenyl) cyclohexanol, Scheme 1). In this work, we have studied not only spectral properties of tramadol–␤-CD complex by UV–visible and fluorescence measurements, but also prepared its solid inclusion complex by co-precipitation method and determined its formation by means of FT-IR, 1 H NMR, SEM and DSC. The formation constant of the inclusion complex was obtained according to the data of UV–visible and fluorescence measurements using modified Benesi–Hildebrand equation. The studies on the solid inclusion complex of ␤-CD with guest molecule have been performed to obtain direct evidence for the formation of the inclusion complexes [8]. The obtained results indicate that the solid structure of these complexes is designable by appropriately selecting type, length and functional substituent group in the guest. Previously, Chan et al. and Rudaz [8] separated the tramadol derivatives by using ␤-CD inclusion method. Tramadol drug is a typical opioid which is a centrally acting analgesic used for treating severe pain. It is a synthetic agent, as a 4-phenyl-piperidine analogue of codeine [9a] and appears to have actions on the GABAergic, noradrenergic and serotonergic systems [9b]. Tramadol is a fine white powder which is practically insoluble in water. Tramadol is marketed as a racemic mixture with a weak affinity for the g-opioid receptor (approximately 1/6th that of morphine). The (+) enantiomer is approximately four times more potent than the (+) enantiomer in terms of 1-opioid receptor affinity and 5-HT reuptake, whereas the (−) enantiomer is responsible for noradrenalin reuptake effects [10a]. These actions appear to produce a

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A. Anton Smith et al. / Spectrochimica Acta Part A 74 (2009) 469–477

Scheme 1. CAche structure of (a) tramadol and (b) anisole.

synergistic analgesic effect with (+) tramadol exhibiting 1–10-fold higher analgesic activity than (−) tramadol [10b]. 2. Experimental 2.1. Instruments Absorption spectral measurements were carried out with a Shimadzu UV 1601 PC model UV–visible spectrophotometer and fluorescence measurements were made by using a Perkin spectrofluorimeter model LS 55. FT-IR spectra were obtained with an Avatar-330 FT-IR spectrometer using KBr pelleting. The range of spectra was from 500 to 4000 cm−1 . A Bruker Advance DRX 400 MHz superconducting NMR spectrophotometer was used to study 1 H NMR spectra. Microscopic morphological structure measurements were performed with a JEOL JSM 5610 LV scanning electron microscope (SEM) and a Shimadzu-60 differential scanning colorimeter (DSC) was used to measure the thermal curves. 2.2. Reagents and materials

Table 1 along with the spectral data of anisole. Tramadol and anisole show absorption maxima around 278, 271 and 217 nm in all solvents. The molar extinction co-efficient of the above maxima is medium (∼10−4 cm−1 ). These results imply that this band is attributed to the (␲, ␲*) transition of aromatic ring. In all solvents both molecules give one broad structureless fluorescence maxima. The fluorescence spectrum of tramadol drug is displayed in Fig. 1. When the solvent polarity and proton donor capacity increase, no significant shift is observed both in the absorption/fluorescence maxima. However, the spectral shifts of both molecules observed in the absorption and fluorescence spectrum in protic and aprotic solvents are consistent with the characteristic behaviour of amino [11] and hydroxyl groups [12–14]. In any one solvent, when compared to anisole no significant spectral shift (absorption and fluorescence) is observed in tramadol. This shows the addition of other groups (cyclohexanol and dimethylamino group) in anisole molecule has not effectively increased the resonance interaction in tramadol drug; i.e., the spectral behaviour of tramadol is similar to anisole molecule suggesting that, the –CH– group in cyclohexane group is not effectively conjugated with the lone pair of electrons from

Tramadol and ␤-CD were obtained from E-Merck and recrystallized from aqueous ethanol. The purity of the compound was checked by similar fluorescence spectra when excited with different wavelengths. All used solvents of the highest grade (spectrograde) were commercially available. Triply distilled water was used for the preparation of aqueous solutions. The concentration of ␤-CD was varied from 1 × 10−3 to 1.2 × 10−2 mol dm−3 . The experiments were carried out at room temperature 303 K. The solid inclusion complex of tramadol and ␤-CD was prepared by co-precipitation method and it was analyzed by using FT-IR, 1 H NMR, DSC and SEM methods. 3. Results and discussion 3.1. Effect of solvents The absorption maxima, log ε and fluorescence maxima of tramadol drug are obtained in solvents with various polarities and hydrogen bonding abilities. The relevant data are complied in

Fig. 1. Fluorescence spectra of tramadol in different solvents: (1) cyclohexane, (2) acetonitrile, (3) methanol, and (4) water.

