Time resolved fluorescence and anisotropy of 1-pyrene butyric acid and pyranine as probes of solvent organization in sucrose solutions

Journal of Crystal Growth 130 (1993) 587—599 North-Holland

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Time resolved fluorescence and anisotropy of 1-pyrene butyric acid and pyranine as probes of solvent organization in sucrose solutions Borlan Pan, Reena Chakraborty and Kris A. Bergiund

1

Departments of Chemical Engineering and Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824, USA Received 31 July 1992; manuscript received in final form 12 January 1993

The microenvironment of solute—solvent interactions in aqueous sucrose solutions, ranging from 0 to 80 wt% in concentration, was investigated using time resolved fluorescence techniques. The fluorescence lifetimes and rotational correlation times of the trace fluorescent probes, PBA (1-pyrene butyric acid) and pyranine (trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate), were measured in sucrose solutions. The behavior of the fluorescence lifetimes and the increase in the rotational correlation times with increasing sucrose concentration provided no evidence for the formation of water exclusive solute clusters in the metastable solutions. Instead, the results indicated the formation of a network of hydrogen bonding interactions between dispersed sucrose molecules.

1. Introduction The characterization of the molecular interactions and dynamics of concentrated sucrose solutions is crucial to understanding the crystallization behavior. Processes including hydrogen bonding, hydrophobic interactions and electrostatic interactions, which generally occur on the picosecond to nanosecond time scales, are manifested in physical properties such as diffusion and viscosity as well as on the thermodynamic properties of phase formation. The behavior in solution, as dictated by the forces and dynamics operating at the molecular space and time scales, can be inferred by measuring interactions with carefully selected fluorescence probe molecules. Previous investigators have studied the nature of molecular associations and solute organization in saturated aqueous solutions by using a variety of means. Myerson and coworkers [1,2] gave evidence of molecular aggregation, where the diffusivities of urea, glycine, potassium chloride and sodium chloride solutions were seen to decrease drastically in the supersaturated region. Larson 1

To whom all correspondence should be addressed,

0022-0248/93/$06.00 © 1993

—

and Garside [3,4] measured density gradients in columns of citric acid, urea, sodium nitrate and potassium nitrate solutions, confirming the formation of solute clusters in supersaturated solutions. Similar results in column studies of sucrose solutions were reported by Allen et a!. [5]. Such results supplement the work of Bergiund and coworkers who used Raman spectroscopy [6,7] to show that species exhibiting the spectra of the solid phase exist in alkali nitrate solutions. Although the studies of Narayanan and Youngquist [81 showed little effect of supersaturation on measurements of the density, viscosity and electrical conductivity of solutions of inorganic ions, the formation of solute aggregates in supersaturated solutions is evident. In sucrose solutions, the viscosity and diffusivity are strongly influenced by an increase in the concentration of sugar and reflect the microscopic structuring of the solvent. Dielectric relaxation and nuclear magnetic resonance (NMR) methods of Suggett and coworkers [9—11]have identified two distinct relaxation processes for water in sugar solutions. The two forms were designated as associated water, hydrating the sugar, and bulk water, comprising the remaining

Elsevier Science Publishers B.V. All rights reserved

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Time resolved fluorescence and anisotropy of PBA and pyranine

water in solution. Richardson et al.’s [13,14] 2H and ~O NMR studies on the mobility of water in sucrose solutions indicated that associated water has a longer relaxation time than the bulk water. Decreasing rotational mobilities of water molecules were observed as sucrose concentration was increased into supersaturated regions. These effects were attributed to the progressive development of a network of hydrogen bonds consisting of (1) water to sucrose, (2) water bridging of the sucrose and (3) direct sucrose to sucrose interactions. X-ray diffraction studies and Raman spectroscopy of sucrose solutions had demonstrated similar effects [15—171. Such investigations demonstrate the influence of microscopic solvent interactions on the crystallization behavior, The fluorescence properties of probe molecules in sucrose solutions offer another perspective on the role of solvent organization in crystallization, Recently, steady state fluorescence investigations using pyranine, as a trace fluorescence probe, demonstrated that the relative amounts of associated and bulk water can be measured by monitoring the relative peak intensities of the steady state fluorescence [181. In these experiments, the steady state emission intensity corresponding to protonated pyranine increased while the intensity corresponding to deprotonated pyranine decreased with increasing sucrose concentration. By relating the emission intensities of the protonated and deprotonated forms of pyranine to sucrose associated water and bulk water, respectively, the relative amounts of the two forms of water could be quantified. To gain further understanding of the nature of the solvent structure, characterization of the microscopic interactions responsible for the steady state emission behavior of pyranine is essential. Time resolved fluorescence techniques are able to resolve additional detail on the organization and dynamics of the solute—solvent interactions in the microenvironment of a probe molecule. The emission lifetime of a fluorescent probe is highly sensitive to its interactions with the solvation microenvironment and provides information on the solvent polarity as well as diffusive processes such as collisional quenching and proton transport. Dynamic aspects of molecular interac-

