Molecular mobility in water and glycerol plasticized cold- and hot-cast gelatin films

Food Hydrocolloids 20 (2006) 96–105 www.elsevier.com/locate/foodhyd

Molecular mobility in water and glycerol plasticized cold- and hot-cast gelatin films Kristine V. Lukasik, Richard D. Ludescher* Department of Food Science, Cook College, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901-8520, USA Received 3 January 2005; accepted 15 March 2005

Abstract The molecular mechanisms that control functionality in gelatin films are poorly understood. We have used phosphorescence emission from erythrosin covalently attached to lysine residues to investigate the effect of plasticizer and physical cross-links on molecular mobility and oxygen diffusion in amorphous gelatin thin films. Phosphorescence emission energy and red-edge effects, which monitor the extent of matrix relaxation during the w0.5 ms probe lifetime, were modulated only slightly by either water or glycerol; the extent of matrix relaxation, however, was significantly greater in cold-cast films with, than in hot-cast films without physical cross-links. The phosphorescence intensity was well described using a stretched exponential in which the lifetime and the stretching factor, a measure of the lifetime heterogeneity, were the fit parameters. Lifetimes, which provide a measure of the rate of matrix collisional quenching, were essentially unaffected by hydration from 0 to 50% RH, slightly decreased from 50 to 84% RH, and greatly decreased at higher RH; rates of matrix collisional quenching were higher in cold-cast than in hot-cast films at all RH values. Glycerol, varied up to 50% by weight of gelatin, only slightly increased the rate of matrix collisional quenching. The rates of oxygen diffusion were higher in cold-cast than hot-cast films at 75 and 84% RH; there was no detectable oxygen diffusion at any glycerol content in dry films. Physical cross-links thus actually increased the molecular mobility of gelatin on the millisecond time scale and water and glycerol, despite their similarity as hydrogen-bonding molecules, had quantitatively different effects on the gelatin mobility. q 2005 Elsevier Ltd. All rights reserved. Keywords: Glass; Amorphous solid; Phosphorescence

1. Introduction The practical utility of gelatin, perhaps the most broadly functional and widely used polymer of natural origin, has been recognized for millennia. Gelatin was used as an adhesive and perhaps as a sizing agent to prepare stone and plaster surfaces for painting as early as the First Dynasty (w2900 BC) in Egypt (Singer, Holmyard, & Hall, 1954) and a cast gelatin ‘glue stick’, suggestive of centralized gelatin manufacture, was discovered by Howard Carter in a rock chamber over the 18th Dynasty (w1400 BC) mortuary temple of Hatshepsut at Deir el Bahari (Lucas, 1962). The list of uses for gelatin developed since then is extensive and diverse (Jones, 1977; Wood, 1977). Despite this long * Corresponding author. Tel.: C1 732 932 9611x231; fax: C1 732 932 6776. E-mail address: [email protected] (R.D. Ludescher).

0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2005.03.007

familiarity, our understanding of the molecular mechanisms underlying much of gelatin’s functionality remains inadequate. Gelatin’s diverse functionality reflects the physical properties of a linear, flexible polymer and the chemical interactions possible in a polypeptide containing 20 different amino acid residues (Veis, 1964). Much of gelatin’s versatility reflects the fact that it is the only food protein that undergoes a thermally reversible helix-coil transition (Guenet, 1992; Stainsby, 1977). As gelatin gels and solutions age, aqueous solvent is excluded from the protein network, which collapses into a rubbery film that vitrifies upon drying. Amorphous solid gelatin is an entangled polymer network that, depending upon the specific time and temperature of drying, is interspersed with a variable number of physical cross-links. This so-called ‘fringed micelle’ model for amorphous gelatin (Slade, Levine, & Finley, 1989) originated with X-ray diffraction studies of dry gelatin gels by Herrmann, Gerngross, and Abitz (1930); these early researchers posited

