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Transition moment directions and selected spectroscopic properties of Ivabradine
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 162–166
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Transition moment directions and selected spectroscopic properties of Ivabradine A. Synak a, P. Pikul b, P. Bojarski a,⇑, J. Nowakowska b, W. Wiczk c, A. Łukaszewicz a, A.A. Kubicki a a
´ sk, Wita Stwosza 57, 80-952 Gdan ´ sk, Poland Institute of Experimental Physics, Department of Mathematics, Physics and Informatics, University of Gdan ´ sk, Faculty of Pharmacy, Department of Physical Chemistry, Hallera 107, 80-416 Gdan ´ sk, Poland Medical University of Gdan c ´ sk, Sobieskiego 18, 80-952 Gdan ´ sk, Poland Faculty of Chemistry, University of Gdan b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" First spectroscopic results for
Ivabradine in rigid PVA matrix are provided. " TRES reveal that there is single emitting conformation of Ivabradine in PVA. " Low emission anisotropy of Ivabradine in PVA is due to large angle between the transition moments. " The angle b = 38° has been determined form Kawski– Gryczynski model in stretched PVA matrix.
a r t i c l e
i n f o
Article history: Received 18 June 2012 Received in revised form 28 August 2012 Accepted 20 September 2012 Available online 28 September 2012 Keywords: Ivabradine Transition moments directions Kawski–Gryczynski method Linear dichroism Emission anisotropy Time resolved emission spectra
a b s t r a c t Based on the Kawski–Gryczynski method the value of angle b = 38° between absorption and fluorescence transition moments of Ivabradine was determined. Such a high value of b is responsible for low emission anisotropy of Ivabradine in a rigid polyvinyl alcohol matrix and in anhydrous glycerol despite the elongated shape of the fluorophore. Selected steady-state and time-resolved spectroscopic results support the analysis. Ó 2012 Elsevier B.V. All rights reserved.
Introduction Ivabradine (3-(3-{[((7S)-3,4-dimethoxy-bicyclo[4.2.0]octa1,3,5-trien-7-yl)methyl]-methyl-amino}propyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2H-3-benzazepin-2-one hydrochloride) is a modern drug, which releases selectively the heart rate, improves the energy balance of heart and reduces its demand for oxygen and energy [1–3]. It is suspected to work by inhibiting selectively
⇑ Corresponding author. Fax: +48 58 341 31 75. E-mail address: fi[email protected] (P. Bojarski). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.09.061
the channels responsible for the creation of current in sinus node cells. This action is used in the treatment of ischemic heart disease, especially in the case of patients with angina pectoris [4]. The molecular structure of Ivabradine suggests that the absorption of this important molecule should occur in ultraviolet region. Furthermore, the molecule should be fluorescent. However, despite its important medical applications, not only the molecular basis of its action has not been well understood, but even its basic spectroscopic properties including transition moments directions have not been studied until now. The aim of this work is to fill partly this gap by reporting selected UV–Vis spectroscopic properties of Ivabradine. Our preliminary results confirmed
A. Synak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 162–166
that Ivabradine is fluorescent. Simultaneously untypically low fluorescence anisotropy was measured in rigid matrices and viscous solutions of Ivabradine. This prompted us to explain this unusual spectroscopic property of Ivabradine and to determine transition moments directions in absorption and fluorescence. One of the most effective methods to determine the directions of transition moments in absorption and fluorescence as well as the angle between them is based on the measurements of linear dichroism and emission anisotropy in a partly organized medium of controllable order degree [5]. Upon uniaxial stretching of initially disordered polymer matrix the long axes of elongated molecules (and their transition moments) become mechanically oriented towards the direction of stretching. The uniaxial mechanical stretching of the polymer film leads to the significant change of linear dichroism ratio of the measured samples and it ensures the control over the orientation of elongated guest molecules (in this case Ivabradine). The use of specific distribution function correlates the degree of stretching (linear dichroism ratio) with the angular distribution of transition moment in absorption in the partly ordered matrix [5–8]. The knowledge on the orientation of transition moment in emission compared to that in absorption can be, in turn, obtained from the Kawski–Gryczynski model [7,8]. In particular, use can be made of the expression for emission anisotropy as a function of stretching degree (or linear dichroism ratio). A very convenient medium to realize the task planned is polyvinyl alcohol thin film due to the simple preparation procedure, good solubility of guest molecule and its pliancy for uniaxial stretching. Theoretical basis Elongated fluorescent molecules embedded in the matrix can be mechanically oriented in a rigid medium by its uniaxial stretching. The control over the orientation degree is provided by the measurements of linear dichroism ratio [6]:
RD ¼
Ak A?