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Fig. 1. Structures of PBA (1-pyrene butyric acid, left) and pyranine (trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate, right) showing the dimensions between hydrogen atoms along the long axis of pyrene.

tions can be deduced from the measurement of the time resolved fluorescence anisotropy where the solvent environment influences the rotational mobility of the probe. Here, we use time resolved fluorescence techniques to investigate the influence of the microscopic structure and dynamics of sucrose solutions on the fluorescent properties of PBA (1-pyrene butyric acid, fig. 1) and pyranine (trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate, fig. 1). PBA is a well-characterized fluorescent probe that has been utilized as an oxygen sensor [19], for studies of the segmental flexibility of proteins [201, the fluidity of membranes [211 and polymer association [22—251.The PBA molecule is hydrophobic and readily forms excimers in water at concentrations above 1 x iO~ M. A blue emission is observed in the steady state fluorescence spectra with peaks at 380 and 398 nm (fig. 2). Furthermore, the unusually long lifetime of this probe (—~100 ns) in aqueous solutions makes the accurate measurement of long rotational correlation times possible. Pyranine, with its dissociable proton, is highly sensitive to proton transfer processes and has been applied to the study of reverse micelles [261, sol-gel polymerization [271, and as a source of protons for pH jump experiments [281.It is highly soluble in aqueous solutions where the sodium ions are probably completely dissociated. The photochemistry of pyranine has been studied and is described elsewhere [261.The protonated form emits with a wavelength maxima at 440 nm, while the dissociated form emits with a wavelength maxima at 511 nm. With these two probes, nanosecond time resolved fluorescence lifetimes and anisotropic rotational correlation times were

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resol redfluorescence and ani.sotropy of PBA and pyranine

not deoxygenated before the measurements. Suprasil were used to contain the samples quartz during cuvettes the measurements.

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2.2. Procedure lowed to cool to 22°Cfor at least 3 h and were Vertically polarized laser pulses of 10 ns full width at half-maximum, from a Nd3~: YAG Qswitched Quanta-Ray DCR-1 laser were used to

~ Wt.% Sucrose: 70 60

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0 I

-

380

400

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I

420

440

460

480

500

Emission Wavelength (nm) Fig. 2. Steady state fluorescence emission of PBA in sucrose solutions with excitation at 355 nm. The spectra are normalized to the maximum peak intensity near 380 nm.

measured in sucrose solutions with the results interpreted in terms of the microscopic solution structure.

2. Experimental methods 2.1. Materials The PBA and pyranine, obtained from Eastman Kodak, and ultrapure sucrose, obtained from Boehringer Mannhein, were used without further purification. PBA is sparingly soluble in aqueous solutions, but at higher pH values the solubility increases presumably because of the deprotonation of the carboxyl group. For this reason, and because of the low reactivity of borate, the sucrose solutions with PBA were prepared at 80°Cby dilution of an 80 wt% solution with a 10mM sodium borate buffer at pH 8.5, to make 10 g samples with concentrations ranging from 0 to 80 wt%. For samples with pyranine, reverse osmosis water was used for dilution. The PBA concentration used was 2 x 106 mol/kg solution, while the pyranine concentration was 1 X iO~ mol/kg solution. The fluorescent probes were added to both the 80 wt% solution and water before dilution. All samples were then a!-

excite the samples at a rate of 10 Hz during the time resolved fluorescence measurements. A harmonic generator produced pulses with a wavelength of 355 nm from the fundamental wavelength of 1064 nm. Using an L-format fluorimeter to measure the fluorescence decays [29], the fluo.

rescence emission was passed through a dichroic polarizing filter, a pair of collimating lenses and a depolarizer at a right angle to the excitation path before entering the monochromator. The depolarizer eliminated the need for intensity correction factors in the analysis of the fluorescence decays. A fast photo-multiplier tube collected the emission profile from a dual-grating monochromator with adjustable input and exit slit widths. Triggering was accomplished by detecting the partially reflected excitation pulse entering the sample chamber. Time resolved decay waveforms were collected and averaged by a Tektronix DSA6O2A digital oscilloscope and transferred to a computer for fitting to exponential decay functions. Laser pulse profiles were measured at 355 nm from a dilute suspension of scattering particles. Alignment of the laser path ensured that the scattered intensity from the horizontal orientation was less than 2% of the intensity from the vertical orientation. Total fluorescence lifetime measurements were collected with the polarizer oriented at the magic angle, 55°from the vertical, to eliminate polarization effects. The final waveforms used in the analysis consisted of an average from 128 waveforms. The vertical and horizontal components of the emission were collected by alternating the orientation of the emission polarizer to counteract intensity fluctuations in the laser power. Waveforms used for each polarization also consisted of an average from about 128

