Excited-State Proton Transfer of Weak Photoacids Adsorbed on

Article pubs.acs.org/JPCB

Excited-State Proton Transfer of Weak Photoacids Adsorbed on Biomaterials: Proton Transfer on Starch Ron Simkovitch and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel S Supporting Information *

ABSTRACT: Steady-state and time-resolved fluorescence techniques were employed to study the excited-state proton transfer (ESPT) from a photoacid adsorbed on starch to a nearby water molecule. Starch is composed of ∼30% amylose and ∼70% amylopectin. We found that the ESPT rate of adsorbed 8hydroxy-1,3,6-pyrenetrisulfonate (HPTS) on starch arises from two time constants of 300 ps and ∼3 ns. We explain these results by assigning the two different ESPT rates to HPTS adsorbed on amylose and on amylopectin. When adsorbed on amylose, the ESPT rate is ∼3 × 109 s−1, whereas on amylopectin, it is only ∼3 × 108 s−1.

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INTRODUCTION Photoacids are aromatic organic molecules that have a higher acidity in their first excited electronic state than in their ground electronic state. Thus, short UV−vis laser pulses that cause photoexcitation to the excited state enable one to follow the photoprotolytic processes. Intermolecular excited-state proton transfer (ESPT) from the acidic group of the excited photoacid to a nearby solvent molecule has been researched extensively.1−15 Biopolymers surround us in all living things, from plant life to the human body, and they exist in an extremely large variety. Proton mobility is one of the key processes that support life. In the current study, the proton mobility of water next to biopolymers through excited-state proton transfer of adsorbed photoacids on the biopolymers was studied. We have studied proton transfer adjacent to cellulose,16,17 chitin,18 and chitosan,19 which are polysaccharides composed of glucose molecules with β (1−4) glycoside bonds. Cellulose can be found in the walls of green plants,20,21 and chitin exists in the exoskeleton of arthropods such as crabs and shrimps and of many insects.22 In this article we report on the excited-state proton transfer (ESPT) from 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) to water next to starch. Starch comprises two biopolymers: branched amylopectin (70−80%) and linear and helical amylose (20−30%). These two polysaccharides are composed of glucose units connected through α (1−4) glycoside bonds (see Scheme 1). Starch can be found in most green plants and is used for energy storage. It is a main ingredient in the human diet and is abundant in potatoes, wheat, maize, rice, and cassava. In amylopectin, glucose units are linearly linked with α(1 → 4) glycosidic bonds. However, the presence of α(1 → 6) bonds © 2015 American Chemical Society

causes branching every 24−30 glucose units. Amylopectin is formed of 2000−200 000 glucose units. Its inner chains are formed of 20−24 glucose subunits only. This branching causes an open structure, and amylopectin is soluble in water and allows enzymes to attach to its many end points. By contrast, amylose contains very few α(1 → 6) bonds or none at all. The number of glucose subunits in amylose is much less than in amylopectin and is usually in the range of 300−600 but can be also longer. There are three main forms of amylose chains: a disordered amorphous conformation or two different helical forms. Thus, amylose has a higher density than amylopectin and is insoluble in water. The self-diffusion of water in porous polysaccharides was studied by Topgaard and Söderman using NMR diffusometry.23 Besides bulk water, two other water fractions with different freezing behavior were found: (a) water with a freezing temperature between approximately −5 and 0 °C and (b) water that is liquid even at −24 °C. The latter nonfreezing water fraction is described by them as a water layer with approximate thickness 1 nm. According to their findings in starch, the low-temperature nonfreezing water layer penetrates into the polysaccharide substructure. They found that in starch the diffusion of water is much slower than in cellulose fibers, and it was attributed to both the smaller amount of freezable water and to the pore geometry. We found that the ESPT rate of HPTS on starch arises from two different ESPT rates. We assign the slow ESPT rate to HPTS molecules adsorbed on amylopectin, while the fast ESPT Received: May 11, 2015 Revised: June 21, 2015 Published: June 30, 2015 9795

DOI: 10.1021/acs.jpcb.5b04510 J. Phys. Chem. B 2015, 119, 9795−9804

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The Journal of Physical Chemistry B Scheme 1. Molecular Structures of the Various Compounds Used in This Study