ð1Þ
where Ak and A\ are the components of the absorbance parallel and perpendicular to the stretching direction of the polymer film. It has been shown that the general relation between the linear dichoism ratio RD and the stretching factor RS [7]:
1 1 þ a2 1 ða2 1Þ2 arcsinð1=aÞ ð3cos2 u 1Þ cos2 u RD ¼ 2 1 1 a2 1 ða2 1Þ2 arcsinð1=aÞ ð3cos2 u 1Þ þ cos2 u
ð2Þ
where
a2 ¼ R2S =ðR2S 1Þ
ð3Þ
u – the angle between the transition moment in absorption and the long axis of the molecule. This formula can be substituted by the following simple linear dependence in the case of elongated molecules [8]: RD ¼
5 1 RS 4 4
ð4Þ
RS = a/b is obtained based on the measurement of parallel and perpendicular linear dimension of the film relative to the axis of stretching, respectively (a = b = 1 cm in the case of usntretched samples used in this work). On the other hand the experimentally obtained linear dependence of RD on RS means that the approximation of elongated molecular structure of the molecule is justified and that the transition moment in absorption is parallel to the long molecular axis [9,10].
163
Mutual orientation of the transition moments in absorption and fluorescence can be determined for elongated molecules like Ivabradine from Kawski–Gryczynski approach [7]. In this method emission anistropy is measured for the set of uniaxially stretched samples, similarly as in the case of linear dichroism measurements. The experimentally obtained emission anisotropy course versus RS is next fitted to the expression obtained by Gryczynski and Kawski. The fit is appropriate only for one specific value of angle b between the transition moment in absorption and fluorescence [7,8]:
rðRS ; bÞ ¼ rðRS Þ
rðRS Þ ¼
3 1 cos2 b 2 2
3 ða2 1Þ1=2 þ 2a2 ða2 1Þ1=2 3a2 arcsinð1=aÞ 1 2 2 2ða2 1Þ1=2 2arcsinð1=aÞ
ð5Þ
ð6Þ
and a is given by Eq. (3). The best fit yields therefore the value of angle b between transition moment in absorption and fluorescence. The advantage of the method described is that the usable range of emission anisotropy:
r¼
I k I? Ik þ 2I?
ð7Þ
in uniaxially stretched PVA films is strongly broadened compared to disordered films or liquid solutions, where emission anisotropy can theoretically change only between 0.2 and 0.4 [11]. However, in a strongly stretched film elongated fluorophores (and of course their transition moments) are preferentially oriented towards the axis of stretching resulting usually in much higher emission anisotropies with the limiting values dependent on the effective order degree [5–10]. It follows from the general definition of emission anisotropy for perfectly ordered medium that r can theoretically change between 0.5 and 1. For the maximal stretching factor used in this work RS = 7 emission anisotropy can theoretically change between 0.85 for b = 0° and 0.42 for b = 90°. Experimental details Ivabradine was purchased from Watson International Ltd. (Kunshan, China). The fluorophore as well as ethylene glycol and polyvinyl alcohol (Fluka) were of analytical grade and their purity was additionally checked by chromatographic methods. Ivabradine was dissolved in 5% water mixture of poly(vinyl alcohol) (PVA, MW 205000) at T = 340 K to obtain homogeneous solution. The samples were left for evaporation for several days. In order to determine the angle between absorption and fluorescence transition moments thin PVA films were uniaxially stretched. Eight samples of different stretching factor were prepared using standard procedure and equipment developed earlier in our laboratory [5,9]. Small overlap between absorption and fluorescence spectrum of Ivabradine could lead to the small contribution of inner filter effects and/or to the nonradiative energy migration between the monomers of Ivabradine. To exclude the inner filter effects the thickness d of oriented samples used in any fluorescence measurements was small enough to fulfill the relation:
2:3emax c d 6 0:1
ð8Þ
where emax is the maximum value of the extinction coefficient and c is the molar concentration of the samples. Upon this condition, reabsorption and reemission effects in the case studied are eliminated [12]. The molar concentration of Ivabradine c = 104 M in the samples prepared by us was much smaller than the critical concentration for excitation energy migration (homotransfer) c0DD 2 101 M. Also no effect of dye concentration on emission anisotropy has been experimentally found below c = 103 M. There-
164
A. Synak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 162–166
fore, the depolarizing effect of excitation energy migration can be upon experimental conditions also neglected [9,13]. The absorption spectra and linear dichroism measurements were carried out with Shimadzu 1650 M spectrophotometer. The use of parallel light beam and the Glan prism enabled a high degree of linear polarization (>99%) in the case of linear dichroism measurements. The fluorescence spectra were measured upon front face excitation (at a magic angle) and front face collection of sample fluorescence using sensitive spectrofluorometer constructed in our laboratory and described earlier [14]. Emission anisotropy was measured with extremely sensitive two-channel single photon counting polarization spectrofluorometer designed earlier in our laboratory and described in detail [15,16]. During the measurements both fluorescence intensity components Ik and I\ were recorded simultaneously, which eliminates the effect of fluctuations of the light source power and speeds up the measurement. Dark counts were independently collected in a similar way and taken into account during the calculations of emission anisotropy. All emission anisotropy results were checked against scattering effects and no contribution of this potential artifact was found. All instrumental factors (including G factor) were automatically
(a)
Fig. 2. Linear dichroism RD versus stretching factor Rs for Ivabradine in PVA film at T = 293 K. Straight line was fitted by the standard least squares method.