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Time resolved fluorescence and anisotropy of PBA and pyranine

waveforms. For decay lifetimes longer than approximately 50 ns, data were collected at a resolution of 0.5 ns per data point. For shorter decay lifetimes, the time interval used was 0.1 ns. Total fluorescence decays of PBA were measured at emission wavelengths of 380 and 400 nm while the emission of pyranine was monitored at 440 and 511 nrn. Anisotropy decays of PBA were measured at 380 nrn only, while anisotropy decays of pyranine were measured at both 440 and 511 nm. For measurements with PBA, each data point collected represents a single sample, while for measurements with pyranine, the data represent averages from at least two samples with the error estimated by the standard deviation.

of the excitation pulse. A Gaussian error distribution is appropriate for measurements employing the digital oscilloscope; therefore the least squares weighting factor is a constant. In order to cornpare different fits, the weighting factor was estimated by averaging the standard deviations for a straight line fit of nine data points centered on each point throughout the waveform. The number of exponential terms used to describe the decay was determined by whether an additional term would decrease the value of the reduced sum of squares by a value of at least 0.5. The time dependent fluorescence anisotropic decay is defined by 2IVh),

A(t)=(I~—IVh)/(IW+

2.3. Analysis of fluorescence decays The fluorescence emission, F(t), from an mitial excited state population, A 1, decays exponentially with characteristic lifetimes, r1, where j represents a single fluorescence component. Typically, the experimental decays can be fit by a sum of one or two exponential terms [29], F(t)

p

=

~ A et,/T,,

(1)

(3)

where I~ and ‘vh are the vertically and horizontally oriented polarization emission intensities, respectively, for a vertically polarized excitation source. The denominator is the total fluorescence decay measured with the magic angle orientation and provides the total fluorescence lifetime, while the numerator is the difference of the vertical and horizontal decays. By independently fitting the difference decay and the total decay to a sum of exponential terms,

i= I

q

where p is the number of exponential terms. When a pulse with a finite duration is used for excitation, the experimentally observed fluorescence decay profile, FObS(t), is distorted by the excitation profile, L(t), tL(t —s)F(s) ds, (2) FOhS(t) = f 0 and the appropriate parameters must be extracted from the convoluted fluorescence profile using numerical means. An iterative least-squares deconvolution procedure was used to obtain the fluorescence parameters from the experimental decays which allows the accurate resolution of single fluorescence lifetimes of up to onetwentieth of the pulse width [30,31]. The Levenberg—Marquardt algorithm [32,33] was used to minimize the reduced sum of squares for the fluorescence parameters and for parameters taking into account the scattered light and time shift

~ D, eh,’a A(t)

=

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For a single exponential thefound. rotathe rotational correlationdecay, times, finding p1, were tional correlation time from this relation is straightforward. However, for multiple difference decays and total decays the situation is complicated by the overlapping contributions of the terms. This situation can be simplified by assuming that each of the exponential terms are independent so that the rotational correlation time for each component is estimated by the relation, =

(5_i

—

r’).

(5)

For large differences in the respective decay times, as is the case here, this procedure gives a

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Emission Wavelength (nm) Fig. 3. Steady state fluorescence emission of pyranine in sucrose solutions with excitation at 342 nm.

reliable approximation to the actual rotational correlation time.

3. Results and discussion 3.1. Steady state fluorescence spectra Steady state fluorescence spectra of PBA are shown in fig. 2. A slight shift to longer wavelengths in the three peaks of the fluorescence spectra is seen as the concentration of sucrose is increased. Also, the intensity of the fluorescence above 400 nm increases relative to the 380 nrn peak. Steady state fluorescence spectra of pyranine in sucrose solutions of various concentrations are shown in fig. 3. The intensity of the 511 nm peak is seen to decrease relative to the peak at 440 nm as the sucrose concentration increases and coincides with a relative increase in the steady state concentration of the excited-state protonated form of pyranine.

50

60

70

I

80

Wt % Sucrose

terms were required at higher concentrations. The value of the longer lifetime increases from 100 ns in the buffer to about 125 ns at 40 wt%. Above this concentration, the longer lifetime remains constant up to 80 wt%. Values for the faster lifetime ranged from 0 to 40 ns. At an emission wavelength of 400 nm, as seen in fig. 5, the faster component is present at all concentra-

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The dependence of the fluorescence lifetimes of PBA on sucrose concentration is shown in fig. 4. The 380 nm fluorescence decay was adequately fit by a single exponential term at sucrose concentrations below 50 wt%, while two exponential

40

Fig. 4. Fluorescence lifetimes of PBA in sucrose solutions, observed at 380 nm with excitation at 355 nm, as a function of sucrose concentration. Squares and triangles represent single measurements of the long lifetime and short lifetime component, respectively.

_

3.2. Time resolved fluorescence lifetimes

30

10

20

30

40

50

60

70

80

Wt % Sucrose Fig. 5. Fluorescence lifetimes of PBA in sucrose solutions, observed at 400 nm with excitation at 355 nm. as a function of

sucrose concentration. Squares and triangles represent single measurements of the long lifetime and short lifetime component, respectively.