(usually water) with a rate constant kPT to form an RO−*···H+ ion pair. The ion-pair intermediate undergoes a second proton transfer to form the free conjugated base RO− and a diffusing proton. The ion pair may recombine with the RO−* to re-form ROH* with a rate constant ka. The diffusive proton in the bulk recombines geminately with RO−. This process is described by the Debye−Smoluchowski equation (DSE).7,26 The fluorescence bands of both the ROH* and the RO−* forms differ in their spectral position, and each form is easily observed either in a steady-state spectrum or by time-resolved fluorescence in two selective spectral regions. The proton-geminate recombination process increases the population the ROH* form. The ROH time-resolved fluorescence at long times is nonexponential because the geminate recombination process is diffusioncontrolled. We used the spherical-symmetric diffusion program (SSDP) of Krissinel’ and Agmon27 to fit the time-resolved fluorescence of HPTS in bulk water and when adsorbed on starch. The SSDP program provides a numerical solution to the Debye− Smoluchowski equation. There are several adjustable parameters; most are known fixed parameters in bulk water.7,25 Figure S2 shows the ROH time-resolved emission signal of HPTS in bulk H2O and D2O as well as the signal computed with the use of the SSDP program. As can be seen, the fit is excellent at short and long times. The fitting parameters are given in Table S1.

process occurs when HPTS molecules are adsorbed on amylose. In commercial flour for home use, the ESPT rate of adsorbed HPTS is slow, similar to that of HPTS on starch.

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MATERIALS AND METHODS Amylose, amylopectin, and cellulose (20 μm powder) were obtained from Sigma-Aldrich, and starch was obtained from Merck (product number 101252). Commercial home-use flour was store-bought. All solvents used in this study were of HPLC grade. The time-correlated single-photon-counting (TCSPC) technique was used in this study. The samples were excited by a cavity-dumped titanium:sapphire femtosecond laser (Mira, Coherent). The second harmonic of the 150 fs laser pulses in the spectral range of 380−440 nm were used to excite the samples. The cavity dumper operated at a rate of 800 kHz. The TCSPC instrument was based on a Hamamatsu 3809U multichannel plate photomultiplier. The TCSPC signals were provided by an Edinburgh Instruments TCC 900 integrated TCSPC system. The full width at half-maximum was approximately 40 ps. Neutral-density filters were used to reduce the excitation pulse energy to about 10 pJ. The steady-state fluorescence spectrum was measured by a Horiba Jobin Yvon FluoroMax-3 fluorescence spectrofluorometer.

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ESPT GEMINATE-RECOMBINATION MODEL The time-resolved fluorescence signal of the HPTS ROH form in H2O and D2O is a textbook example of the diffusioninfluenced reversible geminate-recombination model.24,25 It quantitatively describes the photoprotolytic process that occurs in the excited state of a photoacid. Below, we briefly describe the model: Scheme 2 shows a chemical scheme of a photoprotolytic cycle of photoacids.24,25 In the excited state, a photoacid (ROH*) transfers a proton to a nearby solvent molecule

RESULTS Starch. Figure 1 shows the steady-state excitation and emission spectra of HPTS adsorbed on starch. The starch sample of about 30 mg of powder is glued on a 40 mm diameter quartz plate by first smearing the plate with a thin layer of glycerol and then adding the starch powder. A methanol solution of HPTS is sprayed on the starch. After about half an hour, the methanol evaporates and water is sprayed on the starch sample. The excitation spectrum consists of the strong absorption band of the protonated ROH form at ∼400 nm and a much weaker band of the RO− form at ∼455 nm. The existence of the RO− band in the excitation spectrum indicates that wet starch samples with low water content exhibit proton activity in the ground state, and the effective pH of the wet samples is slightly acidic since the pKa of HPTS in water is ∼7.4. The emission spectrum consists of two emission bands: that of the protonated ROH form at ∼440 nm and of the deprotonated RO− form at ∼512 nm. The band-intensity ratio IFRO−/IFROH depends on the water/starch weight ratio. The semidry sample is a starch sample that had been sprayed by a methanolic solution of HPTS, and the spectra of which were measured after 30 min or longer after the methanol had almost