K.V. Lukasik, R.D. Ludescher / Food Hydrocolloids 20 (2006) 96–105

that gelatin ‘micelles’ were regions of extended parallel polypeptide chains, almost crystalline in structure, whose ends were not closely bound together (Abitz, Gerngross, & Herrmann, 1930), thus generating frayed or fringed micelles. Although often described as crystalline (Slade et al., 1989), gelatin cross-links are not crystals, that is not regions of regularly ordered polypeptide chain that can be replicated infinitely in three dimensions. Rather, they are local regions of protein quaternary structure that are self-limiting in size; whether these regions are purely triple-helical, only partially triple-helical (Guenet, 1992; Ross-Murphy, 1997), or also include b-turn and b sheet motifs (Prystupa & Donald, 1996), is debated. The mechanical properties of films made from gelatin gels cured above and below the triple helix melting temperature differ, with hot-cast films typically being more brittle than cold-cast films (Bradbury & Martin, 1952; Finch & Jobling, 1977; Fraga & Williams, 1985; Menegalli, Sobral, Roques, & Laurent 1999). The glass transition temperature of anhydrous gelatin is routinely reported as O100 8C (Fraga & Williams, 1985; Marshall & Petrie, 1980; Slade et al., 1989; Sobral & Habitante, 2001). Gelatin is readily plasticized by water, the Tg of gelatin films being inversely related to water content (Marshall & Petrie, 1980; Ni & LeFaou, 1993; Slade et al., 1989; Sobral & Habitante, 2001) and both the mechanical and the barrier properties of gelatin films are modulated by additives such as polyols and large molecular weight carbohydrates (Arvanitoyannis, Nakayama, & Aiba, 1998a; 1998b; Arvanitoyannis, Psomiadou, Nakayama, Aiba, & Yamamoto, 1997). Since they often contain plasticizer to give them flexibility, the macroscopic functional properties of edible films made from food proteins are usually modulated by mobility in the rubbery state (Anker, Stading, & Hermansson, 2001; Zhang, Mungara, & Jane, 2000). Despite this, protein films generally have appropriate mechanical properties and sufficiently low permeabilities for molecular oxygen to serve as effective barriers (Krochta & De Mulder-Johnston, 1997). Although there is considerable research on the oxygen permeability and mechanical properties of protein films and the effect of water activity on these properties (Folegatti, Antunes, & Marcondes, 1998; Krochta, 1998; Lim, Mine, & Tung, 1999; Perez-Gago, & Krochta, 2001; Sothornvit & Krochta, 2000; Were, Hettiarachchy, & Coleman, 1999), there is little research correlating macroscopic properties with molecular mobility other than through measurements of Tg (Grevellec, Marquie, Ferry, Crespy, & Vialettes, 2001; Micard & Gilbert, 2000; Mo & Sun, 2001; Ogale, Cunningham, Dawson, & Acton, 2000). In a previous study (Simon-Lukasik & Ludescher, 2004), we reported how phosphorescence emission from the triplet probe erythrosin covalently attached to lysine residues in gelatin can be used to monitor molecular mobility and oxygen diffusion rates in amorphous waterplasticized cold-cast gelatin films. We report here how

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phosphorescence data provide insight into the effects of glycerol and water on molecular mobility in cold and in hotcast gelatin films that differ in content of physical crosslinks. These results provide insight into the molecular mechanisms that underlie the macroscopic effect of these modifications of amorphous solid gelatin.

2. Materials and methods 2.1. Preparation of gelatin VEE GEE Superclear Type A 300 bloom pigskin gelatin obtained from Vyse Gelatin Co. (Schiller Park, IL) was dissolved in distilled deionized water (w100 mg/mL), extensively dialyzed against 0.1 M KCl to replace all salts with KCl, then extensively dialyzed against distilled deionized water to remove excess salt. During dialysis, the temperature was kept above 65 8C whenever possible to ensure the effectiveness of dialysis against ungelled solution. The protein solution was freeze-dried (Labconco Model 4.5, Kansas City, MO) and stored sealed prior to use. Protein was covalently labeled at lysine residues with erythrosin isothiocyanate (ErITC; Molecular Probes, Eugene, OR) as described in Simon-Lukasik and Ludescher (2004); the labeled protein was purified away from unreacted dye by precipitation overnight with w4-fold volume excess of 95% ethanol. The solid was collected by suction filtration onto Whatman #40 filter paper, dispersed in a minimum amount of distilled deionized water, and heated until a clear solution was obtained. The solution was dialyzed (1000 Da MW cutoff dialysis tubing; Spectrum, Houston, TX) extensively dialyzed against distilled deionized water (above 65 8C when possible), and the labeled protein freeze-dried and stored in an airtight and opaque container below 0 8C. The extent of labeling was estimated to be w1 erythrosin:325 residues of gelatin (Simon-Lukasik & Ludescher, 2004). 2.2. Preparation of gelatin films Lyophilized unlabeled gelatin was combined with an appropriate amount of erythrosin-labeled gelatin in deionized water above 65 8C to make 100 mg/mL solutions with a dye content of w9.2!10K6 mol dye/mol residues. This solution was pipetted in 50 mL aliquots onto quartz slides (with approximate dimensions 13 mm!30 mm! 0.6 mm) in a 13 mm!15 mm area. After drying at room temperature (cold-cast films), the slides were placed in a desiccator over P2O5 for 1 week. Hot-cast gelatin films were prepared in the same way, except that slides were equilibrated at w68 8C for 2 h until dry. Films were hydrated by equilibration against saturated salt solutions (Nyqvist, 1983) at room temperature (23.0G1.0 8C) for a minimum of 1 week, protected from light to prevent photobleaching of the probe.