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Time resolved fluorescence and anisoiropy of PBA and pyranine

tions measured. Above 76 wt% fast nucleation of the supersaturated solutions was observed. However, the presence of the crystals did not appear to significantly affect the lifetime measurements. The fluorescence lifetime of PBA is sensitive to the local microenvironment of the probe [19]. Because of the long lifetime of PBA, the diffusional radius in the excited state is also extensive. During the excited state, collisions with quenchers, leading to a decrease in the fluorescence lifetime, can occur. At dilute concentrations of sucrose, the lifetime may be shortened by collisions with dissolved oxygen, but as the solutions become more concentrated, the diffusion of both PBA and oxygen is hindered, causing an increase in the lifetime of PBA. Additionally, PBA is known to have a longer lifetime in more nonpolar solvents [19]. This effect may also explain the shift to shorter wavelengths seen in the steady state spectra. Thus, the increase in the lifetime of PBA is most likely due to the combined effects of the decreased collisional quenching by oxygen and the increased apparent hydrophobicity of the solvent with increasing sucrose concentration. The fluorescence lifetimes of pyranine in sucrose solutions (fig. 6) display greater variation than the lifetimes of PBA. Three regions are present for both wavelengths observed as the concentration is increased to 80 wt%. For the emission at 440 nm, a low intensity long component (not shown), of about 10 ns, is present along with the dominant faster component for concentrations up to about 40 wt%. Because of the relatively large intensity of the 511 nm band at these concentrations, the long lifetime component measured at 440 nm is likely due to overlap from this band. The fluorescence decay at 511 nm, attributed to the deprotonated form, can be fit by a single exponential term throughout the entire range of concentrations. In the dilute sucrose solutions, less than 40 wt%, the apparent lifetimes of both species are relatively constant with the protonated form exhibiting a value of 0.5 ns and the deprotonated form exhibiting a value of 5.5 ns. At 40 wt% the lifetime corresponding to the 440 nm band begins to increase, while the lifetime corresponding to the 511 nm band shows

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Wt % Sucrose Fig. 6. Fluorescence lifetimes of pyranine, excited at 355 nm, in sucrose solutions. Squares and triangles represent the average of the lifetimes observed at 440 and 511 nm, respectively. Error bars indicate the standard deviation of the measurements.

a slight decrease to 5.4 ns and remains relatively constant up to a concentration of 66 wt%. In saturated solutions, the lifetime at 511 nm decreases sharply to a value of 4.4 ns at 80 wt% while at 440 nm, the lifetime continues to increase and plateaus to 3.6 ns near 80 wt% sucrose. As with PBA, these variations in the fluorescence lifetime are due to effects of the local microenvironment of the probe. Changes in the fluorescent lifetimes of pyranine can be attributed to changes in the rates of protonation and deprotonation. It is known that the fluorescence behavior of pyranine is highly sensitive to the proton transport properties of the solution [26]. Fig. 7 depicts the various forms of the pyranine probe as a proton donor in a twopKa=0.5

pyon*

355 nm~

~

440 nm

k

1

-

511 nm

nm

pKa=7.5 ~‘~‘OH PyO + H Fig. 7. Kinetic diagram for the two-state excited-state proton transfer process of pyranine. Excitation at 355 nm leads to the emissions at 440 and 511 nm for the protonated and deprotonated forms, respectively. The pK~’sshown are values in pure water.

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Time resolved fluorescence and anisotropy of PBA and pyranine

state excited-state reaction. In pure water, the protonated and deprotonated ground state forms of pyranine exist at equilibrium with a PKa of 7.5. Upon excitation, the PKa between the two forms decreases to 0.5, causing a nearly complete shift of the excited state pyranine to the deprotonated form [26]. The observed fluorescence lifetime of each form of pyranine is dependent on the intrinsic rate of the excited state decay, k1, the rate of radiationless decay, k~,and the rates of exchange between the protonated and deprotonated forms. The lifetime of the fluorescence emission, excluding the effects of proton exchange, is related to the intrinsic and radiationless decay rates by, =

T

1 1 k~ k~+ kr’ =

‘ ‘

where k~ k~+ kr. A value of 1.6 x iO~5~ for kf corresponds to a lifetime of about 6 ns. This value agrees well with the observed lifetime of the deprotonated form and indicates that the reverse reaction is negligible at bulk water conditions. It has been argued that increases of the steady state fluorescence intensity of the protonated form, due to solvent effects, can be attributed to decreases in k~[26]. Otherwise, the fluorescence decay rate must decrease to account for this effect. It is unlikely that this rate can increase by a factor of six due to an increase in the radiationless rate alone. As a result, a decreased rate of deprotonation causes the ohserved increase in the apparent lifetimes of the protonated form. Similarly, an increase in the rate of protonation leads to the decrease in the apparent lifetimes of the deprotonated form. Together, these rates contribute to the equilibrium observed in the steady state spectra of pyranine and are related to the availability of free water in sucrose solutions. Interpreting the process physically, it can be related to the process of proton transport in solution. It is well known that the extraordinary mobility of a proton in aqueous solution is most likely due to a chain transfer mechanism [34]. The proton does not actually diffuse through the solution, but rather is transferred from a hydronium ion to adjacent water molecules leading to =