determined by our software during the measurement. Emission anisotropy was measured with a high accuracy of 0.002. Time-resolved emission spectra (TRES) and fluorescence decays were obtained simultaneously from a single measurement with the state-of-the-art pulsed spectrofluorometer of picosecond resolution designed in our laboratory and described earlier in detail [17]. The apparatus is equipped with pulsed YAG+ EXPLA laser and optical parametric generator system as the excitation source producing short, spectrally tunable light pulses of about 30 ps FWHM and Hamamatsu 4310 streak camera – Bruker Optics spectrograph system as a heart of detection system. Results and discussion
(b)
Fig. 1. Absorption, fluorescence and emission anisotropy spectra of Ivabradine in PVA film at T = 293 K obtained in (a) disordered (RS = 1) and (b) ordered (RS = 7) polymer matrix. The insert (1a) shows the structure of Ivabradine. The measured fluorescence quantum yield of Ivabradine in PVA is 0.16, the corresponding maximal extinction coefficient is 9400 M1 cm1. The total measurement error of the emission anisotropy is included in the size of the graphic point and does not exceed 0.002.
Ivabradine belongs to the class of elongated molecules, which should orientate well in the uniaxially stretched polyvinyl alcohol thin films along the axis of stretching (Fig. 1a, insert). Fig. 1a and b presents absorption, fluorescence and emission anisotropy spectra of Ivabradine in thin PVA films. Fig. 1a shows absorption and fluorescence spectra in disordered films (RS = 1), whereas Fig. 1b in strongly uniaxially stretched PVA film (RS = 7). No effect of matrix stretching on the shape, location and intensity of absorption and fluorescence spectra was found suggesting the preservation of its
Fig. 3. Emission anisotropy versus stretching factor Rs for Ivabradine in PVA film at T = 293 K (black squares). Solid line has been obtained from Kawski–Gryczynski model.
A. Synak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 162–166
165
Fig. 4. Time-resolved fluorescence image of Ivabradine in PVA taken directly from the apparatus (color image) as well as time –resolved emission spectrum of Ivabradine in PVA and its fluorescence intensity decay extracted from the image. The determined mean fluorescence lifetime of Ivabradine in PVA is 2 ns. (For color interpretation in this figure the reader is referred to see the web version of this article.)
conformation in disordered and ordered matrix. Also no photodegradation effect was found over the time of all fluorescence experiments, which means that Ivabradine is stable in the matrix. However, emission anisotropy spectra depend visibly on stretching when one compares Fig. 1a and b. Emission anisotropy at a given wavelength is about twice higher in the case of seven times stretched film (Fig. 1b) due to the partial rearrangement of Ivabradine transition moments towards the axis of stretching. Simultaneously, it can be seen from the figure that emission anisotropy spectra remain approximately constant in the fluorescence band in both cases, which means that only one type of fluorescent centers contributes to the total emission of Ivabradine. It was a little surprising to find out that the emission anisotropy of Ivabradine in the disordered film is relatively low (about 0.2) and that it increases only to about 0.4 in a strongly ordered matrix (RS = 7). For many elongated molecules with low value of angle b emission anisotropy in such a strongly ordered medium can reach even 0.8 [5,7–9,13,18]. The explanation of different behavior of Ivabradine is that the angle between absorption and fluorescence transition moment is high leading according to Eqs. (5) and (6) to significantly lower fluorescence anisotropy. In order to determine the angle b between transition moments in absorption and fluorescence for Ivabradine, linear dichroism and emission anisotropy studies were carried out for a series of differently stretched samples. Fig. 2 shows the results of linear dichroism RD versus stretching factor RS. It can be seen from the figure that approximately linear dependence between RD and RS was found. This means that the transition moment in absorption is located parallel to the axis of stretching and long molecular axis of Ivabradine. Certain spread
of experimental data around the fitted straight line originates from rather low optical density of the films and is typical for that type of measurement. Fig. 3 shows the results of emission anisotropy measurements for Ivabradine in differently stretched PVA films. With the increase in RS emission anisotropy becomes higher, but even for the largest RS it approaches relatively low value 0.38. Solid line has been obtained from Kawski–Gryczynski model for b = 38° and it represents the best fit to experimental data. Such a high value of b explains low emission anisotropy value observed for Ivabradine in disordered PVA film in the emission band. Indeed, the latter value of can be b compared with the estimation following from the general Perrin formula:
r0 ðbÞ ¼
2 3 1 cos2 b 5 2 2
ð9Þ
The measurement of emission anisotropy in anhydrous ethylene glycol at T = 283 K yields r = 0.192 ± 0.002 which leads to b = 36°, the value close to that following from Kawski–Gryczynski method. Independently, three-dimensional time-resolved emission spectra of Ivabradine in PVA were measured to make sure that there is no other hidden fluorescent component invisible in ordinary steady-state measurement of the emission spectrum. Indeed, no additional emitting component was found both for Ivabradine in disordered and ordered PVA matrix (Fig. 4 shows the result for disordered PVA film). The original measurement result was obtained as a quasi three-dimensional, colourful flat image (wavelength – horizontal axis, time – vertical axis, intensity – colour scale for which blue and red represent minimum and maximum
166
A. Synak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 162–166
values of emission intensity, respectively). By slicing the image along the wavelength axis at a given time moment after the excitation, one obtains emission spectrum corresponding to that time. Further slicing at longer times gives the time evolution of that emission spectrum. In the case of Ivabradine its simple fluorescence spectrum with the one well defined maximum at around 330 nm does not evolve with time, which means that only one fluorescence component is indeed involved in the emission process. By slicing the streak camera image along the time axis at a given wavelength yields, in turn, the fluorescence decay and mean fluorescence lifetime of the fluorophore. It occured, that the fluorescence decay of Ivabradine can be well represented by a simple one exponential curve at any observation wavelength with the constant mean fluorescence lifetime s = 2 ns in disordered and s = 2.03 ns in uniaxially stretched PVA matrix proving the existence of a single emitting center. Similar measurements carried out in ethylene glycol and water lead to qualitatively the same results with the only difference that the mean fluorescence lifetime is longer in PVA matrix compared to liquid solutions (s = 1.65 ns in ethylene glycol and s = 1.35 ns in water). This fact can be attributed to the rigidifying effect of polyvinyl alcohol matrix on molecular structure of Ivabradine. As expected, identical regularity was also observed for the fluorescence quantum yield which is slightly higher in PVA (g = 0.16) than in ethylene glycol or water (g = 0.13).