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Gelatin solutions containing glycerol were made using a second preparation of gelatin labeled at w1 erythrosin:240 residues of gelatin; films were doped at w1.7!10K5 mol dye/mol residues. Glycerol was added to 100 mg/mL gelatin solutions at 10, 30 or 50% (w/w, based on the weight of gelatin in the solution). The films, cold- or hot-cast as described above, were desiccated over P2O5 for approximately 2 weeks prior to use. The thickness of the quartz slides was measured in three places with a digital micrometer (Mitutoyo, Aurora, IL) prior to film casting; the thickness of slide plus adsorbed film was measured after spectroscopic measurements; film thickness was calculated by difference using average values. Film thickness was consistent with other edible film and coating studies (Cuq, Gontard, & Guilbert, 1995). 2.3. Phosphorescence measurements All measurements were made on a Cary Eclipse spectrofluorometer (Varian Instruments, Walnut Creek, CA) equipped with a temperature-controlled sample holder; all measurements were done at 23 8C. Quartz slides were placed on end on the diagonal of a standard 1 cm!1 cm quartz fluorescence cuvette; the cuvette was flushed with a gentle stream of air or N2 at the relative humidity at which samples were equilibrated. The error of the humidity meter (provided by Vaisala, Inc. with the instrument’s calibration) was G1% RH from 0 to 90% RH, and G2% RH above 90% RH. For phosphorescence emission spectra each data point (1 nm interval, 0.099 s averaging time) was collected from a single flash with a 0.1 ms delay, 0.5 ms gate, and 4.0 ms total decay time. Excitation was at 540 or 560 nm; the excitation bandpass varied (5 or 10 nm), depending on the requirements of the experiment. Emission was generally measured in the range from 600 to 800 nm (bandpass of 10 or 20 nm, as appropriate). For most experiments, lifetime measurements of teq (lifetime in air) and t0 (lifetime in nitrogen) were made with excitation at 540 nm and emission at 690 nm, with a 20 nm bandpass for both excitation and emission monochromators. Each intensity decay was the average of 100 cycles; for each cycle, data were collected from a single flash with a delay of 0.1 ms, a 0.04 ms gate, and 4.0 ms total decay time. Phosphorescence intensity decays were collected for each sample in triplicate. For determination of the oxygen diffusion rate, phosphorescence intensity was measured as a function of time during initial equilibration in air (Ieq), upon the introduction of the appropriately hydrated nitrogen stream, and continuously with time during flushing of the cuvette interior. Data were collected using 540 nm excitation and 690 nm emission (20 nm bandpass). Data points were collected from a single flash with 0.2 ms delay, 5.0 ms gate, and 20 ms total decay time.

2.4. Data analysis Emission peak energy (np in cmK1) was determined by fitting spectra plotted versus wavenumber (g(n)) to a lognormal function (Maroncelli & Fleming, 1987) in which bandwidth (D), peak intensity (Ip), and an asymmetry parameter (b) were also adjustable parameters: gðnÞ Z Ip expfKln 2ðln½1 C 2bððn K np Þ=DÞÞ2 =bg

(1)

The full width at half maximum (G) of the emission band was calculated from these fit parameters: G Z D½sinhðbÞ=b

(2)

Phosphorescence lifetimes were determined by nonlinear least-squares analysis of intensity decays with the program NFIT (Island Products, Galveston, TX). Intensity decay data were well-analyzed with a stretched exponential decay model IðtÞ Z Ið0Þ exp½Kðt=tÞb  C c