593

the net translation of protons. The rate limiting step in this process is the field induced reorientation of the acceptor water molecule by the hydronium ion. Consequently, a mobile water molecule unassociated to sucrose is able to accept protons from donor molecules owing to its ability to reorientate. This effect also explains an observed decrease in the conductivity of solutions of alcohols. When water is involved in the hydration of sucrose, interacting indirectly or directly, its ability to reorient may be hindered by interaction with the sucrose molecule and cause the subsequent decrease in the rate of proton transfer. From the peak intensity ratios (PIRs) of the steady state fluorescence spectra, the ratio of the solvation and bulk water molecules was found to decrease as the sucrose concentration increases and at saturation there is one mole of solvation water for every mole of bulk water. Furthermore, the behavior of the fluorescence lifetimes of pyranine provides dynamic information on the structure of sucrose solutions. In the dilute region, the relatively constant lifetimes of both the protonated and deprotonated species indicate that the environment of pyranine does not change appreciably. As the concentration is increased to the 40 to 66 wt% range, the behavior of the lifetimes suggests that the water associated with sucrose becomes increasingly less mobile. The mobility of the bulk water decreases at 40 wt%, but remains relatively mobile up to the saturation concentration at 66 wt%. At saturation, a further decrease in the mobility of bulk water is apparent. 3.3. Time resolved fluorescence anisotropy The rotational correlation time of the probe in solution is a measure of the time required for reorientation of its excited state dipole moment and is related to its rate of rotational diffusion. Rotational correlation times of PBA in sucrose solutions are shown in fig. 8. Although it is clear that the rotational correlation times below 40 wt% are shorter than 1 ns, the accurate determination of these measurements is beyond the limit of the instrumentation used. A more evident increase, in the moderate concentration region, occurs above 40 wt% up to the saturation concen-

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Time resolved fluorescence and anisoiropy of PBA and pyranine

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Fig. 8. Rotational correlation times of PBA in sucrose solutions. Squares and triangles represent single measurements of the rotational correlation times, observed at 380 nm and excited at 355 nm, for the long and fast lifetime components, respectively,

Fig. 10. Rotational correlation times of pyranine in sucrose solutions. Squares and triangles represent the average of the rotational correlation times observed at 440 and 511 nm, respectively, with excitation at 355 nm. Error bars indicate the standard deviation of the measurements.

tration. In the supersaturated region, above about 66 wt%, two components in the anisotropic decay are present. The faster component increases linearly with concentration from a value near zero to about 10 ns. In contrast, a drastic increase of almost three orders of magnitude is seen in the

long component. Although the fast nucleation of sucrose solutions above 76 wt% may cause an increasing variability in the measurement, the trend in the rotational correlation times is evident. Fig. 9 shows the dependence of the rotational correlation time on viscosity up to 74 wt% sucrose [35]. While the faster rotational correlation time increases only slightly as the viscosity increases, a linear relation between the viscosity and the longer rotational correlation time, with a slope of 6.2 X 10-2 ns/cP and an intercept of 0.94 ns, indicates the correspondence between microscopic interactions and the bulk viscosity. Another view of the concentration dependent behavior of fluorescence probes is seen with the rotational correlation times of pyranine (fig. 10). Here, the rotational correlation times of the protonated and deprotonated forms of pyranine are, within the standard deviations of the measurements, the same in the entire concentration range. the low concentration range, below 40 wt%, the rotational correlation times are nearly zero. Above this concentration, the rotational correlation times, as well as the standard deviations of these measurements, increase as the concentra-

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Viscosity (cP) Fig. 9. The dependence of the rotational correlation time of PBA on the viscosity. Squares and triangles represent single measurements of the rotational correlation times, observed at 380 nm and excited at 355 nm, for the long and fast lifetime components, respectively,

.

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tion tncreases. However, the increases observed in the supersaturated region are less than that

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Time resolved fluorescence and anisotropy of PBA and pyranine

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Viscosity (cP) Fig. 11. The dependence of the rotational correlation time of pyranine on the viscosity. Squares and triangles represent the average of the rotational correlation times at 440 and 511 nm, respectively. Error bars indicate the standard deviation of the measurements,

with PBA. This is seen more clearly in fig. 11, where the rotational correlation times are plotted against viscosity [35].Below a concentration of 66 wt%, the rotational correlation time increases linearly in relation to the viscosity, with a slope of 1.5 x 10~ ns/cP, for both the protonated and the deprotonated forms. The intercepts occur at 1.7 and 0.6 ns, for the protonated form and deprotonated form, respectively. These values clearly plateau and show increasing variability, as the solution becomes supersaturated. By examining the rotational behavior of the fluorescent probes, information on the microscopic solution structure can be deduced. According to hydrodynamic theory, the rotational correlation time, p, of a spherical rotor with stick boundary conditions is related to the rotational diffusion coefficient, D, by the Stokes—Einstein— Debye equation [36], p