Scheme 2. Photoprotolytic Cycle of Photoacidsa

a

DSE stands for Debye−Smoluchowski equation (see text). 9796


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molecules are adsorbed on the surface of large objects. In a nonviscous solution of symmetric molecules like HPTS, the fluorescence intensity at t = 0 after pulse excitation is polarized. The transition dipole moment is along one of the major axes of the molecular structure. When excited by a polarized laser pulse, the molecules are preferentially aligned along a certain laboratory axis. In solution, molecular orientational relaxation occurs over a time scale of tens to several hundred picoseconds, depending on the size of the molecule and the viscosity of the solvent. The orientational relaxation can be measured by monitoring the time-resolved fluorescence at two polarizations: parallel and perpendicular to the laser excitation polarization. When a molecule is adsorbed on a large inflexible surface, the orientational relaxation is prevented and the decay of the timeresolved fluorescence signal is independent of the state of polarization. Figure 2a shows the time-resolved fluorescence of 8methoxy-1,3,6-pyrenetrisulfonate (MPTS) in ethanol, measured at two orthogonal polarizations (parallel and perpendicular to the laser polarization). In MPTS, shown in Scheme 1, the hydroxyl group of HPTS is replaced by a methoxy group. MPTS is not a photoacid, and therefore the ESPT process does not take place. In water, the orientational relaxation and the ESPT process in HPTS occur with about the same time constant, and therefore it is difficult to measure the orientational relaxation. We used MPTS instead of HPTS to determine if HPTS is adsorbed on starch. As seen in Figure 2a, the MPTS fluorescence signals in ethanol measured at parallel and perpendicular polarizations show that orientational relaxation occurs in ethanol with a time constant of ∼180 ps. Figure 2b shows the time-resolved fluorescence of MPTS adsorbed on starch, measured at two orthogonal polarizations: parallel and perpendicular to the laser polarization. The two time-resolved signals are identical in their shape and do not show a fast decay or rise component as expected when orientational relaxation occurs within the time window of the fluorescence decay. We therefore conclude that the MPTS molecules are strongly adsorbed on the starch polysaccharide backbone and that orientational motion does not take place within a time window of about 20 ns. We assume that this is also true for HPTS which has almost the same chemical structure. Figures 3a and 3b show, on a linear and semilogarithmic scale, the time-resolved emission at 440 nm of the ROH form of HPTS adsorbed on starch.

Figure 1. Steady-state emission and excitation spectra of HPTS adsorbed on starch. HPTS was excited at 390 nm for the emission spectra, in the excitation spectrum fluorescence was measured at the RO− form peak at 520 nm.

completely evaporated. In the other samples, water is added in ratios of 0.5, 1, 1.5, and 2 times that of the weight of starch. As seen in Figure 1, the emission-band ratio depends on the water content up to water saturation which occurs at about the 2:1 H2O/starch weight ratio. The greater the water content of the sample, the higher the IFRO−/IFROH ratio. The excited-state proton transfer (ESPT) rate of a photoacid in solution can be deduced from the fluorescence band-intensity ratio. The following equation provides an approximation to kPT, the ESPT rate constant: F F −1 −/ I kPT = (IRO ROH)τF

(1) −

where τF is the fluorescence lifetime of the RO form of the photoacid. For HPTS in water, τF = 5.4 ns. The fluorescence lifetime of the ROH form in methanol or ethanol in which ESPT does not take place is about the same as that of the RO− form, τ = 5.2 ns. The similar radiative lifetimes justify the use of this simple equation.1,28 In water, kPT ≈ 1010 s−1 and the fluorescence intensity ratio is about 30. In saturated water/starch samples, the ratio is close to 2.5. This indicates that the effective ESPT rate of HPTS adsorbed on starch is much smaller than in water. Timeresolved fluorescence measurements provide much more accurate information on the photoprotolytic process of HPTS adsorbed on starch. Time-Resolved Measurements. Time-resolved polarization fluorescence measurements can be used to determine if

Figure 2. Time-resolved fluorescence of MPTS, measured at parallel and perpendicular polarizations to the laser polarization: (a) in ethanol; (b) adsorbed on starch. 9797


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Figure 3. Time-resolved fluorescence of the ROH form of HPTS adsorbed on starch, measured at 440 nm.