(3)

in which I(t) is intensity as a function of time following pulsed excitation, I(0) the intensity at time zero, t the phosphorescence lifetime, b a stretching factor that characterizes the distribution of decay times (Richert, 1997), and c a constant. As b tends from unity to zero in the expression above, the expression is less single valued (less a single exponential) and the distribution of decay times becomes broader (Lindsey & Patterson, 1980); the fit t essentially describes the value of the lifetime at the peak of the distribution. The stretched exponential, or Kohlrausch–Williams–Watts, decay function is particularly appropriate for describing a complex glass-like system in which there is a distribution of relaxation times for the dynamic molecular processes that depopulate the triplet state in the millisecond timescale of probe emission (Angell, 1995; Richert, 2000). Fits were judged acceptable if they had satisfactory fit errors and if data points were randomly distributed about the fit curve; all data sets had c2%1.0 and most had r2 in the range of 0.99–1.0. Phosphorescence lifetimes were interpreted in terms of the rate constants associated with the various processes that contribute to de-excitation of the excited triplet state of the probe (Duchowicz, Ferrer, & Acuna, 1998). tK1 Z kP Z kRP C kTS1 C kTS0 C kQ ½O2 

(4)

In this expression, kRP is the rate of radiative decay from the triplet state (equal to 41 sK1 for erythrosin phosphorescence; Duchowicz et al., 1998; Lettinga, Zuilhof, & van Zandvoort, 2000). kTS1 is the rate for thermally activated reverse intersystem crossing from the triplet to the singlet excited state (S1); it has an exponential dependence on the 0 energy gap (DE) between S1 and T1: kTS1 ðTÞZ kTS1 expðKDE=RTÞ. The value of kTS1 at 23 8C was estimated 0 as 49 sK1 using kTS1 Z 6:5 !107 sK1 (calculated from data in Duchowicz et al., 1998) and a value of DEZ 34.7 kJ molK1 calculated from the temperature dependence of the ratio of delayed fluorescence to phosphorescence

K.V. Lukasik, R.D. Ludescher / Food Hydrocolloids 20 (2006) 96–105

where     4 p2 Dt px rðx; tÞ Z exp K 2 sin p 2L 4L and 0

BZ

I 4pNA R Pht ip K1 Z Ieq 1000 eff 0 O2

In Eq. (5), I(t) represents intensity as a function of time following the transfer to nitrogen, rather than a decay transient as in the stretched exponential model (Eq. (3)). I0 and Ieq are the phosphorescence intensity values of erythrosin equilibrated in nitrogen and in air, respectively. L represents average film thickness. Reff, the effective interaction distance necessary for quenching to occur in the erythrosin–O2 complex, is assumed to be 1 nm (Jayarajah et al., 2000). NA is Avogadro’s number and pO2 is the partial pressure of oxygen (Z0.21 atm.). ht0i is the average stretched exponential lifetime of erythrosin in the absence of oxygen. The diffusion coefficient D was determined by iterative fit of Eq. (5) to phosphorescence intensity data; values of the diffusion coefficient were input to generate a series of fit curves; the quantity S[(DataKFit)2/Data2] was minimized to find the best value of D for each sample.

3. Results 3.1. Phosphorescence emission energy and red-edge effect in plasticized gelatin films Gelatin films doped with erythrosin-labeled gelatin to a final dye concentration of 9.2!10K6 erythrosin/mol residues were prepared by drying gelatin solutions at either 23 (coldcast) or 68 8C (hot-cast), that is, at temperatures below and far

normalized phosphorescence intensity

(a) 0% RH 58% RH 84% RH 94% RH 97.5% RH

1.0

0.8

0.6

0.4

0.2

0.0 600

650

700

750

800

emission wavelength / nm

(b) normalized phosphorescence intensity

(Duchowicz et al., 1998; Lukasik, 2004). The term kTS0, the rate of non-radiative quenching due to intersystem crossing to the ground state S0, is sensitive to collisions between the local matrix and the probe (Papp & Vanderkooi, 1989; Pravinata, You, & Ludescher, 2005; Schauerte, Steele, & Gafni, 1997; Simon-Lukasik & Ludescher, 2004). The term kQ[O2] describes the rate of collisional quenching of the excited triplet state of the probe by oxygen. The sum of the latter two terms, kTS0CkQ[O2], which is the total rate constant for non-radiative decay (kNR) of the triplet state, was calculated using Eq. (4) and the values of kRP and kTS1 cited above. Intensity versus time data collected in oxygen desorption experiments (I(t)) were analyzed for the diffusion coefficient (D) of oxygen in gelatin films using the following equation (Jayarajah, Yekta, Manners, & Winnik, 2000; Yekta, Masoumi, & Winnik, 1995) ð I0 L dx IðtÞ Z (5) L 0 1 C Brðx; tÞ