=

1/6D

=

r

7V/kT

(7)

where ~ is the viscosity, T is the temperature, V is the hydrodynamic volume of the rotor, and k is Boltzmann’s constant. At constant temperature, the rotational correlation time is expected to increase linearly as the viscosity increases. A linear

595

relationship between the rotational correlation time and viscosity is found for PBA. With pyranine, the relationship is linear below 66 wt% and becomes nonlinear above this concentration. The two probe molecules are complementary in that PBA can form hydrogen bonds only at the carboxyl group and has minimal interactions with the solvent while pyranine has three negatively charged sulfonate groups directly on the pyrene group and may exert a greater effect on its solvation shell. From the slopes of the rotational correlation time versus the viscosity, the diameter of the spherical rotational cavity for PBA is found to o be 8 A. This corresponds well to the actual 9 A value for the largest diameter of a pyrene group and implies that the bulk viscosity is directly related to the interactions at the molecular scales. At concentrations below 60 wt%, the diameter for the spherical rotation of pyranine is found from the slope of the rotational correlation times to be slightly larger at 11 A and agrees well with the molecular dimension of this molecule. This may reflect the slightly larger size and greater electrostatic interactions due to the sulfonate groups of the pyranine molecule. Hydrodynamic theory may account for the anomalous behavior of pyranine in the supersaturated region. The assumption of stick boundary conditions implies a zero fluid velocity at the interface between the rotor and the solution. For molecules much larger than the solvent molecules, such as proteins and polymers, the stick boundary condition is satisfied. Smaller molecules that do not obey the stick boundary condition have a greater degree of rotational mobility and have been described as experiencing slip boundary conditions. Several theoretical treatments have attempted to describe this phenomenon. Molecules in solution have been described as rotating spheroids that displace the solvent molecules as they rotate [37]. Accordingly, nonspherical particles cause a greater drag force to be exerted on .

.

.

.

the particle so that the stick boundary condition is approached as the particles become increasingly nonspherical. Another view accounts for substick boundary conditions by allowing cavities within the solvent in which the molecule can rotate [38]. Both nonsphericity and solvent inter-

596

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Time resolved fluorescence and anisotropy of PBA and pyranine

actions are likely to contribute to the rotational behavior of the probe A molecule of PBA in aqueous solution can be approximated by an oblate spheroid within its solvation shell. However, above saturation, the measured rotational correlation time for pyranine does not vary linearly with viscosity and appar ently, partial slip boundary conditions are ohserved Because of the additional polar groups the solvent may be ordered in a more spherical cavity around pyranine and thus leading to faster rotatation Since the rotational correlation times of the pyranine are long relative to its fluores cence lifetime the time constant of the difference decay (in eq. (5)) is nearly equal to the fluorescence lifetime. Therefore, such measurements are not conclusive and further studies are needed to eliminate any errors in the measurements Uti lization of a picosecond excitation pulse and a single photon counting apparatus is a promising alternative. Regardless of the specific explanations for the relative rotational mobilities it is clear that the structure of the sucrose solutions becomes increasingly less mobile on the microscopic scale as the concentration is increased. 3.4. Structural characteristics of sucrose solutions By relating the results obtained from these time resolved fluorescence measurements with previous investigations using other methods, a more complete description of the solution structure can be made. Mathlouthi [17] used X-ray diffraction to describe three regions of organization in sucrose solutions where the molecular associations changed from a “free-water” organization, below 22.3 wt%, to an intermediate, short-range ordering of bound water, and then a short-range ordering of solute molecules at concentrations greater than 65.3 wt%. They interpreted their data as evidence of direct associations between sucrose molecules in supersaturated solution. Moreover, Tikhomiroff [39,40] and Pidoux [41] interpreted their data in terms of proto-nuclei or clusters of water exclusive sucrose molecules. Using NMR methods, Richardson et al. [14] described the progressive formation of a hydrogen bonding network as the concentration

(a)

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Fig. 12. Schematic illustration of the hypothetical formation of a hydrogen bonding network in sucrose solutions. (a) In dilute . solutions, the bulk water remains relatively unperturbed. (b) Between 40 and 66 wt%, the network of associated water progressively extends into the solution. (c) Above the saturation concentration, a less mobile network of associated water extends throughout the solution with more mobile bulk water located in the interstices of the network.