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Table 1 Absorption and fluorescence maxima (nm) and Stokes shift of tramadol HCl and anisole in different solvents (temperature ∼303 K). S. No.

Tramadol HCl

Anisole

Solvents

abs

log ε

flu

Stokes shift

abs

log ε

flu

Stokes shift

1

Cyclohexane

278.3 271.4 217.2

3.38 3.35 4.27

302.1

2830

278.2 271.3 217.1

3.48 3.37 4.25

302.3

2865

2

Diethyl ether

279.3 272.4 217.4

3.25 3.36 4.22

303.2

2822

279.1 272.2 217.2

3.22 3.31 4.20

303.2

2847

3

1,4-Dioxane

279.5 272.2 217.2

3.24 3.33 4.25

303.2

2796

279.3 272.2 217.1

3.14 3.23 4.15

303.2

2822

4

Acetonitrile

279.2 272.6 217.6

3.23 3.32 4.31

303.2

2835

279.3 272.2 217.2

3.20 3.30 4.21

303.2

2822

5

Ethylacetate

279.2 271.8 217.2

3.15 3.26 4.12

303.2

2835

279.1 271.6 217.1

3.14 3.22 4.10

303.2

2847

6

Dichloromethane

278.6 271.2 216.6

3.28 3.30 4.15

303.2

2912

278.2 271.1 216.3

3.25 3.20 4.11

303.2

2963

7

t-Butyl alcohol

279.2 272.2 216.5

3.23 3.27 4.18

303.2

2835

279.1 272.2 216.4

3.21 3.22 4.19

303.2

2847

8

2-Butanol

279.3 272.0 216.3

3.28 3.31 4.10

303.2

2822

279.2 272.2 216.6

3.18 3.21 4.11

303.2

2835

9

2-Propanol

279.4 272.6 216.8

3.22 3.30 4.14

303.2

2809

279.3 272.5 216.6

3.24 3.31 4.12

303.2

2822

10

1-Butanol

279.0 272.4 216.5

3.20 3.21 4.15

303.2

2860

279.1 272.3 216.4

3.10 3.22 4.11

303.2

2847

11

Ethanol

279.4 272.2 216.6

3.30 3.31 4.21

303.2

2809

279.3 272.3 216.4

3.28 3.32 4.20

303.2

2822

12

Methanol

279.4 272.6 216.2

3.23 3.21 4.22

303.2

2809

279.2 272.1 216.0

3.21 3.20 4.24

303.2

2835

13

Water

278.0 270.5 216.2

3.21 3.25 4.19

303.2

2989

278.2 270.2 216.1

3.23 3.15 4.16

303.2

2963

14 (a) (b) (c)

Correlation co-efficient ET (30) vs ¯vss BK vs ¯vss (D,n) vs ¯vss

hydroxy or dimethylamino group. The above results indicate that, even though the anisole part linked with cyclohexanol and dimethyl amino group in tramadol, no conjugation is present in between the above groups; hence in all solvents, tramadol drug behaves like anisole molecule. 3.2. Correlation of solvatochromic shift with the solvent polarity When a solute is placed in a solvent, one observes the combined effects of general and specific interactions. The separation of these interactions is often difficult. Empirically or theoretically derived solvent parameters like Reichardt’s–Dimroth, ET (30) [15], Bilot–Kawasaki (BK) [16] and Lippert f(D,n) [17] values as accurate registers of solvent polarity have been used by several authors to correlate molecular spectroscopic properties [18–20].