99

0% glycerol 10% glycerol 30% glycerol 50% glycerol

1.0 0.8

0.6 0.4

0.2

0.0 600

650

700

750

800

emission wavelength / nm

Fig. 1. The effect of hydration (a) and glycerol content (b) on the phosphorescence emission spectrum of erythrosin-labeled gelatin in coldcast gelatin films.

above the melting point of the gelatin triple helix (Veis, 1964). Phosphorescence emission spectra at 23 8C of cold-cast N2-equilibrated films varied slightly with hydration (varied by equilibration over the range from 0 to 97.5% RH) and with glycerol content (varied from 0 to 50 wt% of the gelatin) (Fig. 1). The phosphorescence emission peak energy (determined by fitting the phosphorescence emission spectrum to a lognormal function, Eq. (1) in Section 2) for both cold- and hot-cast films at 23 8C are plotted as a function of hydration and glycerol content in Fig. 2. Despite the increase in the polarity of the gelatin environment due to an increase in the content of polar plasticizer, the emission energy of the probe varied only slightly with an increase in either hydration or glycerol content. Under all conditions, however, the emission peak energy was lower in cold-cast films than in hot-cast films, although this difference decreased at higher RH. The lower emission energy reflects an increase in the extent of dipolar relaxation around the excited triplet state of the probe (Pravinata et al., 2005; Richert, 2000); since the probe phosphorescence lifetime is actually lower in cold-cast than in hot-cast gelatin (see below), this suggests that the rate of

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(a) 14650

(a) 280 cold-cast hot-cast

260 240

14600

∆ν / cm–1

ν540 / cm–1

220 14550

200 180 160 140

14500 cold-cast hot-cast

120 100

14450 0

20

40

60 RH / %

80

80

100

(b) 14650

0

20

40

60 RH / %

80

(b) 280 cold-cast hot-cast

260 240

∆ν / cm–1

14600 ν540 / cm–1

100

14550

220 200 180 160 140

14500 cold-cast hot-cast

120 100

14450 0

10

20

30

40

50

Glycerol / wt%

80 0

10

20

30

40

50

60

Glycerol / wt %

Fig. 2. The effect of hydration (a) and glycerol content (b) on the peak phosphorescence emission energy of erythrosin-labeled gelatin in cold-cast (B) and hot-cast (C) gelatin films.

Fig. 3. The effect of hydration (a) and glycerol content (b) on the red-edge effect in the phosphorescence emission spectra of erythrosin-labeled gelatin in cold-cast (B) and hot-cast (C) gelatin films.

dipolar relaxation was faster in cold-cast than in hot-cast gelatin films. The red-edge effect (REE) reflects the difference in emission energy with excitation at the peak and the red-edge (540 and 560 nm, respectively, for erythrosin) of the absorption band. This difference is an indicator of the rigidity of the local solvent environment; a decrease in magnitude of the REE reflects an increase in the rate of matrix relaxation around the excited triplet state (Demchenko, 2002; Pravinata et al., 2005). This energy difference generally decreased as the content of plasticizer increased in films with both water (Fig. 3a) and glycerol (Fig. 3b). Hydration caused little if any decrease in the magnitude of the REE in hot-cast and a small decrease in cold-cast films while glycerol induced a small decrease in the magnitude in hot-cast and a more significant decrease in the cold-cast films.

(Simon-Lukasik & Ludescher, 2004). Comparable measurements of intensity decays at 23 8C of erythrosinlabeled gelatin in hot-cast films as a function of hydration were also well fit using a stretched exponential function. Lifetimes (t) from these analyses are plotted versus %RH in Fig. 4a; the corresponding stretching factors (b) are plotted in Fig. 4b. In N2-equilibrated hot-cast films, the lifetimes were constant at 0.557G0.004 ms over the range from 0 to 84% RH, and decreased about 14% (to 0.477 ms) at 97.5% RH. These values were significantly higher than those seen in N2-equilibrated cold-cast films, where the lifetimes were constant at 0.490G0.013 ms over the range from 0 to 84% RH and then decreased to 0.304 ms at 97.5% RH (Simon-Lukasik & Ludescher, 2004). The lifetime was lower in air-equilibrated hot-cast films only at 58% RH and above (Fig. 4a). The effect of oxygen was slight (4% decrease in lifetime) at 58 and 75% RH and then increased dramatically in films equilibrated at higher RH. The influence of oxygen was considerably less in hotcast than in cold-cast films at these intermediate RH values; oxygen decreased the lifetime 12 and 18% in cold-cast films at 58 and 75% RH (Simon-Lukasik & Ludescher, 2004);