Figure 4. Time-resolved fluorescence of HPTS RO− form adsorbed on starch, measured at 525 nm.

Figure 5. Time-resolved fluorescence emission of HPTS ROH, measured at 435 nm, and RO−, measured at 525 nm in H2O and D2O shown on (a) linear scale 0−3 ns and (b) semilogarithmic scale 0−20 ns.

There are several signals in the figures that differ in the amount of water/starch weight ratio. The signals are bimodal with a short-time component with a decay time of about ∼200 ps, followed by a longer nonexponential decay with an average time constant of about 3 ns. The greater the water content, the higher the amplitude of the rapid component. The decay times of both components are only slightly dependent on the water content. For comparison, the figures also contain the fluorescence signals of the ROH form of HPTS in H2O solution. As seen in the figure, the decay of HPTS in water is also bimodal, but the major time component is the rapid-decay component with an amplitude of about 0.97, whereas the amplitude of the long-time fluorescence tail is rather small. The rapid component in bulk water is assigned to the ESPT rate with a time constant in water of ∼100 ps, and the long tail is a consequence of an efficient geminate recombination of the

proton from the solvent to re-form the excited-state ROH* form of HPTS.24,25 Figures 4a and 4b show, on linear and semilogarithmic time scales, the time-resolved fluorescence signals of the RO− form of HPTS in water/starch samples, measured at 525 nm, a position slightly to the red of the band peak at 512 nm, in order to reduce the overlap between the ROH and RO− emissions. The fluorescence signals of the RO− form show a rise component that has a time constant of about that of the fast decay component of the ROH form. The long-time decay of the RO− form is exponential, and the decay time is about 5.4 ns, equal to the fluorescence decay time of the RO− form of HPTS in aqueous solution. The signal-to-noise ratio of the risetime component of the RO− signals of the various water−starch samples is not as good as that of the decay of the ROH form, and therefore its analysis is limited in obtaining fine-detail 9798


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The Journal of Physical Chemistry B information. The long-time rise component of the RO− form fits the rapid decay of the ROH fluorescence and thus enables confirmation that an ESPT process did indeed occur in these samples. Kinetic Isotope Effect. The kinetic isotope effect of the ESPT process of HPTS in H2O and D2O solutions is about 3.15 ± 0.15. Figure 5a shows the time-resolved emission of HPTS ROH and RO− forms in H2O and D2O bulk solutions. The initial decay time of the ROH signals shown in the figure provides the ESPT time constants of 100 and 310 ps for H2O and D2O, respectively. Figure 5b shows the time-resolved fluorescence of both the ROH and RO− forms of HPTS adsorbed on starch in water−starch samples of 2:1 weight ratio. The short-time decay of ROH and the RO− rise time differ in H2O and D2O starch samples, as expected for an ESPT process. In complex systems such as HPTS adsorbed on starch, it may occur that the decay of the fluorescence signals may not be simple single-exponent because of nonradiative processes. The kinetic isotope effect seen in Figure 5b clearly shows that the rapid component of the signals of the ROH form of HPTS adsorbed on the starch sample is due to the ESPT process. Figures S1a and S1b in the Supporting Information show the signals shown in Figures 5a and 5b on a semilogarithmic scale up to 20 ns, while in Figures 5a and 5b we show only the first 3 ns of the fluorescence signals. Starch consists of two types of polysaccharides: linear and helical amylose and branched amylopectin. It generally contains about 25% amylose and 75% amylopectin. The ROH timeresolved fluorescence signal of HPTS adsorbed on the starch used in this study consists of two time components with short and long decay times. Their relative amplitude, ai, is about the same (ai ≈ 0.5) in saturated water samples. In a sample of water/starch of weight ratio less than 1, the amplitude of the short-time component is smaller than that of the long-time decay component. In commercial home-use flour we found that the ROH signal consists of only the long-time decay component. To further explore the ESPT process of photoacids adsorbed on starch, we measured the ESPT process of HPTS adsorbed on amylose from potatoes and adsorbed on amylopectin from maize. ESPT in Amylose. Amylose is the nonbranched polysaccharide of starch. It is in amorphous or helical forms and is made of α-D-glucose units bound through α(1−4) glycoside bonds. Its content in starch is 20−30% of the structure. The number of monomers usually ranges between 300 and 3000 but can be much larger. As described above, the time-resolved fluorescence of the ROH form of HPTS adsorbed on starch in a 1:1 water−starch sample is bimodal with short and long decay-time components. We analyze the complex ROH signal by suggesting two contributions to it. Figure 6 shows the excitation and emission spectra of HPTS adsorbed on amylose. The sample contained water at weight ratios of 1:1 and 2:1 water-to-starch. As seen in the figure, the excitation spectrum shows that HPTS was in the ROH form in the ground state. The emission spectrum consists of two emission bands: that of the ROH form with a band peak at 440 nm and a stronger band of the RO− form with a band peak at ∼512 nm. We therefore conclude that efficient ESPT process occurs when HPTS is adsorbed on amylose since the band intensity ratio IFRO−/IFROH is much greater than that in starch, as seen in Figure 1. This indicates that the effective ESPT rate is larger in amylose than in starch as given in eq 1.