of sucrose increased into supersaturation. Refinements to these models, as illustrated in fig. 12, are provided by incorporating results from the time resolved fluorescence measurements. In dilute solutions, the solution structure up to a concentration of about 40 wt% has been described with an isotropic two-state model [42], where the rotational mobility of water is described by a weighted average of the rotational mobilities of solvation water and bulk water. The PIR calculated from the steady state fluorescence spectra of pyranine [18], along with the fluorescence lifetimes of the protonated and deprotonated forms of pyranine and the rotational correlation times of PBA and pyranine, exhibit relatively small changes in this region. Thus, as the solution becomes more concentrated and more water is associated to the sucrose, the structure of

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Time resolved fluorescence and anisotropy of PBA and pyranine

the bulk water does not change appreciably from that of pure water in the dilute concentration range. As the concentration of sucrose increases from 40 wt% to about 66 wt%, the behavior of the fluorescence probes indicates a nonlinear structural transformation where the solvent becomes less fluid. This structural transformation has been characterized by NMR as the formation of a cooperative [14] network structure of hydrogen bonding throughout the solution. As the number of solute molecules increases, the water molecules associated directly with the sucrose interact, through the hydrogen bonding networks, with water molecules not directly participating in the solvation of sucrose. These hydrogen bonds were postulated to be comprised of (1) water—water interactions, (2) hydrogen bond bridging between sucrose molecules and (3) sucrose—sucrose interaction. However, it was not possible to relate these microscopic interactions to specific locations relative to the sucrose molecule. Near the saturation point, another structural transformation is evident from the decrease in the lifetime of the deprotonated pyranine, the rapid increase in the rotational correlation time of PBA and the apparent leveling of the rotational correlation times of pyranine. Also, the steady state fluorescence results indicate that the molar ratio of bulk to solvation water per molecule of sucrose is approximately unity [18]. As the number of molecules of bulk water decreases relative to the number of sucrose molecules with increasing supersaturation, the structure of the remaining bulk water is likely to be considerably different from that of pure water. Thus, the transport properties transform from those governed essentially by the interaction of probe with bulk water in dilute solution to those resulting from the interaction of the probe with increasingly more solute associated water. Considering the similar rotational correlation times of the deprotonated and protonated forms of pyranine, a change in the solvent organization is evident. The development of a less mobile network of hydrogen bonds between the sucrose molecules with relatively more mobile bulk water located in the interstitial spaces provides an ex-

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planation for the observations. The hydrogen bonding network would limit the rotational mobility of the probe, but allow the exchange of protons with the remaining interstitial bulk water molecules. Although the immediate solvation environment of the two forms of the pyranine hydroxyl group differs, the solvation environments of the molecule, as a whole, are similar. Hence, the solution is isotropic at the length and time scale of the probe rotational motion so that any partitioning of the probe into more hindered solvent exclusive sucrose clusters is not directly observed. Although these results do not show any evidence of solute aggregation in supersaturated solutions, they do not preclude the formation of such clusters as crystallization progresses. Formation of a hydrogen bonded network may occur between solvent exclusive clusters of sucrose as well. Indeed, solvent structuring could explain the stability of supersaturated solutions in the absence of nucleating agents. In supersaturated solutions, the mobility of water appears to become sufficiently restricted so that its entropic contribution to the free energy is inadequate to cornpensate for the loss of entropy due to the crystallization of sucrose. It is evident that the dynamic properties of microscopic molecular interactions in the solution are responsible for the thermodynamic and kinetic properties of crystallization.

4. Conclusions Fluorescence techniques provide means by which transport and microenvironmental properties can be measured and consequently, the molecular structure of solutions can be deduced. As a result of the time resolved measurements with PBA and pyranine in sucrose solutions, further evidence is available suggesting that the structure and composition of bulk water changes with an increase in sucrose concentration from a water structure to a highly ordered structure in supersaturated solutions. Analysis of the relation between rotational correlation times and viscosities for the two different probe molecules confirms the significance

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of understanding the short range interactions that govern transport. Information on the localized structure can be extracted from this data and incorporated with information obtained by other means. As the solution becomes more concentrated, the structure of the water transforms from a relatively unperturbed form to one in which a hydrogen bonding network forms between dispersed sucrose molecules with minimal effect on the bulk water. In supersaturated solutions, however, the remaining bulk water also becomes mdirectly associated to the sucrose forming a higher order of solvent interactions. In concentrated sucrose solutions, the solution is described as a homogeneous network of solvated solute molecules connected by more mobile and dynamic bulk solvent molecules. Evidence of both bulk and solvation water is present in solutions of all concentrations examined, but no clear indications of solvent exclusive clusters in solution were ohserved.

Acknowledgments We wish to thank the Michigan State University LASER Laboratory for the use of their facilities. Funding was provided by the National Needs fellowship program and the Cooperative State Research Service of the United States Department of Agriculture. Additional support was provided by the Department of Chemical Engineering and the Crop Bioprocessing Center/ Research Excellence Fund at Michigan State University.