Conclusion Based on linear dichroism and emission anisotropy experimental studies in PVA thin films of controllable order degree followed by the analysis resulting from Kawski–Gryczynski model, transition moment directions in absorption and fluorescence of Ivabradine were determined. The obtained relatively low emission anisotropy values even in strongly uniaxially stretched PVA films can be quantitatively explained only by the large angle b = 38° formed between the transition moments in absorption and emission. The lack of time evolution of emission spectrum as well as the independence of mean fluorescence lifetime of observation wavelength demonstrate the presence of a single emitting conformation of Ivabradine in PVA films. However, slight mean lifetime dependence on the solvent viscosity was found which is probably
a result of the rigidifying effect of the matrix on molecular structure of Ivabradine. This and further spectroscopic properties of Ivabradine require further detailed studies and will be reported shortly together with respective quantum chemical calculations. Acknowledgments This work has been supported by the following projects: BW538-5200-0634-1 (A.S.), the Polish Ministry of Science and Higher Education grants DS-8441-4-0132-11 (W.W.), DS-5200-4-0024-11 (P.B. and A.K.), the Human Capital Operational Programme, Action 4.1.1 ‘‘Educators for the Elite – integrated training program for Ph.D. students, Post-docs and Professors as academic teachers at University of Gdan´sk’’ (A.S. and A.Ł.). References [1] M. François-Bouchard, G. Simonin, M.-J. Bossant, C. Boursier-Neyret, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 745 (2000) 261–269. [2] K. Fox, I. Ford, P.G. Steg, M. Tendera, R. Ferrari, Lancet 372 (2008) 807–816. [3] K. Fox, I. Ford, P.G. Steg, M. Tendera, M. Robertson, R. Ferrari, Lancet 372 (2008) 817–821. [4] J.C. Tardif, I. Ford, M. Tendera, M.G. Bourassa, K. Fox, Eur. Heart J. 26 (2005) 2529–2536. [5] J. Michl, E.W. Thulstrup, Spectroscopy with Polarized Light, VCH Publ. Inc., New York, 1986. [6] Y. Tanizaki, Bull. Chem. Soc. Jpn. 32 (1959) 1362; Y. Tanizaki, Bull. Chem. Soc. Jpn. 38 (1965) 1798–1799. [7] A. Kawski, Z. Gryczynski, Z. Naturforsch. 41a (1986) 1195; A. Kawski, Z. Gryczynski, Z. Naturforsch. 42a (1987) 808–812. [8] Z. Gryczynski, A. Kawski, Z. Naturforsch. 42a (1987) 1396; Z. Gryczynski, A. Kawski, Z. Naturforsch. 43a (1988) 193–195. [9] A. Synak, P. Bojarski, Chem. Phys. Lett. 416 (2005) 300–304. [10] A. Kawski, P. Bojarski, A J. Spectrosc. 11 (2007) 67–94. [11] F. Perrin, Ann. Physique 12 (1929) 169. [12] I. Ketskemety, J. Dombi, R. Horvai, J. Hevesi, L. Kozma, Acta Phys. Chem. (Szeged) 7 (1961) 17–21. [13] P. Bojarski, A. Kaminska, L. Kulak, M. Sadownik, Chem. Phys. Lett. 375 (2003) 547–552. [14] A. Kawski, G. Piszczek, B. Kuklinski, T. Nowosielski, Z. Naturforsch. 49a (1994) 824–828. [15] A. Kubicki, Exp. Tech. Phys. 37 (1989) 329–333. [16] P. Bojarski, A. Kawski, J. Fluoresc. 2 (1992) 133–139. [17] Kubicki, P. Bojarski, M. Grinberg, M. Sadownik, B. Kuklin´ski, Opt. Commun. 263 (2006) 275–280. [18] A. Kawski, Z. Gryczynski, I. Gryczynski, J.R. Lakowicz, G. Piszczek, Z. Naturforsch. 51 (1996) 1037–1041.