Figure 6. Steady-state emission spectrum (excitation at 390 nm) and excitation spectrum (fluorescence measured at the RO− peak at 530 nm) of HPTS adsorbed on amylose.

Figures 7a and 7b show, on linear and semilogarithmic scales, the time-resolved emission signals of HPTS adsorbed on amylose in a 2:1 weight-ratio water−amylose sample, measured at 440 nm (the ROH band maximum) and at 525 nm, slightly red-shifted to the RO− band maximum (512 nm). As seen in the figures, the decay of the ROH signal is bimodal, with fast- and slow-decaying components. On a semilogarithmic scale, the long-time tail of the ROH fluorescence is nonexponential and qualitatively fits the concept of reversible proton-geminate recombination as described in the model section above and in more detail, also in refs 24 and25. For comparison, we added to Figure 7b the ROH and RO− HPTS signals in bulk solution H2O. The amplitude of the long-time fluorescence tail in H2O is much smaller than that of HPTS adsorbed on amylose. The initial decay time of ROH and the signal rise time of RO− in amylose are longer than in water. The slower decay time of the rapid component of the ROH signal is a consequence of a slower ESPT rate in the amylose sample. We used the SSDP program to analyze the ROH signal of HPTS adsorbed on amylose. We were able to obtain an excellent fit to the experimental signal as shown in Figure S3. The fitting parameters are given in Table 1. The ESPT rate is about one-third that of HPTS in bulk water. The intrinsic rate constant, ka, of the geminate recombination is about that of water.24 Proton diffusion in the water adjacent to the amylose is about one-fourth that in bulk water. We used the dielectric constant of water (ε = 78) to determine the Coulomb potential between the proton and the RO− form of HPTS. Equation 2 provides the Debye radius, RD, which is the gauge for the Coulomb potential of the proton−RO− ion pair. At a distance RD between the ions, the thermal energy is equal to the Coulomb potential

RD =

ze 2 4πεε0kBT

(2) −

where z is the ionic charge of RO in electron-charge units, e is the electron charge, kB is the Boltzmann constant, and 4πε0 is the permittivity. RD = 28 Å for the RO− form of HPTS. The long-time nonexponential fluorescence tail is indicative of proton diffusion adjacent to the amylose polysaccharide structure. We obtain a value of D = 2.5 × 10−5 cm2/s from the 9799


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Figure 7. Time-resolved fluorescence emission of HPTS adsorbed on amylose ROH and RO− signals: (a) shown on a linear scale; (b) shown on a semilog scale.

Table 1. SSDP Fitting Parameters of HPTS on Starch kPT [ns−1] Merck starch H2O amylose amylopectin commercial home-use flour