References [1] AS. Myerson and L.S. Sorrel, AIChE J. 28 (1982) 778. [2] AS. Myerson and Y.C. Chang, in: Industrial Crystallizalion ‘84, Eds. Si. Jan~ié and E.J. de Jong (Elsevier, Amsterdam, 1984) p. 27. [3] MA. Larson and J. Garside, Chem. Eng. Sci. 41(1986) 1285. [4] MA. Larson and J. Garside, J. Crystal Growth 76 (1986) 88. [5] AT. Allen M.P. McDonald, W.M. Nicol and R.M. Wood, Nature 235 (1972) 236.

[6] G.A. Hussman, MA. Larson and K.A. Bergiund, Industrial Crystallization ‘84, Eds. S.J. Jañcié and E.J. Jong Amsterdam, 1984) p. [7] P.M. (Elsevier, McMahon, K.A. Berglund and21.MA. Larson, Industrial Crystallization ‘84, Eds. S.J. Janèié and E.J.

in: de in: de

Jong (Elsevier, Amsterdam, 1984) p. 229. [8] H. Narayanan and G.R. Youngquist, AIChE Symp. Ser. 253 (1987) 1. [9] A. Suggett and A.H. Clark, J. Solution Chem. 5 (1976) 1. [10] A. Suggett, S. Ablett and P.J. Lillford, J. Solution Chem. 5 (1976) 17. [11] A. Suggett, J. Solution Chem. 5 (1976) 33. [12] M.J. Tait, A. Suggett, F. Franks, S. Ablett and PA. Quickenden J. Solution Chem. 1 (1972)131.

[131Theory Si. Richardson and M.P. to Steinberg, in: and Applications Food, Eds.

Water Activity: LB. Rockland and L.R. Beauchat (Dekker, New York, 1987) p. 235 [14] Si. Richardson, IC. Baianu and M.P. Steinberg, J. Food Sci 52 (1987) 806. [15] M. Mathlouthi and DV. Luu, Carbohydrate Res. 81 (1980) 203. [161 M. Mathlouthi, C. Luu, M. Meffrov-Biget and DV. Luu, Carbohydrate Res. 81(1980) 213. [17] M. Mathlouthi, Carbohydrate Res. 91(1981)113. [18] R. Chakraborty and K.A. Berglund, AIChE Symposium Series No. 284, 83 (1991) 114; J. Crystal Growth 125 (1992) 81. [19] W.M. Vaughan and G. Weber, Biochemistry 9 (1970) 464. [20] D.J. Arndt-Jovin, S.A. Latt, G. Stringkler and TM. Jovin, J. Histochem. CI/tochem. 27 (1979) 87. [21] S.A. Latt and S. Brodie, in: Excited States of Biological Molecules, Ed. J.B. Burks (Wiley, New York, 1976) p. 178. [22] F.M. Winnik, MA. Winnik and S. Tazuke, Macromolecules 20 (1987) 38. [23] F.M. Winnik, Macromolecules 20 (1987) 2745. [24] F.M. Winnik, Macromolecules 22 (1989) 734. [25] F.M. Winnik, Langmuir 6 (1990) 522. [26] H. Kondo, I. Miwa and J. Sunamoto, J. Phys. Chem. 86 (1982) 4826. [27] J.C. Pouxviel, B. Dunn and J.I. Zink, J. Phys. Chem. 93 (1989) 2134. [28] E. Pines and D. Huppert, J. Phys. Chem. 87 (1984) 4471. [29] JR. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum, New York, 1983). 130] A. Grinvald and I.Z. Steinburg, Anal. Biochem. 59 (1974) 583. [31] A. Grinvald, Anal. Biochem. 75 (1976) 260. [32] W.H. Press, B.P. Flannery, S.A. Teukolsky and W.T. Vetterling, in: Numerical Recipes in C: The Art of Scientific Computing (Cambridge University Press, Cambridge. 1988) p. 540. [33] D.W. Marquardt, J. Soc. Ind. AppI. Math. 11(1963) 431. [34] J. O’M. Bockris and A.K.N. Reddy, Modern Electrochemistry, Vol. 1 (Plenum, New York, 1970) p. 461.

B. Pan eta!.

/

Time resolved fluorescence and anisotropy of PBA and pyranine

[35] R.C. Weast, Ed., CRC Handbook of Chemistry and Physics, 69th ed. (CRC Press, Boca Raton, FL, 1988) p. D-262. 136] P. Debye, Polar Molecules (Leipzig and Rheinhold, New York, 1929). [37] C.M. Hu and R. Zwanzig, J. Chem. Phys. 60 (1974) 4354. [38] iL. Dote, D. Kivelson and RN. Schwartz, J. Phys. Chem. 85 (1981) 2169.

[39] [40] [41] [42]

599

N. Tikhomiroff, Ind. Aliment. Agr. (Paris) 82 (1965) 755. N. Tikhomiroff, Zucker 18 (1965) 226, 257. G. Pidoux, Zucker 16 (1972) 523. JR. Zimmerman and W.E. Brittin, J. Phys. Chem. 61 (1957) 1328.