0.558 0.382 0.368

0.617 0.405 0.397

Among these parameters BK and f(D,n) take into account of solvent polarity alone, whereas ET (30) incorporates both solvent polarity and hydrogen bonding effects. From the correlation of Stokes shift with any one of these parameters and an idea of about the type of interaction between the solute and solvent can be obtained. The Stokes shift of tramadol measured in different solvents is correlated with ET (30), BK and f(D,n) parameters (Table 1). The Stokes shift observed in tramadol is similar to those observed in other hydroxyl molecules [11–14]. Fig. 2 shows the plots of ¯vss vs the ET (30), BK and f(D,n) parameters. The Stokes shift slightly increasing from cyclohexane to water in tramadol is found to be more in accordance with ET (30) than with BK and f(D,n) values. Since tramadol is not having hydroxy or amino group in the aromatic ring, the interaction between this drug with any one solvent

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Fig. 3. Absorption spectra of tramadol in different ␤-CD concentrations (×10−3 ) (mol dm−3 ): (1) 0, (2) 1, (3) 2, (4) 3, (5) 4, (6) 5, (7) 6, (8) 7, (9) 8, (10) 9, and (11) 10. Fig. 2. Plot of Stokes shifts (cm−1 ) of tramadol vs ET (30), BK and f(D,n) solvent parameters: (1) cyclohexane, (2) diethyl ether, (3) 1,4-dioxane, (4) ethyl acetate, (5) dichloro methane, (6) acetonitrile, (7) t-butyl alcohol, (8) 2-butanol, (9) 2-propanol, (10) 1-butanol, (11) ethanol, (12) methanol, and (13) water.

is very small. Hence, the above three parameters gave small correlation co-efficient values. Moreover, no significant spectral shift is observed in the absorption and emission spectra supported this implication.

3.3. ˇ-Cyclodextrin effects Table 2 shows the absorption and fluorescence maxima of tramadol and anisole (4 × 10−5 mol dm−3 ) in pH ∼ 7.0 solutions containing various concentrations of ␤-CD. Upon increasing the concentration of ␤-CD in both compounds the absorption maximum is not changed but the absorbance is gradually decreased (Fig. 3). No isosbestic point could be noticed which rules out

Table 2 Absorption and fluorescence maxima (nm) of tramadol and anisole at different concentrations of ␤-CD (temperature ∼303 K). No.

Tramadol Concentration of ␤-CD (M)

Anisole abs

log ε

flu

IF

abs

log ε

flu

IF

1

Water

276.6 271.7 216.0

3.39 3.36 4.25

303

525

276.9 271.8 216.1

3.48 3.45 4.27

303

540

2

0.001

276.6 271.5 216.0

3.25 3.31 4.22

303

452

276.7 271.6 216.1

3.36 3.37 4.26

303

435

3

0.002

278.0 271.5 217.5

3.21 3.27 4.18

303

407

278.1 271.2 217.7

3.33 3.34 4.23

303

401

4

0.004

278.0 271.7 216.3

3.11 3.16 4.05

303

398

278.2 271.9 216.5

3.22 3.26 4.15

303

385

5

0.006

277.7 271.4 216.3

3.05 3.07 3.92

303

388

277.9 271.7 216.5

3.15 3.15 4.08

303

376

6

0.008

278.6 271.7 217.8

2.98 2.96 3.84

303

374

278.9 271.8 217.9

3.08 3.06 4.00

303

362

7

0.010

278.3 271.7 216.4

2.91 2.90 3.80

303

361

278.8 271.9 216.6

3.01 3.01 3.95

303

342

8

Correlation co-efficient 1:1 1:2

9

Binding constant 1:1 1:2

0.7022 0.9330 154 12242

10

H (kcal/mol)

−75.30

11

G (kcal/mol) 1:1 1:2

−12.68 −23.70

0.6721 0.9412 186 38915

0.9573

198

0.9672

227

−15.95 −13.16 −26.62

−10.41

−13.66

A. Anton Smith et al. / Spectrochimica Acta Part A 74 (2009) 469–477

Fig. 4. Benesi–Hildebrand plot for the complexation of tramadol with ␤-CD: (a) 1:2 and (b) 1:1.