3.2. Phosphorescence quenching in water-plasticized gelatin films In a previous study, we reported phosphorescence emission lifetimes in air and N2-equilibrated erythrosinlabeled cold-cast gelatin films as a function of hydration

K.V. Lukasik, R.D. Ludescher / Food Hydrocolloids 20 (2006) 96–105

(a) 0.6

4.0

log (kNR / s–1)

0.5

τ / ms

0.4 0.3

cold-cast/Air cold-cast/N2 hot-cast/Air hot-cast/N2

3.6

3.4

0.2 0.1

3.8

101

hot-cast/Air hot-cast/N2

3.2

0.0 0

20

40

60

80

100

120

0

20

40

60

80

100

120

RH / %

RH / %

Fig. 5. The effect of hydration on the non-radiative decay rate (kNR) for phosphorescence of erythrosin-labeled gelatin in hot-cast gelatin films in the presence of air (:) and nitrogen (C) (calculated from lifetime data in Fig. 4); comparable data for erythrosin-labeled gelatin in cold-cast gelatin films in the presence of air (6) and nitrogen (B) are from Simon-Lukasik and Ludescher (2004).

(b) 1.00 0.95 0.90

β

0.85 0.80 0.75 0.70

hot-cast/Air hot-cast/N2

0.65 0

20

40

60

80

100

120

RH / %

Fig. 4. The phosphorescence lifetime (a) and stretching factor b (b) of erythrosin-labeled gelatin in hot-cast gelatin films as a function of hydration in the presence of air (:) and nitrogen (C).

the influence of oxygen was similar in hot- and cold-cast films at 84% RH and above. The effect of hydration on the rate constant for nonradiative decay (kNR) of the erythrosin triplet state, calculated from the lifetime data, is plotted in Fig. 5 along with comparable data from cold-cast films reported in Simon-Lukasik and Ludescher (2004). In N2-equilibrated hot-cast films, kNR at 23 8C reflected only the rate of intersystem crossing to the ground state; that is, kNRZkTS0. In these films, kNR was 1705 sK1 and constant over the range from 0 to 84% RH and increased above 84% RH, rising gradually to 2007 sK1 at 97.5% RH. kNR was the same within error in air and N2-equilibrated hot-cast films at RH%21%, slightly higher in air-equilibrated films at 58 and 75% RH, and significantly higher in air-equilibrated films above 84% RH. These differences reflected the effect of hydration on the oxygen quenching rate; that is, kNRZ kTS0CkQ[O2] in air-equilibrated films at 58% RH and above and thus the difference between kNR in air and in N2-equilibrated films reflected the effect of hydration on kQ[O2]. The effect of hydration on kNR in hot-cast films was similar to that seen in cold-cast films, except that kNR

was significantly lower in hot than in cold-cast films at all hydration values. The stretching factor b provides a measure of the width of the distribution of lifetimes required to adequately fit the intensity decay data; values near 1 indicate narrow distributions (the distribution has a single lifetime) and the width of the distribution increases dramatically as b decreases to zero (Lindsey & Patterson, 1980). The value of b was constant and identical in both N2 (0.882G0.003) and air-equilibrated (0.882G0.006) hot-cast films over the range from 0 to 84% RH (Fig. 4b); above 84% RH, however, the values of b decreased significantly in both films. This trend with hydration was essentially identical to that seen in cold-cast films (Simon-Lukasik & Ludescher, 2004), although the values of b were slightly lower in N2 and air-equilibrated cold-cast films over the range from 0 to 84% (0.840G0.007 and 0.841G0.006, respectively) and also at higher RH. 3.3. Phosphorescence quenching in glycerol-plasticized gelatin films Intensity decays of erythrosin-labeled gelatin were measured at 23 8C in dry cold- and hot-cast films as a function of the glycerol content from 0 to 50 wt% (films equilibrated at 0% RH); the lifetimes and stretching factors from fits to these data sets are plotted as a function of glycerol content in Fig. 6. The lifetime decreased slightly with an increase in glycerol content in both coldand hot-cast films. Oxygen had no effect on the erythrosin lifetime at any glycerol concentration; the lifetimes were identical within error in N2 and air-equilibrated films at all glycerol contents. The lifetimes, however, were significantly lower in cold-cast than in hot-cast films at all glycerol contents.