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Transition moment directions and selected spectroscopic properties of Ivabradine A. Synak a, P. Pikul b, P. Bojarski a,⇑, J. Nowakowska b, W. Wiczk c, A. Łukaszewicz a, A.A. Kubicki a a
´ sk, Wita Stwosza 57, 80-952 Gdan ´ sk, Poland Institute of Experimental Physics, Department of Mathematics, Physics and Informatics, University of Gdan ´ sk, Faculty of Pharmacy, Department of Physical Chemistry, Hallera 107, 80-416 Gdan ´ sk, Poland Medical University of Gdan c ´ sk, Sobieskiego 18, 80-952 Gdan ´ sk, Poland Faculty of Chemistry, University of Gdan b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" First spectroscopic results for
Ivabradine in rigid PVA matrix are provided. " TRES reveal that there is single emitting conformation of Ivabradine in PVA. " Low emission anisotropy of Ivabradine in PVA is due to large angle between the transition moments. " The angle b = 38° has been determined form Kawski– Gryczynski model in stretched PVA matrix.
a r t i c l e
i n f o
Article history: Received 18 June 2012 Received in revised form 28 August 2012 Accepted 20 September 2012 Available online 28 September 2012 Keywords: Ivabradine Transition moments directions Kawski–Gryczynski method Linear dichroism Emission anisotropy Time resolved emission spectra
a b s t r a c t Based on the Kawski–Gryczynski method the value of angle b = 38° between absorption and fluorescence transition moments of Ivabradine was determined. Such a high value of b is responsible for low emission anisotropy of Ivabradine in a rigid polyvinyl alcohol matrix and in anhydrous glycerol despite the elongated shape of the fluorophore. Selected steady-state and time-resolved spectroscopic results support the analysis. Ó 2012 Elsevier B.V. All rights reserved.
Introduction Ivabradine (3-(3-{[((7S)-3,4-dimethoxy-bicyclo[4.2.0]octa1,3,5-trien-7-yl)methyl]-methyl-amino}propyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2H-3-benzazepin-2-one hydrochloride) is a modern drug, which releases selectively the heart rate, improves the energy balance of heart and reduces its demand for oxygen and energy [1–3]. It is suspected to work by inhibiting selectively
⇑ Corresponding author. Fax: +48 58 341 31 75. E-mail address: fi[email protected] (P. Bojarski). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.09.061
the channels responsible for the creation of current in sinus node cells. This action is used in the treatment of ischemic heart disease, especially in the case of patients with angina pectoris [4]. The molecular structure of Ivabradine suggests that the absorption of this important molecule should occur in ultraviolet region. Furthermore, the molecule should be fluorescent. However, despite its important medical applications, not only the molecular basis of its action has not been well understood, but even its basic spectroscopic properties including transition moments directions have not been studied until now. The aim of this work is to fill partly this gap by reporting selected UV–Vis spectroscopic properties of Ivabradine. Our preliminary results confirmed
A. Synak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 162–166
that Ivabradine is fluorescent. Simultaneously untypically low fluorescence anisotropy was measured in rigid matrices and viscous solutions of Ivabradine. This prompted us to explain this unusual spectroscopic property of Ivabradine and to determine transition moments directions in absorption and fluorescence. One of the most effective methods to determine the directions of transition moments in absorption and fluorescence as well as the angle between them is based on the measurements of linear dichroism and emission anisotropy in a partly organized medium of controllable order degree [5]. Upon uniaxial stretching of initially disordered polymer matrix the long axes of elongated molecules (and their transition moments) become mechanically oriented towards the direction of stretching. The uniaxial mechanical stretching of the polymer film leads to the significant change of linear dichroism ratio of the measured samples and it ensures the control over the orientation of elongated guest molecules (in this case Ivabradine). The use of specific distribution function correlates the degree of stretching (linear dichroism ratio) with the angular distribution of transition moment in absorption in the partly ordered matrix [5–8]. The knowledge on the orientation of transition moment in emission compared to that in absorption can be, in turn, obtained from the Kawski–Gryczynski model [7,8]. In particular, use can be made of the expression for emission anisotropy as a function of stretching degree (or linear dichroism ratio). A very convenient medium to realize the task planned is polyvinyl alcohol thin film due to the simple preparation procedure, good solubility of guest molecule and its pliancy for uniaxial stretching. Theoretical basis Elongated fluorescent molecules embedded in the matrix can be mechanically oriented in a rigid medium by its uniaxial stretching. The control over the orientation degree is provided by the measurements of linear dichroism ratio [6]:
RD ¼
Ak A?