4 10 3 0.4 0.4

ka [Å ps−1] 13 8 7.35 0.7 0.7

D [cm2 s−1] 1.4 9.3 2.5 1.7 1.7

data fit. This value is 3.5 times smaller than that in water; however, it is high when proton diffusion is considered along a thin layer of water next to biomaterials. The diffusion coefficient of sodium ion in bulk water is about 7 times smaller than that of the proton.29 We emphasize that proton conductivity next to amylose is not negligible. Most biomaterials are considered electrical insulators when compared to semiconductors. Proton conductance is much lower than that of electronic materials. However, we claim that proton conductance is fairly high in wetted biomaterials, as we have found in the current study. ESPT in Amylopectin. Figure 8 shows the steady-state excitation and emission spectra of HPTS adsorbed on amylopectin from maize. In the presence of methanol, the fluorescence spectrum consists mainly of the ROH band and only about 2% of the RO− band. The weak RO− band may be due to the low water content of the amylopectin powder. In the presence of a 1:1 weight ratio of water and amylopectin, the emission spectrum consists of both the ROH and the RO− bands, with a band ratio, IFRO−/IFROH of ∼1/2. The excitation spectrum of both methanol and water samples measured at 520 nm is similar to the absorption spectrum of the ROH form. The excitation and fluorescence spectra of HPTS adsorbed on amylopectin indicate that the ground state HPTS is in the protonated form, as found in a solution of pH ∼ 6 and that the ESPT rate in the water sample is rather low compared to HPTS in bulk water or amylose samples. This statement is based on eq 1 in which the IFRO−/IFROH ratio is used to estimate the ESPT rate constant, kPT. Figure 9 shows the time-resolved emission of HPTS ROH measured at 440 nm and the RO− form measured at 520 nm, when adsorbed on amylopectin. The TCSPC ROH and RO− signals in the presence of 0.5:1 water:amylopectin weight ratio show that the ESPT occurs at

× × × × ×

−5

10 10−5 10−5 10−5 10−5

RD [Å]

a0 [Å]

dim

τf−1 [109 s−1]

25 28 28 25 25

6 6 6 6 6

3 3 2.9 3 3

0.22 0.18 0.14 0.20 0.20

Figure 8. Steady-state emission and excitation spectra of HPTS adsorbed on amylopectin. HPTS was excited at 400 nm for the emission spectrum, in the excitation spectrum fluorescence was measured at the RO− peak at 520 nm

the rather slow rate of about 3 × 108 s−1. This rate is about 30 times slower than the ESPT rate in water of 9 × 109 s−1 or 10 times slower than that for HPTS on amylose. In methanol amylopectin, the decay rate of the ROH form measured at 440 nm is slightly slower than that of the water samples. However, the signal intensity (counts/second) measured at 520 nm is about 5 times smaller and is that of the fluorescence-band tail of the ROH form. The rise of the signal at 520 nm in methanol solution does not match the ROH decay rate. In the water sample, the rise of the RO− signal consists of 50% slow-rise component with a rise time that fits the ROH decay time. The slow-rise component measured at 520 nm that matches the decay rate of the ROH form is the signature of the ESPT process that HPTS undergoes. 9800


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Figure 9. Time-resolved fluorescence emission of HPTS adsorbed on amylopectin. ROH and RO− signals, shown on (a) linear scale and (b) semilog scale.

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by the SSDP program. The fit of the fast decay component shown in red in Figure 10 is satisfactory. The SSDP programfitting parameters are given in Table 1. Commercial Home-Use Flour. We found that when we used commercial home-use flour, the time-resolved fluorescence of the ROH form of HPTS showed only the slow-decay time component. This decay is in excellent agreement with the fluorescence decay of the ROH signal of amylopectin and fits the signal at long times (t > 5 ns) of the HPTS adsorbed on the Merck starch. Figure S4 shows the time-resolved fluorescence of the ROH form of HPTS adsorbed on commercial home-use flour and on amylopectin. As seen in the figure, the fluorescence rise and decay of both samples are identical within the signal-to-noise ratio of the measurements.