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(a) 0.6

cold-cast/Air cold-cast/N2 hot-cast/Air hot-cast/N2

3.35 log (kNR / s–1)

0.5

τ / ms

0.4 0.3

3.30 3.25 3.20

0.2

cold-cast/Air cold-cast/N2 hot-cast/Air hot-cast/N2

0.1

3.15 3.10

0.0 0

10

20

30

40

0

50

10

20

30

40

50

Glycerol / wt%

Glycerol / wt %

Fig. 7. The effect of glycerol content on the non-radiative decay rate (kNR) for phosphorescence of erythrosin-labeled gelatin in cold-cast (6,B) and hot-cast (:,C) gelatin films in the presence of air (6,:) and nitrogen (B,C). Calculated from lifetime data in Fig. 6.

(b) 1.0

β

0.9

0.8 cold-cast/Air cold-cast/N2 hot-cast/Air hot-cast/N2

0.7

0

10

20

30

40

50

Glycerol / wt %

Fig. 6. The phosphorescence lifetime t (a) and stretching factor b (b) of erythrosin-labeled gelatin in cold-cast (6,B) and hot-cast (:,C) gelatin films as a function of glycerol in the presence of air (6,:) and nitrogen (B,C).

The non-radiative decay rates calculated from these lifetime data are plotted versus glycerol content in Fig. 7. The identity in kNR between N2 and air-equilibrated samples indicated that kNRZkTS0 for all glycerol-containing films at 0% RH and 23 8C; that is, kQ[O2]z0 at all glycerol contents. kNR was w1790 sK1 in cold-cast films at low glycerol content (0–10 wt%) and increased to w2220 sK1 at 50 wt% glycerol; kNR was significantly smaller in hot-cast films, rising from w1550 sK1 at low glycerol content (0–10 wt%) to w1690 sK1 at 50 wt% glycerol. Glycerol had essentially no effect on the magnitude of the stretching factor in both cold- and hot-cast films (Fig. 6b). However, b was consistently, albeit only slightly, lower in cold-cast than in hot-cast films at all glycerol contents. 3.4. Oxygen diffusion in plasticized gelatin films The rate of oxygen diffusion in hot-cast films at 53% RH and above was determined from analysis of the rate of

increase in the phosphorescence intensity that accompanied out-gassing from an oxygen saturated film following transfer to a N2 atmosphere (Simon-Lukasik & Ludescher, 2004). Oxygen diffusion rates for hot-cast films equilibrated to RH values between 53 and 84% are listed in Table 1; oxygen diffusion was not detectable by our phosphorescence technique at lower RH values. The diffusion rate increased slightly from 1.1 to 3.1!108 cm2 sK1 on increasing RH from 53 to 75% RH and did not increase further at 84% RH. The diffusion rates in hot-cast films at low RH were the same as those measured in cold-cast films (Table 1; Simon-Lukasik & Ludescher, 2004) but DO2 at 75 and 84% RH was about 2-fold smaller in hot than in coldcast gelatin. Out-gassing in hot-cast films at RHO84% was complex and characterized by multiple diffusion processes; consequently diffusion constants could not be extracted using our analysis procedure; a similar situation also applies in cold-cast films (see Fig. 7 in Simon-Lukasik & Ludescher, 2004). No out-gassing of oxygen was detectable in either coldor hot-cast films at any glycerol content at 0% RH; consequently the rate of oxygen diffusion as monitored by our protocol was indistinguishable from zero in dry glycerol-containing films at 23 8C. Table 1 Estimated oxygen diffusion coefficient in water-plasticized films of erythrosin-labeled hot-cast gelatin RH/%

53 58 75 84 a b

DO2 (10K8cm2 sK1) Hot-cast filmsa

Cold-cast filmsb

1.1G0.6 1.9G0.8 3.1G0.9 2.1G0.9

2.0G1.1 6.1G1.4 5.0G2.0

Data are the average for n samples, where 4%n%5. Data from Simon-Lukasik and Ludescher (2004).