ð1Þ
where Ak and A\ are the components of the absorbance parallel and perpendicular to the stretching direction of the polymer film. It has been shown that the general relation between the linear dichoism ratio RD and the stretching factor RS [7]:
1 1 þ a2 1 ða2 1Þ2 arcsinð1=aÞ ð3cos2 u 1Þ cos2 u RD ¼ 2 1 1 a2 1 ða2 1Þ2 arcsinð1=aÞ ð3cos2 u 1Þ þ cos2 u
ð2Þ
where
a2 ¼ R2S =ðR2S 1Þ
ð3Þ
u – the angle between the transition moment in absorption and the long axis of the molecule. This formula can be substituted by the following simple linear dependence in the case of elongated molecules [8]: RD ¼
5 1 RS 4 4
ð4Þ
RS = a/b is obtained based on the measurement of parallel and perpendicular linear dimension of the film relative to the axis of stretching, respectively (a = b = 1 cm in the case of usntretched samples used in this work). On the other hand the experimentally obtained linear dependence of RD on RS means that the approximation of elongated molecular structure of the molecule is justified and that the transition moment in absorption is parallel to the long molecular axis [9,10].
163
Mutual orientation of the transition moments in absorption and fluorescence can be determined for elongated molecules like Ivabradine from Kawski–Gryczynski approach [7]. In this method emission anistropy is measured for the set of uniaxially stretched samples, similarly as in the case of linear dichroism measurements. The experimentally obtained emission anisotropy course versus RS is next fitted to the expression obtained by Gryczynski and Kawski. The fit is appropriate only for one specific value of angle b between the transition moment in absorption and fluorescence [7,8]:
rðRS ; bÞ ¼ rðRS Þ
rðRS Þ ¼
3 1 cos2 b 2 2
3 ða2 1Þ1=2 þ 2a2 ða2 1Þ1=2 3a2 arcsinð1=aÞ 1 2 2 2ða2 1Þ1=2 2arcsinð1=aÞ
ð5Þ
ð6Þ
and a is given by Eq. (3). The best fit yields therefore the value of angle b between transition moment in absorption and fluorescence. The advantage of the method described is that the usable range of emission anisotropy:
r¼
I k I? Ik þ 2I?
ð7Þ
in uniaxially stretched PVA films is strongly broadened compared to disordered films or liquid solutions, where emission anisotropy can theoretically change only between 0.2 and 0.4 [11]. However, in a strongly stretched film elongated fluorophores (and of course their transition moments) are preferentially oriented towards the axis of stretching resulting usually in much higher emission anisotropies with the limiting values dependent on the effective order degree [5–10]. It follows from the general definition of emission anisotropy for perfectly ordered medium that r can theoretically change between 0.5 and 1. For the maximal stretching factor used in this work RS = 7 emission anisotropy can theoretically change between 0.85 for b = 0° and 0.42 for b = 90°. Experimental details Ivabradine was purchased from Watson International Ltd. (Kunshan, China). The fluorophore as well as ethylene glycol and polyvinyl alcohol (Fluka) were of analytical grade and their purity was additionally checked by chromatographic methods. Ivabradine was dissolved in 5% water mixture of poly(vinyl alcohol) (PVA, MW 205000) at T = 340 K to obtain homogeneous solution. The samples were left for evaporation for several days. In order to determine the angle between absorption and fluorescence transition moments thin PVA films were uniaxially stretched. Eight samples of different stretching factor were prepared using standard procedure and equipment developed earlier in our laboratory [5,9]. Small overlap between absorption and fluorescence spectrum of Ivabradine could lead to the small contribution of inner filter effects and/or to the nonradiative energy migration between the monomers of Ivabradine. To exclude the inner filter effects the thickness d of oriented samples used in any fluorescence measurements was small enough to fulfill the relation:
2:3emax c d 6 0:1
ð8Þ
where emax is the maximum value of the extinction coefficient and c is the molar concentration of the samples. Upon this condition, reabsorption and reemission effects in the case studied are eliminated [12]. The molar concentration of Ivabradine c = 104 M in the samples prepared by us was much smaller than the critical concentration for excitation energy migration (homotransfer) c0DD 2 101 M. Also no effect of dye concentration on emission anisotropy has been experimentally found below c = 103 M. There-
164
A. Synak et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 162–166
fore, the depolarizing effect of excitation energy migration can be upon experimental conditions also neglected [9,13]. The absorption spectra and linear dichroism measurements were carried out with Shimadzu 1650 M spectrophotometer. The use of parallel light beam and the Glan prism enabled a high degree of linear polarization (>99%) in the case of linear dichroism measurements. The fluorescence spectra were measured upon front face excitation (at a magic angle) and front face collection of sample fluorescence using sensitive spectrofluorometer constructed in our laboratory and described earlier [14]. Emission anisotropy was measured with extremely sensitive two-channel single photon counting polarization spectrofluorometer designed earlier in our laboratory and described in detail [15,16]. During the measurements both fluorescence intensity components Ik and I\ were recorded simultaneously, which eliminates the effect of fluctuations of the light source power and speeds up the measurement. Dark counts were independently collected in a similar way and taken into account during the calculations of emission anisotropy. All emission anisotropy results were checked against scattering effects and no contribution of this potential artifact was found. All instrumental factors (including G factor) were automatically
(a)
Fig. 2. Linear dichroism RD versus stretching factor Rs for Ivabradine in PVA film at T = 293 K. Straight line was fitted by the standard least squares method.