the possibility of a single equilibrium involving 1:1 complexation between tramadol with ␤-CD. The possibilities are proposed for this deviation: (i) 1:2 inclusion complex may be formed, (ii) due to the ␤-CD cavity space restriction more than one type of complex each having 1:1 stoichiometry may be formed, and (iii) when ␤-CD concentration is increased, the changes detected in the absorption spectra may be tramadol solution containing 1% methanol can also make the interaction between both components. Since in this experiment the concentration of methanol is practically constant with respect to ␤-CD concentration it may affect the isosbestic point. In the following discussions proposing two distinct complexes, one with tramadol forms 1:2 complex with ␤-CD cavity and the other is more than one type of complex each having 1:1 stoichiometry may be formed. The above results are due to both molecules that are transferred from more protic environments (bulk aqueous phases) to less protic environments (␤-CD cavity). The above results indicate that tramadol molecule is entrapped into the ␤-CD to form inclusion complex. Further, the increase in the absorbance in ␤-CD solutions indicates, the aromatic ring is encapsulated in the nonpolar ␤-CD cavity. In order to determine the stoichiometry of the inclusion complex, the dependence on ␤-CD of the tramadol absorbance and fluorescence was analysed by using the Benesi–Hildebrand equation [21] for 1:1 complex (Eq. (1)) and the 1:2 complex (Eq. (2)) between tramadol and ␤-CD as shown below: 1 1 1 =  + I − I0 I − I0 K(I − I0 ) [ˇ − CD]

(1)

1 1 1 =  + 2 I − I0 I − I0 K(I − I0 ) [ˇ − CD]

(2)

473

Fig. 5. Fluorescence spectra of tramadol in different ␤-CD concentrations (×10−3 ) (mol dm−3 ): (1) 0, (2) 1, (3) 2, (4) 3, (5) 4, (6) 5, (7) 6, (8) 7, (9) 8, (10) 9, and (11) 10.

Here, tramadol-[␤-CD]2 represents the 1:2 inclusion complex, and K1 and K2 are the equilibrium constants for the complex formation. Fig. 5 depicts the emission spectra of tramadol (excited at 270 nm) with varying concentration of ␤-CD. The effect of ␤-CD on the fluorescence spectra of tramadol is different to the corresponding effect on the absorption spectra. As in absorption spectra, both molecules fluorescence intensities decrease at 330–350 nm accompanying the addition of ␤-CD. However in tramadol, the fluorescence intensity is increased around 330–350 nm (Fig. 6). The decrease in fluorescence intensity at 302 nm suggests the formation of an inclusion complex between guest and host [13]. The complexation is complete at 8 × 10−3 mol dm−3 ␤-CD concentration and further addition of ␤-CD no significant changes observed in the fluorescence maximum. There are three important thermodynamic parameters in the inclusion process [22]. The free energy change can be calculated from the formation constant ‘K’ by Eq. (5): G = −RT ln K

(5)

The thermodynamic parameter G for the binding of the guest molecule to ␤-CD is given in Table 2. As can be seen from Table 2, G is negative which suggests that the inclusion process proceeded simultaneously at 303 K. The experimental results indicate that the inclusion reaction of ␤-CD with guest was an exothermic process.

where K is the formation constant, I0 is the initial absorption/fluorescence intensity of free tramadol, I is the absorption/fluorescence intensity of ␤-CD inclusion complex and I is the observed absorption/fluorescence intensity. According to Eqs. (1) and (2), a plot of 1/I − I0 vs 1/[␤-CD] (both absorption and fluorescence) gives an upward curves but 1/I − I0 vs 1/[␤-CD]2 gives a linear line as shown in Fig. 4. This analysis reflects the formation of 1:2 inclusion complex between tramadol and ␤-CD complex. Hence, the concentration of the 1:2 complex may be expressed by using the formation constants of the 1:1 and 1:2 complexes. K1

Tramadol + ˇ − CDtramadol–ˇ − CD K2

Tramadol + ˇ − CDtramadol–[ˇ − CD]2

(3) (4)

Fig. 6. Benesi–Hildebrand plot for the complexation of tramadol with ␤-CD: (a) 1:2 and (b) 1:1.

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3.4. Possible inclusion complex of tramadol Considering the above results and discussions, the possible inclusion mechanism is proposed as follows: three different types of inclusion complex formation between tramadol and ␤-CD are possible. In type I arrangement, the cyclohexanol or dimethyl amino group of tramadol is entrapped within the ␤-CD cavity, in type-II arrangement the anisole part of tramadol is encapsulated within the ␤-CD cavity and in type III, tramadol forms 1:2 inclusion complex with ␤-CD (Scheme 1). If anisole part is encapsulated in the

␤-CD cavity the absorbance and fluorescence intensity of tramadol should be similar to anisole molecule (1:1 inclusion complex is formed between anisole and ␤-CD). On the other hand, if cyclohexanol or dimethyl amino group encapsulated in the ␤-CD cavity, we expect the absorbance and fluorescence intensity of tramadol should be similar to aqueous medium. Our results indicate that, in ␤-CD solutions the absorbance and fluorescence intensity of tramadol is decreased with an increasing ␤-CD concentration at 303 nm (which is similar to anisole). However, around 330–350 nm without any spectral maximum, the

Scheme 2. Proposed inclusion complex structure of tramadol with ␤-CD.