DISCUSSION Data Analysis. We were unable to obtain a reasonable fit with the use of the SSDP program for the ROH time-resolved emission signal of HPTS adsorbed on starch. We therefore suggest that the ROH signal is composed of two contributions, since HPTS is adsorbed on two types of sites. On site 1 the adsorbed HPTS is capable of transferring a proton to a nearby water molecule at a rate that is approximately that of HPTS in bulk water. The probability of geminate recombination is higher than in bulk water. For the second site the ESPT rate is rather slow, about 20 times slower. The ESPT time constant is about 2.2 ns rather than about 100 ps as in bulk water. We used the following procedure to fit the ROH timeresolved signal of HPTS adsorbed on starch purchased from Merck. In step 1, we fit the HPTS ROH signal of the amylopectin by the SSDP program. In step 2, we then subtract from the ROH signal of the Merck starch the curve fit of the slow time component obtained in step 1. The amplitude of the slow-component curve was determined by matching the computed signal with the starch signal at the long time of 15 ns. Figure 10 shows the ROH experimental signals of HPTS adsorbed on Merck starch and on amylopectin. In step 3, we subtracted the fit of the amylopectin signal from the bimodal experimental ROH signal of HPTS adsorbed on Merck starch. The residual curve, marked in red, was then fitted

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COMPARISON OF THE PHOTOPROTOLYTIC PROCESS IN STARCH AND IN CELLULOSE The main difference in the polymeric structure between cellulose and starch is that the glucose monomers are covalently linked to each other in starch by an α(1−4) glycoside bond and in cellulose by a β(1−4) glycoside bond. Both biopolymers have a rather complex spatial structure that includes branching, helical structure, and also amorphous and crystalline regions. Figure 11 shows the time-resolved fluorescence of the ROH form of HPTS adsorbed on cellulose and on starch at a 1:1 weight ratio of water. The signal of cellulose, like that of starch, is bimodal with a high-amplitude fast-decaying component followed by a lowamplitude long-time decaying component. The main difference between the ROH HPTS signal of cellulose and that of the starch sample is the relative amplitude of the rapid-decay component versus that of the slow-decay time component. As seen in the figure, the amplitude of the rapid component in cellulose is higher than in starch. The lower amplitude of about one-tenth of the long-time component in cellulose enables the use of the SSDP program to fit the whole signal. This is in contrast with our analysis of the signals of HPTS adsorbed on starch in which we used a rather complex procedure. As described above, the rapid decay-rate component of ROH HPTS adsorbed on starch is about one-third of that of bulk water. This component we assign to the ESPT rate. The water in both starch and cellulose is the proton acceptor and the proton-diffusing space. In water:amylopectin samples of 1:1 weight ratio, the configuration of water next to the polysaccharide is different

Figure 10. Time-resolved fluorescence emission of HPTS adsorbed on Merck starch and on amylopectin as well as the fit of the ROH signal of HPTS adsorbed on amylopectin. 9801


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Figure 11. Time-resolved fluorescence emission of HPTS adsorbed on starch/cellulose at a 1:1 weight ratio of water, shown on (a) a normalized linear scale and (b) a normalized semilog scale.

skeleton, the ESPT rate is slow and the ESPT rate from HPTS to this type of water is 10 times slower or more than in bulk water.

from that next to cellulose. It is known that in cellulose water pools are formed.30 When adsorbed HPTS is next to a water pool, the ESPT and the geminate-recombination processes are similar to those of bulk water. The long-time component of the ROH signal for starch appears to be the same as that found when HPTS is adsorbed on commercial home-use flour and on amylopectin. These facts hint that the long tail of the ROH fluorescence in starch arises from a polymeric structure in which the water molecules are incapable of accepting the proton at the same rate as in bulk water. HPTS studies of concentrated aqueous solutions of MgCl2 and LiCl reveal that the ESPT rate strongly depends on the salt concentration.31,32 It decreases exponentially as the concentration of salt is increased. In 1 M MgCl2, about ∼0.38 of the water molecules are bound to a solvated ion. The dielectric constant of these water molecules is small, ε ∼ 4. Only ∼0.62 of the water molecules are free and therefore have a regular large dielectric constant of 78. The minimum Mg2+ ion hydration number of H+ obtained from the measurements of Hasted and co-workers33 is 10. This solvation number can also be deduced from the enthalpy of formation of H+(H2O)n determined by Kebarle for gas-phase H+/water clusters.31 The proton-transfer rate will be strongly dependent on the average proton-hydrate size. The smaller the proton hydrate, the slower the ESPT rate. The ESPT rate of adsorbed HPTS on amylose is large 3 × 109 s−1, whereas ESPT of HPTS adsorbed by amylopectin is smaller by a factor of about 9:4 × 108 s−1. The water solubility in cold water of amylopectin is large, whereas that of amylose is rather small. These are two water related properties that seem to behave in opposite directions. Cellulose, like amylose, is insoluble in water; in cellulose water pools are formed when water is added to cellulose.34,35,23 In our previous study, we used this property to explain the large ESPT rate in the cellulose−water samples kPT ∼ 7 × 109 s−1. If water pools are also formed in amylose−water samples as in cellulose, it can of course explain the large ESPT rate we found in amylose. Unfortunately, we were unable to find literature results that show that water pools are indeed formed in amylose water samples. The results of the NMR water diffusivity study23 show that a large fraction of nonfreezable water in water−starch samples has a slow diffusion rate when compared to cellulose. We propose that slow diffusion water also have slow rotation relaxation, where fast relaxing water is unique in ESPT processes. In the slow diffusing water next to the starch