determined by our software during the measurement. Emission anisotropy was measured with a high accuracy of 0.002. Time-resolved emission spectra (TRES) and fluorescence decays were obtained simultaneously from a single measurement with the state-of-the-art pulsed spectrofluorometer of picosecond resolution designed in our laboratory and described earlier in detail [17]. The apparatus is equipped with pulsed YAG+ EXPLA laser and optical parametric generator system as the excitation source producing short, spectrally tunable light pulses of about 30 ps FWHM and Hamamatsu 4310 streak camera – Bruker Optics spectrograph system as a heart of detection system. Results and discussion
(b)
Fig. 1. Absorption, fluorescence and emission anisotropy spectra of Ivabradine in PVA film at T = 293 K obtained in (a) disordered (RS = 1) and (b) ordered (RS = 7) polymer matrix. The insert (1a) shows the structure of Ivabradine. The measured fluorescence quantum yield of Ivabradine in PVA is 0.16, the corresponding maximal extinction coefficient is 9400 M1 cm1. The total measurement error of the emission anisotropy is included in the size of the graphic point and does not exceed 0.002.
Ivabradine belongs to the class of elongated molecules, which should orientate well in the uniaxially stretched polyvinyl alcohol thin films along the axis of stretching (Fig. 1a, insert). Fig. 1a and b presents absorption, fluorescence and emission anisotropy spectra of Ivabradine in thin PVA films. Fig. 1a shows absorption and fluorescence spectra in disordered films (RS = 1), whereas Fig. 1b in strongly uniaxially stretched PVA film (RS = 7). No effect of matrix stretching on the shape, location and intensity of absorption and fluorescence spectra was found suggesting the preservation of its
Fig. 3. Emission anisotropy versus stretching factor Rs for Ivabradine in PVA film at T = 293 K (black squares). Solid line has been obtained from Kawski–Gryczynski model.
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Fig. 4. Time-resolved fluorescence image of Ivabradine in PVA taken directly from the apparatus (color image) as well as time –resolved emission spectrum of Ivabradine in PVA and its fluorescence intensity decay extracted from the image. The determined mean fluorescence lifetime of Ivabradine in PVA is 2 ns. (For color interpretation in this figure the reader is referred to see the web version of this article.)
conformation in disordered and ordered matrix. Also no photodegradation effect was found over the time of all fluorescence experiments, which means that Ivabradine is stable in the matrix. However, emission anisotropy spectra depend visibly on stretching when one compares Fig. 1a and b. Emission anisotropy at a given wavelength is about twice higher in the case of seven times stretched film (Fig. 1b) due to the partial rearrangement of Ivabradine transition moments towards the axis of stretching. Simultaneously, it can be seen from the figure that emission anisotropy spectra remain approximately constant in the fluorescence band in both cases, which means that only one type of fluorescent centers contributes to the total emission of Ivabradine. It was a little surprising to find out that the emission anisotropy of Ivabradine in the disordered film is relatively low (about 0.2) and that it increases only to about 0.4 in a strongly ordered matrix (RS = 7). For many elongated molecules with low value of angle b emission anisotropy in such a strongly ordered medium can reach even 0.8 [5,7–9,13,18]. The explanation of different behavior of Ivabradine is that the angle between absorption and fluorescence transition moment is high leading according to Eqs. (5) and (6) to significantly lower fluorescence anisotropy. In order to determine the angle b between transition moments in absorption and fluorescence for Ivabradine, linear dichroism and emission anisotropy studies were carried out for a series of differently stretched samples. Fig. 2 shows the results of linear dichroism RD versus stretching factor RS. It can be seen from the figure that approximately linear dependence between RD and RS was found. This means that the transition moment in absorption is located parallel to the axis of stretching and long molecular axis of Ivabradine. Certain spread
of experimental data around the fitted straight line originates from rather low optical density of the films and is typical for that type of measurement. Fig. 3 shows the results of emission anisotropy measurements for Ivabradine in differently stretched PVA films. With the increase in RS emission anisotropy becomes higher, but even for the largest RS it approaches relatively low value 0.38. Solid line has been obtained from Kawski–Gryczynski model for b = 38° and it represents the best fit to experimental data. Such a high value of b explains low emission anisotropy value observed for Ivabradine in disordered PVA film in the emission band. Indeed, the latter value of can be b compared with the estimation following from the general Perrin formula:
r0 ðbÞ ¼
2 3 1 cos2 b 5 2 2
ð9Þ
The measurement of emission anisotropy in anhydrous ethylene glycol at T = 283 K yields r = 0.192 ± 0.002 which leads to b = 36°, the value close to that following from Kawski–Gryczynski method. Independently, three-dimensional time-resolved emission spectra of Ivabradine in PVA were measured to make sure that there is no other hidden fluorescent component invisible in ordinary steady-state measurement of the emission spectrum. Indeed, no additional emitting component was found both for Ivabradine in disordered and ordered PVA matrix (Fig. 4 shows the result for disordered PVA film). The original measurement result was obtained as a quasi three-dimensional, colourful flat image (wavelength – horizontal axis, time – vertical axis, intensity – colour scale for which blue and red represent minimum and maximum
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values of emission intensity, respectively). By slicing the image along the wavelength axis at a given time moment after the excitation, one obtains emission spectrum corresponding to that time. Further slicing at longer times gives the time evolution of that emission spectrum. In the case of Ivabradine its simple fluorescence spectrum with the one well defined maximum at around 330 nm does not evolve with time, which means that only one fluorescence component is indeed involved in the emission process. By slicing the streak camera image along the time axis at a given wavelength yields, in turn, the fluorescence decay and mean fluorescence lifetime of the fluorophore. It occured, that the fluorescence decay of Ivabradine can be well represented by a simple one exponential curve at any observation wavelength with the constant mean fluorescence lifetime s = 2 ns in disordered and s = 2.03 ns in uniaxially stretched PVA matrix proving the existence of a single emitting center. Similar measurements carried out in ethylene glycol and water lead to qualitatively the same results with the only difference that the mean fluorescence lifetime is longer in PVA matrix compared to liquid solutions (s = 1.65 ns in ethylene glycol and s = 1.35 ns in water). This fact can be attributed to the rigidifying effect of polyvinyl alcohol matrix on molecular structure of Ivabradine. As expected, identical regularity was also observed for the fluorescence quantum yield which is slightly higher in PVA (g = 0.16) than in ethylene glycol or water (g = 0.13).