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fluorescence intensity is increased along with ␤-CD concentration. In ␤-CD, both tramadol and anisole exhibit different spectral trends which show both molecules form different types of inclusion complex. It is already reported [12] when the aromatic molecule is completely entrapped within the ␤-CD cavity, the absorbance and fluorescence intensity is decreased in ␤-CD than aqueous medium. This confirms the environment around the anisole group in tramadol with ␤-CD cavity is same as in the anisole molecule. Further, an increase in fluorescence intensity around 330–350 nm indicates cyclohexanol or dimethylamino group also encapsulated in the ␤-CD cavity. The question may arise, if 1:2 inclusion complex is formed in tramadol, why any significant spectral maxima are not observed in the longer wavelength (around 340 nm)? The answer is (i) as discussed in solvents, the –CH– group in cyclohexanol group is not effectively conjugated with anisole group and (ii) due to space restriction in ␤-CD cavity a small interaction is present between cyclohexanol and anisole groups in tramadol. These features indicate that in Scheme 2, tramadol forms 1:2 inclusion complex with ␤-CD cavity. Further, the Benesi–Hildebrand 1:1 plot on both absorption and fluorescence spectral values gives an upward curves but 1:2 plot gives a linear line as shown in Fig. 4. This

475

analysis confirms the formation of 1:2 inclusion complex between tramadol and ␤-CD complex. This is further supported by using semiempirical quantum mechanical calculations. The internal diameter of the ␤-CD is approximately 6.65 Å and its height is 7.8 Å (Scheme 2). To determine the dimensions of tramadol the geometry of the ground state was optimized by using AM1 (MOPAC 6.0 version using PC model). The distance between H3 and H6 is 7.09 Å, H6 and H13 is 8.28 Å, H6 and H18 is 8.65 Å, H6 and H11 is 10.02 Å and H7 and H10 is 9.30 Å. This calculation revealed that the length of the two methyl groups is higher than ␤-CD. Since the length between dimethyl amino group and cyclohexanol in tramadol is larger than that of the upper rim of ␤-CD, the methyl group attached at cyclohexanol ring should be encapsulated in one ␤-CD cavity and the anisole part should be present in the other ␤-CD cavity. 3.5. FT-IR spectral studies FT-IR spectra of tramadol, ␤-CD and the solid inclusion complex are also studied (Fig. 7). Tramadol drug examined in KBr pellet, displays one absorption band at 3306 cm−1 . This band

Fig. 7. FT-IR spectrum of TRA in KBr (a) tramadol and (b) tramadol–␤-CD complex.

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Fig. 8. Thermal spectra of (a) ␤-CD, (b) tramadol, and (c) tramadol–␤-CD inclusion complex.

represents the intermolecular hydrogen bond and in the inclusion complex it moves to 3368 cm−1 longer wavenumber. The aromatic C–H stretching frequency of the pure drug appears around 2930 and 2861 cm−1 and these frequencies are lost in the inclusion complex. The aromatic ring stretching frequency of the pure drug appears around 1607, 1578, 1480, 1462,1444, and

1433 cm−1 whereas these frequencies are very weak 1637, 1608, and 1584 cm−1 in the inclusion complex. The O–CH3 stretching modes 3066, 3018, and 3004 cm−1 in the pure drug are lost in the inclusion complex. N–CH3 HCl stretching frequency at 2629 and 2861 cm−1 of the tramadol is also lost in the inclusion complex. N–CH3 HCl stretching and bending frequencies at 1218, 1194 cm−1 and 510 cm−1 of the tramadol are moved in the inclusion complex to 1291, 1245, 1156 and 529 cm−1 respectively. Ar–O–CH3 symmetric stretching at 1087 and 1073 cm−1 moves to 1079 and 1028 cm−1 in the inclusion complex respectively. Ar–O–CH3 asymmetric stretching at 1289 and 1242 cm−1 is shifted to 1291 and 1245 cm−1 in the inclusion complex respectively. In tramadol, Ar–C–H bending (out of plane) absorption band 777 and 791 cm−1 moves to 777 and 757 cm−1 in this inclusion complex respectively. The out of plane ring bending of the tramadol also shifts to the shorter wavenumber in the complex (702 and 704 cm−1 ). The cyclohexane bending vibrations at 1444 cm−1 of the tramadol move in the inclusion complex to 1458 and 1431 cm−1 . Further, the ␤-CD absorption band (2928 cm−1 ) is also appearing in the inclusion complex. Moreover, the absorption intensity of most of the frequencies is moved in the inclusion complex that is significantly weaker (10–60%) than tramadol molecule. These results indicate the amino group is present in hydrophilic part and the anisole group is encapsulated in the ␤-CD cavity.