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SUMMARY AND CONCLUSIONS In the current work, we studied the excited-state proton transfer (ESPT) from a common photoacid, 8-hydroxy-1,3,6pyrenetrisulfonate (HPTS), adsorbed on starch to water situated next to the scaffold of the starch. Starch is a polysaccharide composed of glucose units that are linked through an α(1−4) glycoside bond (see Scheme 1). Starch is composed of two similar polysaccharides: about 25−30% linear amylose and mainly branched amylopectin that also forms α(1−6) bonds which cause branching every 24−30 glucose units. We found that an ESPT process indeed occurs from adsorbed HPTS on the glucose monomers to adjacent water molecules. The decay of the time-resolved fluorescence signal of the ROH form of protonated HPTS is bimodal with shortand long-time components with similar amplitudes. The time constant of the short decay-time component is about 300 ps and is assigned to proton transfer to water. The long decay time is about 2.5 ns and is also assigned to a proton-transfer process that is about 10 times slower than the previously mentioned ESPT. To resolve the two complex time components of the fluorescence decay of the ROH form of HPTS adsorbed on starch, we also measured the ESPT process of HPTS adsorbed on pure amylose and pure amylopectin. We found that the ESPT process of the amylose sample is fast and fits the fastdecaying component of the ROH form of HPTS on starch. The ESPT process of HPTS on amylopectin is rather slow and qualitatively fits the slow-decaying component of the ROH fluorescence of HPTS adsorbed on starch. On comparing the ESPT rates of HPTS adsorbed on amylose, amylopectin and starch we conclude that the ESPT rate of HPTS on starch occurs on two time scales. The fast ESPT rate occurs at HPTS adsorbed on the amylose scaffold. This ESPT rate constant, kPT, is about 3 times slower than that found for HPTS in water. The slow ESPT process takes place from HPTS adsorbed on the amylopectin of starch. The ESPT rate of HPTS on amylopectin is 10 times slower than on the amylose. In previous studies of the ESPT process in concentrated aqueous ionic solutions of LiClO4 and MgCl2, we found that the ESPT rate decreases exponentially with ionic concentration. 9802


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We explain this phenomenon by the high sensitivity of the ESPT process to the amount of free water in solution. The ESPT rate is rather slow when the proton acceptor is a small cluster of water molecules. When the average water cluster falls to less than five water molecules, the rate decreases by a factor of about 10. In amylopectin the available free water cluster is rather small, and the ESPT rate is 30 times smaller than in water. However, in amylose the availability of large water clusters is high and the ESPT rate is high as well.

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ASSOCIATED CONTENT

S Supporting Information *

Time-resolved fluorescence emission of HPTS; time-resolved fluorescence comparison of commercial home-use flour and amylopectin. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jpcb.5b04510.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph 972-3-6407012; Fax 9723-6407491 (D.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Raphael Lamed for his valuable contribution to this study. This work was supported by the Israel Science Foundation.

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REFERENCES

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The Journal of Physical Chemistry B (35) Watanabe, A.; Morita, S.; Kokot, S.; Matsubara, M.; Fukai, K.; Ozaki, Y. Drying Process of Microcrystalline Cellulose Studied by Attenuated Total Reflection IR Spectroscopy with Two-Dimensional Correlation Spectroscopy and Principal Component Analysis. J. Mol. Struct. 2006, 799, 102−110.

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Excited-State Proton Transfer of Weak Photoacids Adsorbed on

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