Conclusion Based on linear dichroism and emission anisotropy experimental studies in PVA thin films of controllable order degree followed by the analysis resulting from Kawski–Gryczynski model, transition moment directions in absorption and fluorescence of Ivabradine were determined. The obtained relatively low emission anisotropy values even in strongly uniaxially stretched PVA films can be quantitatively explained only by the large angle b = 38° formed between the transition moments in absorption and emission. The lack of time evolution of emission spectrum as well as the independence of mean fluorescence lifetime of observation wavelength demonstrate the presence of a single emitting conformation of Ivabradine in PVA films. However, slight mean lifetime dependence on the solvent viscosity was found which is probably
a result of the rigidifying effect of the matrix on molecular structure of Ivabradine. This and further spectroscopic properties of Ivabradine require further detailed studies and will be reported shortly together with respective quantum chemical calculations. Acknowledgments This work has been supported by the following projects: BW538-5200-0634-1 (A.S.), the Polish Ministry of Science and Higher Education grants DS-8441-4-0132-11 (W.W.), DS-5200-4-0024-11 (P.B. and A.K.), the Human Capital Operational Programme, Action 4.1.1 ‘‘Educators for the Elite – integrated training program for Ph.D. students, Post-docs and Professors as academic teachers at University of Gdan´sk’’ (A.S. and A.Ł.). References [1] M. François-Bouchard, G. Simonin, M.-J. Bossant, C. Boursier-Neyret, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 745 (2000) 261–269. [2] K. Fox, I. Ford, P.G. Steg, M. Tendera, R. Ferrari, Lancet 372 (2008) 807–816. [3] K. Fox, I. Ford, P.G. Steg, M. Tendera, M. Robertson, R. Ferrari, Lancet 372 (2008) 817–821. [4] J.C. Tardif, I. Ford, M. Tendera, M.G. Bourassa, K. Fox, Eur. Heart J. 26 (2005) 2529–2536. [5] J. Michl, E.W. Thulstrup, Spectroscopy with Polarized Light, VCH Publ. Inc., New York, 1986. [6] Y. Tanizaki, Bull. Chem. Soc. Jpn. 32 (1959) 1362; Y. Tanizaki, Bull. Chem. Soc. Jpn. 38 (1965) 1798–1799. [7] A. Kawski, Z. Gryczynski, Z. Naturforsch. 41a (1986) 1195; A. Kawski, Z. Gryczynski, Z. Naturforsch. 42a (1987) 808–812. [8] Z. Gryczynski, A. Kawski, Z. Naturforsch. 42a (1987) 1396; Z. Gryczynski, A. Kawski, Z. Naturforsch. 43a (1988) 193–195. [9] A. Synak, P. Bojarski, Chem. Phys. Lett. 416 (2005) 300–304. [10] A. Kawski, P. Bojarski, A J. Spectrosc. 11 (2007) 67–94. [11] F. Perrin, Ann. Physique 12 (1929) 169. [12] I. Ketskemety, J. Dombi, R. Horvai, J. Hevesi, L. Kozma, Acta Phys. Chem. (Szeged) 7 (1961) 17–21. [13] P. Bojarski, A. Kaminska, L. Kulak, M. Sadownik, Chem. Phys. Lett. 375 (2003) 547–552. [14] A. Kawski, G. Piszczek, B. Kuklinski, T. Nowosielski, Z. Naturforsch. 49a (1994) 824–828. [15] A. Kubicki, Exp. Tech. Phys. 37 (1989) 329–333. [16] P. Bojarski, A. Kawski, J. Fluoresc. 2 (1992) 133–139. [17] Kubicki, P. Bojarski, M. Grinberg, M. Sadownik, B. Kuklin´ski, Opt. Commun. 263 (2006) 275–280. [18] A. Kawski, Z. Gryczynski, I. Gryczynski, J.R. Lakowicz, G. Piszczek, Z. Naturforsch. 51 (1996) 1037–1041.