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4. Discussion Phosphorescence from erythrosin-labeled gelatin provides information about three different aspects of molecular mobility and dynamic heterogeneity within the gelatin matrix. First, the emission energy and red-edge effect sense changes in the rate of dipolar relaxation around the excited triplet state (Demchenko, 2002; Ludescher, Shah, McCaul, & Simon, 2001; Pravinata et al., 2005; Richert, 2000); both the emission energy (np) and the magnitude of the REE decrease as the solvent relaxation rate increases. Second, the emission lifetime (t) provides a direct measure of the rate of non-radiative coupling to the ground state (kTS0) due to matrix collisions with the probe (Papp & Vanderkooi, 1989; Pravinata et al., 2005; Schauerte et al., 1997; Vanderkooi & Berger, 1989), while the stretching factor (b) provides a measure of the extent of dynamic heterogeneity due to local variations in kTS0 within the matrix. And, third, the kinetics of oxygen out-gassing provide a direct measure of the rate of oxygen diffusion (DO2 ) through the matrix (Lu, Manners, & Winnik, 2001; Masoumi et al., 1996; Simon-Lukasik & Ludescher, 2004). This study evaluated the effect of plasticizer (water or glycerol) and physical cross-links (triple helices) on each of these modes of molecular mobility within the gelatin matrix. 4.1. Influence of plasticizer on gelatin mobility Water is a ubiquitous plasticizer of biomaterials. In both hot-cast (this study) and cold-cast gelatin (Simon-Lukasik & Ludescher, 2004), hydration had complex effects on the molecular mobility of the matrix. The influence of water can be divided into three different hydration regimes (SimonLukasik & Ludescher, 2004). Hydration at low RH (!w50%) only slightly increased the rate of solvent relaxation; otherwise, the matrix mobility on the submillisecond time scale (kTS0) was unaffected by water. At intermediate RH (from w50 to 84%) the mobility of the gelatin matrix increased slightly; although the rate of solvent relaxation was unaffected beyond that seen at lower hydration, kTS0 increased slightly and oxygen diffusion increased to detectable levels. At high RH (O84%), however, there was a significant increase in molecular mobility in the matrix; although the rate of solvent relaxation was unaffected by additional water, the rate of non-radiative quenching increased dramatically and oxygen diffusion became a complex process describable by multiple diffusion coefficients. The dynamic heterogeneity of the gelatin matrix (as measured by b) was unaffected by hydration over the range from 0 to 84% RH and increased dramatically at higher RH. The dynamic consequences of hydration were thus quite complex at high water contents (corresponding to RHO84%) in both cold- and hot-cast films; the matrix was not only more mobile but the breadth of the distribution of matrix sites due to differences in local matrix mobility (differences in kTS0) was also much broader

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than that seen at lower hydration levels. It is reasonable to speculate that the more complex oxygen diffusion process at high hydration reflects this underlying dynamic heterogeneity. Glycerol, on the other hand, had very little effect on the molecular mobility of the matrix in either cold- or hot-cast gelatin films at 0% RH and 23 8C; increasing glycerol content induced only small increases in the extent of solvent relaxation and the rate of collisional quenching (and no change in the matrix heterogeneity) and had no detectable effect on the rate of oxygen diffusion. Thus, despite its importance as a mechanical plasticizer of gelatin (Arvanitoyannis et al., 1997, 1998a), glycerol had no significant effect on the local molecular mobility of gelatin on the sub-millisecond time scale. 4.2. Influence of physical cross-links on gelatin mobility In contrast to the effect of plasticizers, the presence of physical cross-links (due to casting gelatin films below the melting temperature of the collagen-like triple helices) had a significant influence on the molecular mobility of the gelatin matrix. At all levels of both water and glycerol examined, the rates of solvent relaxation and of collisional quenching (kTS0) were significantly lower in hot-cast than in cold-cast films; the extent of matrix heterogeneity was also significantly lower in hot than in cold-cast films. The rate of oxygen diffusion was unaffected by the presence of crosslinks at 53 and 58% RH, but was w2-fold lower in hot-cast films at higher RH levels. Gelatin films containing crosslinks thus had significantly higher molecular mobility, as well as a broader distribution in mobility due to matrix heterogeneities (lower b), than films without cross-links. Since the effect of cross-links is to increase the macroscopic stability and strength of gelatin films (Bradbury & Martin, 1952; Finch & Jobling, 1977; Fraga & Williams, 1985; Menegalli et al., 1999), we speculate that formation of cross-links in the gelatin solution prior to film formation introduces physical constraints within the dehydrating gel that interfere with the formation of a well-packed polymer matrix held together by strong interactions among the polymer segments; the absence of strong interactions may thus give rise to a more mobile matrix. Whatever the mechanism, it is clear that local molecular mobility and global mechanical properties are not directly coupled in these gelatin films. No information is available on how many physical cross-links are required to modulate the molecular mobility of the polymer matrix, as it was not possible to determine the triple-helix content of these films. Detailed correlations between the measured molecular mobility and the triple helix content are clearly required to determine the specifics of how triple helix formation modulates matrix mobility.

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Acknowledgements This research was supported by a grant ((2002-01585) from the National Research Initiative of the US Department of Agriculture.

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