Fig. 9. Scanning electron microscope photographs (Pt. coated) of (a) ␤-CD (500×), (b) ␤-CD (3000×), (c) tramadol (100×), (d) TRA (200×) and (e and f) tramadol-␤-CD inclusion complex (100× and 200×).

A. Anton Smith et al. / Spectrochimica Acta Part A 74 (2009) 469–477

3.6. Conformation analysis by 1 H NMR spectroscopy NMR spectroscopy is one of the most powerful tools for the study of formation of inclusion complex between CDs and a variety of guest molecule [23,24]. In general, the resonance of the ␤-CD protons located within or near the cavity (H-3, 5, and 6) shows remarkably large shifts in the mixture. A minor shift is observed for the resonance of H-1, 2, and 4 located on the exterior of ␤-CD. As can be seen from the obtained results, the chemical shifts data for the inclusion complex were different from those for the free compound. The resonance of all protons of tramadol within the ␤-CD cavity was shifted upfield in the inclusion complex, which provided that the preferred fixed orientation was formed with the ␤-CD cavity. The addition of tramadol into the ␤-CD results in a downfield chemical shift for the tramadol protons in DMSO. These values are given below: tramadol (inclusion complex) ppm: OH ∼9.925 (8.995), aromatic H ∼6.805, 7.079, 7.288 (6.817, 7.068, 7.268), OCH3 ∼3.754 (3.574), N-CH3 ∼2.418, 2.503, 2.570 (3.496, 3.632, 3.751), cyclohexane ∼ 1.441–1.757 (1.489–1.805). In particular, the resonance of the protons of ␤-CD, located within or near the cavity showed remarkably large downfield shift (one OCH3 = +0.059, OH = 0.040 ppm) in the tramadol–␤CD complex, which suggested that the resonance of OH and OCH3 groups are shielded largely in the complex and the aromatic group must penetrate deeply into the cavity. A small shift of (CH = 0.01 ppm and other OCH3 = 0.002 ppm) was observed for resonance of hydrogens located on the exterior part of the ␤-CD cavity. These features indicate that tramadol molecule is included in ␤-CD cavity. 3.7. Differential scanning colorimetry (DSC) study The DSC curves of tramadol, ␤-CD and inclusion complex are shown in Fig. 8. It can be shown from Fig. 8. The DSC curves of inclusion complex with the DSC curves of tramadol and inclusion complex is different from each other, i.e., ␤-CD ∼260 ◦ C, time ∼19.15 min; tramadol ∼191.06 ◦ C, time ∼15.6 min; tramadol–␤-CD ∼215.0 ◦ C, time ∼18.1 min. These values prove and suggest that a new inclusion complex is formed. 3.8. Microscopic morphological observation Firstly, we observed a powder form of tramadol and ␤-CD by scanning electron microscope and then we saw a powder form of the inclusion complex (Fig. 9). Pictures clearly elucidated the difference in each case. ␤-CD shows sheeted/plated structure, tramadol shows angular and rectangular shape structure and the inclusion complex structure is surrounded to subangular structure. The inclusion complex structure is different from ␤-CD and tramadol. Modification of crystals and powder can be assumed as a proof of the formation of new inclusion complex. 4. Conclusion The following conclusions can be drawn from the above studies: (i) solvent studies show, the spectral behaviour of tramadol and anisole is same both in the So and S1 states, (ii) ␤-CD studies indicate that, due to space restriction of the ␤-CD cavity, a weak interaction is present between the anisole and cyclohexanol groups in tramadol, (iii) tramadol forms 1:2 inclusion complex with ␤-CD and (iv) FT-

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